WO2026006166A1

WO2026006166A1 - Production of pancreatic beta cells in perfusion cultures

Info

Publication number: WO2026006166A1
Application number: PCT/US2025/034760
Authority: WO
Inventors: Stephanie Jinyi DOONG; Thomas Matthew GAGLIARDI; Omar Mohamed ABDILLAHI
Original assignee: Vertex Pharmaceuticals Inc
Current assignee: Vertex Pharmaceuticals Inc
Priority date: 2024-06-24
Filing date: 2025-06-23
Publication date: 2026-01-02
Anticipated expiration: 2026-12-24

Abstract

Disclosed herein are compositions and methods related to differentiation of stem cells into pancreatic islet cells. In some embodiments, the methods provided herein relate to generation of pancreatic β cell, α cell, δ cells, and EC cells in vitro. In some embodiments, the disclosure provides pharmaceutical compositions including the cells generated according to the methods disclosed herein, as well as methods of treatment making use thereof.

Description

PRODUCTION OF PANCREATIC BETA CELLS IN PERFUSION CULTURES

RELATED APPLICATIONS

This application claims the benefit of priority under U.S.C. § 119(e) of U.S. Provisional Application No. 63/663,451, filed June 24, 2024, U.S. Provisional Application No. 63/684,045, filed August 16, 2024 and U.S. Provisional Application No. 63/712,377, filed October 25, 2024, the disclosures of each of which are incorporated herein by reference in their entirety.

BACKGROUND

Transplantation of pancreas or pancreatic islets has been used for treating diabetes, such as type I diabetes. Pancreatic islet transplantation does not need major surgery and the function of the islet grafts can be maintained for years in a recipient. However, a shortage of pancreatic islets donors prevents this therapy from being effectively implemented. Artificial pancreas or pancreatic islets provide an alternative source of transplantable islets. Thus, there is a need for methods of in vitro restitution of pancreatic islets whose function and characteristics resemble endogenous pancreatic islets.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (V013870099WO00-SEQ-JSH.xml; Size: 35,925 bytes; and Date of Creation: June 18, 2025) are herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.

SUMMARY

Tangential flow filtration (TFF) compositions and systems including a cell culture, wherein the cell culture comprise a liquid media and/or a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor are provided herein, along with methods of making and using the same.

Also provided are methods that include culturing a cell culture in a bioreactor, wherein the cell culture comprises a liquid media and a plurality of cell clusters, transporting a portion of the cell culture from the bioreactor into a TFF system, removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system, returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor, and/or replacing the removed portion of the liquid media with a new portion of liquid media. Compositions and systems for performing these methods are also disclosed.

In one aspect, a method is provided. According to some embodiments, the method comprises the steps of: (a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; (b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; (c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; (d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; and (e) replacing the removed portion of the liquid media with a new portion of liquid media.

In another aspect, a tangential flow filtration (TFF) system is provided. According to some embodiments, the TFF system comprises: a cell culture, wherein the cell culture comprises a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor.

In yet another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 400.

In still another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a shear rate of greater than or equal to 400 s'¹.

In another aspect, a method for culturing cells is provided. According to some embodiments the method comprises: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; and controlling the flow of the portion of the cell culture through the TFF system to shear the plurality of cell clusters to provide an average maximum transverse dimension of the plurality of cell clusters that is between or equal to 75 pm and 600 pm.

In one aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a Reynold’s number (Re) of less than or equal to 400 to the portion of the portion of the cell culture pumped to the TFF system.

In another aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a shear rate of greater than or equal to 400 s'¹ to the portion of the portion of the cell culture pumped to the TFF system.

In yet another aspect, a system for culturing cells is provided. According to some embodiments, the system comprises: a bioreactor configured to contain a cell culture, the bioreactor comprising: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

In still another aspect, a method for culturing cells is provided. According to some embodiments, the method comprises: alternatingly performing the steps of: drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; and wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor. The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1C are exemplary diagrams of media exchange systems. FIG. 1A depicts an exemplary alternating tangential flow filtration (ATF) media exchange system, where the ATF system comprises a column having a plurality of hollow fibers. In this exemplary ATF system, cell culture is pumped out of the bioreactor by the diaphragm pump into the ATF system, where liquid media is removed through the pores of the hollow fibers as permeate and cell clusters are retained as retentate within the hollow fibers. The retentate is then returned back to the bioreactor by the diaphragm pump. Media that was lost from the bioreactor (as a result of the permeate removed by the ATF system) is replenished in the bioreactor as fresh media by the “Feed in.” FIG. IB depicts an exemplary settling media exchange system. FIG. 1C depicts an exemplary continuous flow tangential flow filtration (ATF) media exchange system according to some embodiments.

FIG. ID schematically illustrates a method of exchanging spent media using centrifugation, according to some embodiments.

FIGs. 2A-2C provide schematic cross-sectional views of various non-limiting cell culture systems, according to some embodiments.

FIG. 3 provides a non-limiting schematic flow diagram of a method of agitating fluid during perfusion culture, according to some embodiments.

FIGs. 4A-4B demonstrate expansion of human embryonic stem cells (hESCs) using a continuous perfusion system. FIG. 4A depicts fold expansion 3 days after adaptation into R01- R06 (1st bar in each pair) or 3 days after in-vessel-passage (IVP) (2nd bar in each pair), as described in Example 1. FIG. 4B depicts the percentage of SOX17-negative and Oct4-positive cells in each reactor as measured by flow cytometry.

FIGs. 5A-5B show clusters from reactors R13 and R14. FIG. 5A depicts reactor R13 clusters three days post IVP. FIG. 5B depicts reactor R14 clusters six days post-adaptation, without IVP. The clusters continued to grow significantly from 3 days, post-adaptation, to 6 days post- adaptation.

FIG. 6 shows the percentage of NKX6.1-positive, ISLl-positive cells in each reactor after completion of a 5-stage differentiation protocol using different media exchange methods. NKX6.1 and ISL1 were assessed by flow cytometry. Reactor conditions can be found in Table 2. Labels R14-1, R14-2, and R14-3 refer to Biotts from R14 seeded on days 4, 5, and 6, respectively, post adaptation.

FIG. 7 shows the yield of NKX6.1-positive/ISLl-positive cells produced in exemplary rapid media exchange in reactors compared with centrifugation exchange methods, according to some embodiments.

FIG. 8 compares the growth curves throughout the differentiation process for cell culture using rapid media exchange and cell culture using centrifugation, according to some embodiments.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

For purposes of this description, certain embodiments, advantages, and novel features of the embodiments of this disclosure are described herein. The disclosed methods, apparatus, and systems should not be construed as being limiting in any way. Instead, the present disclosure is directed toward all novel and nonobvious features and embodiments of the various disclosed embodiments or examples, alone and in various combinations and sub-combinations with one another. The methods, apparatus, and systems are not limited to any specific embodiment or feature or combination thereof, nor do the disclosed embodiments require that any one or more specific advantages be present or problems be solved.

Although the operations of some of the disclosed embodiments are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, unless the context clearly dictates otherwise, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed methods can be used in conjunction with other methods. Additionally, the description sometimes uses terms like “provide” or “achieve” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art.

All features described herein are independent of one another and, except where structurally impossible, can be used in combination with any other feature described herein. For example, a feed in as shown in FIG. 1A can be included in the settling media exchange system depicted in FIG. ID.

FIG. 1A a non-limiting schematic diagram of a cell culture system 100, according to some non-limiting embodiments. Cell culture system 100 comprises a bioreactor 110 including an internal volume configured to contain a liquid cell culture media 120. It should be understood that the system 100 may be used with any of the cells, cell culture media, and other compositions disclosed herein as the disclosure is not limited in this fashion. The bioreactor may be configured to mix the cell culture media 120 using a mixer 112. The mixer in FIG. 1 A is represented as an impeller. However, it should, of course, be understood that any of a variety of mixers (e.g., magnetic mixing bars, ultrasonic mixers, rocker tables, etc.) may also be used as the disclosure is not so limited. In some embodiments, fresh cells and/or cell culture media may be added to the bioreactor via feed inlet channel 114 that is configured to be coupled to one or more suitable sources of cells and/or cell culture media.

FIG. 1A additionally depicts an exemplary alternating tangential flow filtration (ATF) media exchange system 150. In some embodiments, the ATF system 150 comprises a column 152 having a plurality of porous, hollow fibers extending along at least a portion of a length, and in some instances the entire length, of the column 152. The porous hollow fibers, or other appropriate filter, may be configured to retain cells within a retentate while transmitting a permeate out of the fibers or other appropriate filter via the pores. It should, of course, be understood that although the ATF system of FIG. 1 A is primarily described as comprising a plurality of porous fibers including a central lumen extending along their length that is in fluid communication with the bioreactor 110, any of a variety of appropriate columns may be used, including columns with a single lumen through which the cell culture media is passed, as the disclosure is not so limited. In this exemplary ATF system 150, cell culture media 120 is pumped out of the bioreactor by a pump 154 into the ATF system 150, where liquid media is removed through the pores of the hollow fibers as permeate and cell clusters greater than a predetermined size threshold are retained as retentate within the hollow fibers or other filter. In the depicted embodiment with a diaphragm pump, the cell culture media 120 may (reversibly) be passed from the bioreactor 110 to the column 152 via an outlet channel 116 that fluidly connects the bioreactor 110 to the column 152. The reversible flow of the cell culture media 120 into the column 152 from the bioreactor 110 and retentate into the bioreactor 110 from the column 152 is indicated by bidirectional flow arrow 130. Fluid flow into or out of the ATF system 150 may be directed using one or more pumps. In FIG. 1A, pump 154 is represented as a diaphragm pump to provide bidirectional flow along a single flow path into and out of the ATF system. However, it should be understood that any of a variety of pumps may be used, depending on the embodiment. For example, pump 154 may be a diaphragm pump, a positive displacement pump, a peristaltic pump, a lobe pump, a rotary vane pump, or any of a variety of other kinds of pump, depending on the embodiment. Further, while a bidirectional flow arrangement has been shown, as elaborated on further below relative to FIG. 1C, a single directional flow arrangement for the transport of cell culture media and retentate between the bioreactor 110 and the ATF system 150 may also be used.

As noted above, column 152 may be configured to retain a retentate comprising cells or cell clusters greater than a predetermined size threshold, from the cell culture media, while passing a permeate (e.g., comprising spent cell culture media) out of the column 152 to a column outlet 158. The retentate may then be returned to the bioreactor by the diaphragm pump 154. Thus, the portion of the cell culture including cell clusters greater than a threshold size included in the retentate may be returned to the bioreactor from the ATF system 150 or other TFF system as elaborated on further below. During this process of flowing the cell culture media into and out of the ATF or other TFF system, shear stresses may also be applied to the flow of media through the column 152 such that the shear stresses break apart cell clusters greater than the desired cell cluster size range. Media that was lost from the bioreactor (as a result of the permeate removed by the ATF system) may be replenished in the bioreactor by fresh media provided to the interior volume of the bioreactor 110 via feed inlet channel 114 fluidly coupled to a fresh cell culture media source. In some embodiments, the flow paths associated with the media inlet channel 114 may include a unidirectional valve (e.g., a ball valve, duckbill valve, or other appropriate unidirectional valve) to avoid backflow of media. Similarly, the outlet 158 of the ATF system 150 may also include a unidirectional valve (e.g., a ball valve, duckbill valve, or other appropriate unidirectional valve) to prevent backflow of permeate back into the ATF system 150. As discussed above, any of a variety of suitable tangential flow filtration columns may be used including, but not limited to, a tangential flow filtration column comprising a plurality of elongated porous hollow fibers including lumens extending along their length. The lumens may be in fluid communication with the pump 154 and bioreactor 110. FIG. IB depicts a nonlimiting example of a tangential flow filtration column comprising a plurality of fibers, illustrating the fibers as they appear across the transverse cross-section of column 152 taken at line IB- IB in FIG. 1A. The lumens 173 may be in fluid communication with a first port of the column in fluid communication with the bioreactor 110 and the outlet 158 may be in fluid communication with a volume surround the elongated porous hollow fibers of the ATF system 150. Thus, during operation fluid passed to the column may enter the column 152 via the lumens 173 of the fibers 171 and may be separated into a permeate comprising spent cell culture media, which is passed out of the column through the porous walls of the elongated porous hollow fibers, and a retentate comprising concentrated cells, which is retained within the lumen of the fibers. Other embodiments of tangential flow filtration columns are also possible, as the disclosure is not so limited. In either case, after filtration, the retentate may be returned to bioreactor 110 and mixed back into cell culture media 120 via operation of a mixer 112 that is configured to mix the cell culture media 120 within the bioreactor 110.

Although FIG. 1A shows an ATF system, it should of course be understood that ATF is not the only form of tangential flow filtration that may be used. For example, FIG. 1C presents a non-limiting embodiment of a cell culture system 100 comprising a tangential flow filtration (TFF) system that is configured for continuous fluid flow (indicated by unidirectional flow arrows 130) through a TFF column 152 that includes a first inlet port in fluid communication with the bioreactor 110 and a second outlet port that is also in fluid communication with the internal volume of the bioreactor 110. A pump 154 may be associated with either an upstream or downstream portion of the flow path extending between the inlet and outlet of the above noted inlet and outlet ports. Like the ATF column 152 of FIG. 1 A, the TFF column 152 of FIG. 1C is configured to retain cells and/or cell clusters greater than a threshold size within a retentate while passing a permeate comprising spent cell culture media, cell debris, and cell clusters less than the noted size threshold out of column 152 via the outlet 158 of the TFF column 152. The TFF column may have a similar design to the ATF column of FIG. 1 A. Any of a variety of pumps 154 may be used to pump liquid through column 152. For example, pump 154 of FIG. 1C is represented as an in-line pump, but it should of course be understood that other pump configurations are also possible. As indicated by flow arrows 130, cell culture media retained within column 152 may be passed back to the bioreactor 110, where it can be mixed back into cell culture media 120 using the mixer 112, as described above. After a predetermined time period and/or after a desired percentage of the cell clusters are within a desired range of cell cluster size, the cell culture media contained within the bioreactor may be subjected to any other desired subsequent processing steps including flowing the cell culture media 120 out of the bioreactor 110 to one or more other systems and/or containers.

It should be noted that while the above disclosed arrangement may be used to perform the shearing and filtration processes periodically, in other embodiments the disclosed systems and methods may be used to continuously circulate cell culture media 120 through a filtration arrangement during a filtering process rather than an alternating flow as depicted in FIG. 1A. Thus, the disclosed systems may be used to either continuously or periodically transport a cell culture to a TFF system and return the filtered portion including the cell clusters above a desired threshold (i.e., the retentate) to the bioreactor. One embodiment of a continuously circulating system is elaborated on further below relative to FIG. ID.

FIG. ID illustrates one exemplary method of concentrating cells or cell clusters without tangential flow filtration of any kind. FIG. ID illustrates a bioreactor 180 comprising cells 181 and spent media 182, being mixed within an internal volume of the bioreactor 180 using a mixer 183. In a first step 190, cells are concentrated to the bottom of the bioreactor (e.g., by using centrifugation or by allowing the cells to settle after turning off the mixer 183). Spent media 182 can then be removed as indicated by arrow 192 from a portion of the indicated liquid volume vertically above the settled cells and cell clusters relative to a local direction of gravity. In step 194, new media 185 is then added to the volume of the bioreactor 180 as indicated by arrow 196. Optionally, the cells, cell clusters, and new cell culture media can be mixed using mixer 183. Whereas the systems illustrated in FIGs. 1A-1C are configured for continuous operation, methods as illustrated in FIG. ID may be considered a batch rather than continuous production method.

A significant advantage of the systems and methods for perfusion provided herein is that they allow continuous replacement of cell culture media, providing cells with fresh nutrients and removing waste produced by the cultured cells. These benefits are related, in at least some embodiments, to the circulation of fresh cell culture media into the bioreactor, e.g., by adequate mixing and agitation. However, cells that settle within isolated dead zones (e.g., substantially non-circulating zones) of a bioreactor during a cell culturing process may starve or otherwise die as a result of their inability to mix with fresh cell culture media. The existence of dead zones can be very problematic, since over time fresh cells may settle into the dead zone while dead cells cycle out. It has been recognized, in the context of the present disclosure, that the existence of even a small dead-zone of the bioreactor can, in at least some cases, kill as much as 90% of the cells grown during a cell culture process. The present disclosure recognizes the problems associated with dead zones within bioreactors during cell culturing and provides technical and methodological improvements that can prevent their formation, thereby improving culture yield.

It has been recognized herein that dead zones of the cell culture are disproportionately likely to appear at ports (e.g., inlets or outlets) of the bioreactor and/or at the bottom of the bioreactor (with respect to local gravity). This dead zone formation effect may be compounded by the type and location of mixers within a bioreactor, since without wishing to be bound by any particular theory, the ability of a mixer to agitate a fluid depends, at least in part, on the location of the fluid relative to the mixer. In some embodiments, the bioreactor contains a mixer (e.g., an impeller such as impeller 112 discussed above with reference to FIG. 1 A) that mixes the cell culture via rotation around an axis. In some cases, fluid beneath the mixer may be more resistant to rotation and mixing. For example, without wishing to be bound by any particular theory, fluid disposed beneath the mixer may be aligned with the mixer’s axis of rotation, meaning that the fluid is subjected to less centrifugal force than fluid further from the axis of rotation of the mixer. Moreover, without wishing to be bound by any particular theory, in some embodiments a mixer creates a vortex beneath the mixer, and the vortex may contribute to trapping cells within a deadzone formed beneath the mixer.

Both the risk of dead zone formation and the potential harm for cell culture are exaggerated for ports at the bottom of the bioreactor. The bottom of the bioreactor may be prone to dead zone formation, without wishing to be bound by any particular theory, because of the tendency of cells or cell clusters to settle towards the bottom as a result of the action of gravity on the cells. This effect may be amplified in the context of culturing cell clusters, since cell clusters are larger and more massive than individual cells, and may tend to settle more quickly. And the effects of cell settling and dead-zone formation can be compounded by the shape of the reactor vessel itself. For example, in some embodiments a bioreactor comprises a bottom angled towards the port in order to ensure proper drainage of the bioreactor vessel towards the bottom port when the bottom port is open. For example, the bioreactor may have a conical or tapered bottom angled towards the port. But even when the port is closed, the settling of cultured cells can, in some embodiments, be concentrated towards the port by the angled bottom, intensifying the risk of dead zone formation discussed above.

However, the use of a port (e.g., a first port) situated at the bottom of the bioreactor with respect to local gravity (e.g., when the bioreactor is disposed on a level surface) provides technical advantages in at least some embodiments. For example, a port situated at the bottom of the bioreactor may, in some embodiments, be convenient for using gravity to drain the bioreactor (e.g., after a culture process is complete). A dead zone of the cell culture may, in some cases, form adjacent to a port disposed at the bottom of a bioreactor. Several factors may contribute to the formation of such a dead-zone. Without wishing to be bound by any particular theory, in at least some cases, ports may be shaped in a way that creates a dead zone (e.g., because a port is formed in a cavity of the bioreactor, and the cavity acts as a harbor that dampens fluid motion).

A solution for preventing dead zone formation near a port is recirculating fluid through the port and into another part of the bioreactor. This can be accomplished, for example, by pumping the fluid from the port to another port of the bioreactor. However, this solution has certain drawbacks, particularly for culturing cell clusters. In particular, in some embodiments, this type of pumping can stress cells unduly, particularly in the context of cell cluster culturing. The result is that this process can negatively impact culture efficiency, even if it eliminates a dead zone

The present disclosure provides, in some aspects, improved systems and methods for agitating fluid located near a port of a bioreactor to prevent dead zone formation and improve cell culture efficiency by applying low shear pressure oscillations to the fluid via the port to disperse cells in a portion of the bioreactor adjacent to the port back into solution. The agitation does not require recirculation of the fluid from the dead zone to another portion of the bioreactor, which may help to avoid undue stressing of the cells. Rather, the agitation simply ensures that fluid near the port adequately mixes with fluid in the bulk of the bioreactor such that fluid adjacent to the port is routinely mixed with the rest of the cell culture to prevent dead zone formation near the port. The systems and methods provided herein may, in some embodiments, be particularly advantageous when used to agitate fluid near a drain port disposed at the bottom of the bioreactor (with respect to local gravity) and/or beneath a mixing apparatus of the bioreactor to both provide fluid mixing as well as dispersing of cells back into solution.

According to some embodiments, the system is configured to agitate fluid near a port (e.g., a first port disposed at the bottom of a bioreactor) by altematingly flowing cell culture media into and out of the bioreactor via the port. In some embodiments the system comprises a fluid conduit connected to the port such that, in at least some configurations of the port, fluid can flow from the bioreactor to the fluid conduit via the port.

The system comprises a pressure source connected to the port via the fluid conduit, according to some embodiments. The pressure source may be configured to change the pressure within the fluid conduit. A pressure change may be used to draw a portion of cell culture media out of a bioreactor through a port in the bottom of the bioreactor and into the fluid conduit. Likewise, a pressure change may be used to return a portion of the cell culture media from the fluid conduit to the bioreactor through the port. The drawing of the portion of cell culture media out of the bioreactor and the return of the portion of cell culture media to the bioreactor may be alternated in order to agitate cells near the bottom of the bioreactor. For example, in some embodiments, the pressure source is configured to increase the pressure of the fluid conduit to force fluid into the bioreactor from the fluid conduit. Any of a variety of pressure sources may be used, depending on the embodiment. For example, in some embodiments, the pressure source is a pump (e.g., a diaphragm pump, a peristaltic pump) or a compressed gas source (e.g., a compressed air tank, a compressed inert gas tank, pressurized house air or gas, etc.), and/or any other appropriate type of source of pressurized gas and/or vacuum. Depending on the embodiment, the pressure source may be configured to raise or lower pressure in the fluid conduit. For example, the pressure source may be a reversable pump configured to be actuated between a pressure-increasing state and a pressure-decreasing state, e.g., by pumping fluid into or out of the fluid conduit, respectively. Thus, a single pressure source can, in some embodiments, be used to agitate fluid near the port by altematingly pressurizing and depressurizing the fluid conduit to flow cell culture media into and out of the bioreactor, respectively. However, embodiments in which multiple different pressure sources at different pressures may be used as the disclosure is not so limited.

In some embodiments, the pressure source is configured to work in concert with a pressure sink. The pressure sink may be configured to change the pressure within the fluid conduit. For example, in some embodiments, the pressure sink is configured to decrease the pressure of the fluid conduit to allow fluid from the bioreactor to flow into the fluid conduit. For example, the pressure sink may be configured to apply a pressure that is less than a pressure of the pressure source during flow back into the bioreactor from the fluid conduit. In some embodiments, the pressure sink is the pressure source. However, the pressure sink and the pressure source may be a different separate component, depending on the embodiment. For example, in some embodiments, the system further comprises a valve (e.g., a manual valve or a controllable valve) configured to be actuated between a first state, where the fluid conduit is fluidly connected to the pressure source but not the pressure sink, and a second state, where the fluid conduit is fluidly connected to the pressure sink but not the pressure source, in order to altematingly pressurize and depressurize the fluid conduit to flow cell culture into and out of the bioreactor, respectively. Any of a variety of pressure sinks may be used. For example, in some embodiments, the pressure sink is an external atmosphere. In other embodiments, the pressure sink may be a vacuum source configured to apply a pressure that is less than an atmospheric pressure to the fluid conduit during flow out of the bioreactor into the fluid conduit. As another example, in some embodiments, a pressure sink is a pump configured to pump fluid out from the fluid conduit.

It has been discovered that altematingly drawing a portion of cell culture media from the port and returning it to the port to produce low shear pressure oscillations near the port can agitate the cells near the port to ensure that they remain adequately supplied with cell culture media and are dispersed back into solution without unduly stressing the cells, improving cell culture yield. According to some embodiments, it may be desirable to control the applied shear rates to be less than a desired threshold shear rate to avoid risking damage to the cultured cells. Appropriate control of the fluid flow may be particularly important in the context of culturing cell clusters, which can be more sensitive than ordinary cells to inhospitable fluid flow conditions.

Accordingly certain aspects of the present disclosure relates to controlling the shear experienced by the portion of cell culture media flowed into the conduit. According to some embodiments, the system comprises a relief valve. The relief valve may be fluidically connected to the fluid conduit, e.g., such that the pressure at the relief valve will tend to equalize with the pressure of the fluid conduit. In some embodiments, the relief valve is configured to limit the pressure differential between the fluid conduit and an external atmosphere. For example, the relief valve may be configured to permit fluid exchange between the fluid conduit and the external atmosphere, e.g., by passing gas into or out of the relief valve. The relief valve may be used to moderate the pressure of the fluid conduit. For example, in some embodiments, when a pressure source is used to force a portion of culture media out of the fluid conduit and into the bioreactor, the fluid conduit may exceed a desired threshold pressure due to pressurization from the pressure source, with the result that the flow rate of the portion of culture media back into the bioreactor upon reversing the flow direction may be greater than a desired flow rate which may apply too much shear to the cells during return of the cell culture fluid to the bioreactor. Thus, a relief valve may be used to keep the pressure driving flow of the portion of cell culture media below a threshold value that would subject the portion to excessive shear, resulting in improved culture yield. The relief valve may be actuated to vent pressurized gas from the fluid conduit above a threshold pressure by any of a variety of appropriate methods. For example, in some embodiments the relief valve is actuated manually, is electronically actuated in response to pressure sensed in the fluid conduit by a pressure sensor, is configured to passively vent above the threshold pressure, and/or may be configured in any other appropriate manner to permit venting of the gas within the conduit when a pressure is greater than the desired threshold pressure. In either case, according to some embodiments, the relief valve is configured to remain closed until subjected to a threshold pressure, at which point the relieve valve actuates to relieve the excess pressure by venting to the surrounding external atmosphere or other appropriate volume. In some embodiments, the relief valve is configured to stay open, and is configured to maintain but limit a pressure differential between the external atmosphere and the fluid conduit by acting as a fluid flow barrier. For example, in some embodiments, the relief valve may be or comprise a high resistance flow barrier (e.g., an effusion barrier) configured to limit the rate of gas flow through the relief valve, thereby maintaining a pressure differential between the external atmosphere and the fluid conduit. Other embodiments are also possible, as the disclosure is not so limited.

The relief valve may be configured to connect the fluid conduit with any of a variety of appropriate types of external atmosphere. For example, the external atmosphere may be an air atmosphere. However, there is no reason why the bioreactor cannot be used in another external atmosphere (e.g., an inert external atmosphere such as a nitrogen or noble gas atmosphere), depending on the embodiment. Likewise, the external atmosphere could have any of a variety of appropriate temperatures and pressures, depending on the embodiment. The external atmosphere may, in some cases, act as a pressure and/or temperature reservoir that maintains substantially constant pressure and/or temperature during exchange with the cell culture system. For example, when gas flows through the relief valve between the fluid conduit and the external atmosphere, the external atmosphere experiences little, if any, change in pressure or temperature, according to some embodiments. Alternatively, the relief valve may fluidly connect the fluid conduit with a controlled isolated gas volume and/or gas source at a controlled pressure as the disclosure is not limited to connections with the external surrounding atmosphere.

Whether or not a relief valve is used, the cell culture system may be configured, according to some embodiments, to maintain an appropriate average shear rate within the fluid conduit to avoid damaging the cells and/or cell clusters suspended within the liquid cell culture media being moved in and out of the fluid conduit through the associated port. In some embodiments, the cell culture system is configured to maintain an average shear rate within the fluid conduit and port of less than or equal to 4000 sec'¹, less than or equal to 3000 sec'¹, less than or equal to 2000 sec'¹, less than or equal to 1800 sec'¹, less than or equal to 1600 sec'¹, less than or equal to 1400 sec'¹, less than or equal to 1200 sec'¹, less than or equal to 1000 sec'¹, less than or equal to 800 sec'¹, or less than or equal to 600 sec'¹. In some embodiments, the cell culture system is configured to maintain an average shear rate within the fluid conduit and port of greater than or equal to 100 sec'¹, greater than or equal to 200 sec'¹, greater than or equal to 400 sec'¹, greater than or equal to 600 sec'¹, greater than or equal to 800 sec'¹, greater than or equal to 1000 sec'¹, greater than or equal to 1200 sec'¹, greater than or equal to 1400 sec'¹, greater than or equal to 1600 sec'¹, greater than or equal to 1800 sec'¹, greater than or equal to 2000 sec'¹, or greater than or equal to 3000 sec'¹. Combinations of these ranges are also possible (e.g., greater than or equal to 200 sec'¹ and less than or equal to 4000 sec'¹, or greater than or equal to 400 sec'¹ and less than or equal to 2000 sec'¹). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. The cell culture system may be configured to provide pressure agitation by drawing and returning portions of cell culture media with any of a variety of appropriate volumes. In some embodiments, a portion of cell culture media drawn into a fluid conduit and/or returned from the fluid conduit into the bioreactor has a volume of greater than or equal to 1 mL, greater than or equal to 10 mL, greater than or equal to 50 mL, greater than or equal to 100 mL, greater than or equal to 200 mL, greater than or equal to 400 mL, greater than or equal to 600 mL, greater than or equal to 800 mL, greater than or equal to 1000 mL, greater than or equal to 1200 mL, greater than or equal to 1400 mL, greater than or equal to 1600 mL, greater than or equal to 1800 mL, greater than or equal to 2000 mL, greater than or equal to 2200 mL, greater than or equal to 2400 mL, greater than or equal to 2600 mL, or greater than or equal to 2800 mL. In some embodiments, a portion of cell culture media drawn into a fluid conduit and/or returned from the fluid conduit into the bioreactor has a volume of less than or equal to 3000 mL, less than or equal to 2800 mL, less than or equal to 2600 mL, less than or equal to 2400 mL, less than or equal to 2200 mL, less than or equal to 2000 mL, less than or equal to 1800 mL, less than or equal to 1600 mL, less than or equal to 1400 mL, less than or equal to 1200 mL, less than or equal to 1000 mL, less than or equal to 800 mL, less than or equal to 600 mL, less than or equal to 400 mL, less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL, or less than or equal to 10 mL. Combinations of these ranges are also possible (e.g., greater than or equal to 1 mL and less than or equal to 3000 mL, greater than or equal to 10 mL and less than or equal to 500 mL, greater than or equal to 50 mL and less than or equal to 100 mL, or greater than or equal to 600 mL and less than or equal to 100 mL). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

A portion of cell culture media drawn into the fluid conduit and/or a portion of cell culture media returned to the bioreactor may have a volume representing any of a variety of suitable proportions of a total volume of the bioreactor. For example, in some embodiments, a ratio of a volume of a portion of cell culture media to a volume of a bioreactor is greater than or equal to 0.100%, greater than or equal to 0.125%, greater than or equal to 0.150%, greater than or equal to 0.175%, greater than or equal to 0.200%, greater than or equal to 0.225%, greater than or equal to 0.250%, or greater than or equal to 0.275%. In some embodiments, a ratio of a volume of a portion of cell culture media to a volume of a bioreactor is less than or equal to 0.300%, less than or equal to 0.275%, less than or equal to 0.250%, less than or equal to 0.225%, less than or equal to 0.200%, less than or equal to 0.175%, less than or equal to 0.150%, or less than or equal to 0.125%. Combinations of these ranges are also possible (e.g., greater than or equal to 0.100% and less than or equal to 0.300%, or greater than or equal to 0.150% and less than or equal to 0.250%). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

Cell culture media may be drawn into and/or removed from the conduit with any of a variety of suitable flow rates. In some embodiments, cell culture media is drawn into and/or removed from the conduit with a flow rate of greater than or equal to 0.5 L/min, greater than or equal to 1 L/min, greater than or equal to 2 L/min, greater than or equal to 3 L/min, greater than or equal to 4 L/min, greater than or equal to 5 L/min, greater than or equal to 6 L/min, greater than or equal to 7 L/min, greater than or equal to 8 L/min, greater than or equal to 9 L/min, greater than or equal to 10 L/min, greater than or equal to 11 L/min, greater than or equal to 12 L/min, greater than or equal to 13 L/min, or greater than or equal to 14 L/min. In some embodiments, cell culture media is drawn into and/or removed from the conduit with a flow rate of less than or equal to 15 L/min, less than or equal to 14 L/min, less than or equal to 13 L/min, less than or equal to 12 L/min, less than or equal to 11 L/min, less than or equal to 10 L/min, less than or equal to 9 L/min, less than or equal to 8 L/min, less than or equal to 7 L/min, less than or equal to 6 L/min, less than or equal to 5 L/min, less than or equal to 4 L/min, less than or equal to 3 L/min, less than or equal to 2 L/min, or less than or equal to 1 L/min. Combinations of these ranges are also possible (e.g., greater than or equal to 0.5 L/min and less than or equal to 15 L/min, greater than or equal to 0.5 L/min and less than or equal to 1.5 L/min, or greater than or equal to 5 L/min and less than or equal to 15 L/min). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

Cell culture media can be draw into and/or removed from the fluid conduit using any of a variety of appropriate timescales. In some embodiments, a portion of cell culture media is drawn into a fluid conduit and/or returned from the fluid conduit into the bioreactor over a period of greater than or equal to 1 s, greater than or equal to 3 s, greater than or equal to 5 s, greater than or equal to 10 s, greater than or equal to 30 s, greater than or equal to 60 s, greater than or equal to 90 s, greater than or equal to 120 s, greater than or equal to 150 s, greater than or equal to 180 s, greater than or equal to 210 s, greater than or equal to 240 s, greater than or equal to 270 s, greater than or equal to 300 s, greater than or equal to 330 s, greater than or equal to 360 s, greater than or equal to 390 s, greater than or equal to 420 s, greater than or equal to 450 s, greater than or equal to 480 s, greater than or equal to 510 s, greater than or equal to 540 s, or greater than or equal to 570 s. In some embodiments, a portion of cell culture media is drawn into a fluid conduit and/or returned from the fluid conduit into the bioreactor over a period of less than or equal to 600 s, less than or equal to 570 s, less than or equal to 540 s, less than or equal to 510 s, less than or equal to 480 s, less than or equal to 450 s, less than or equal to 420 s, less than or equal to 390 s, less than or equal to 360 s, less than or equal to 330 s, less than or equal to 300 s, less than or equal to 270 s, less than or equal to 240 s, less than or equal to 210 s, less than or equal to 180 s, less than or equal to 150 s, less than or equal to 120 s, less than or equal to 90 s, less than or equal to 60 s, less than or equal to 30 s, less than or equal to 10 s, less than or equal to 5 s, or less than or equal to 3 s. Combinations of these ranges are also possible (e.g., greater than or equal to 1 s and less than or equal to 600 s, greater than or equal to 3 s and less than or equal to 180 s, greater than or equal to 3 s and less than or equal to 5 s, or greater than or equal to 30 s and less than or equal to 150 s). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

The system is, according to some embodiments, configured to maintain the sterility of the cell culture. Maintaining the sterility of the cell culture is important, according to some embodiments, for both cell survival and to the subsequent safe use of cultured cells for biomedical applications. Accordingly, in some embodiments, the systems and methods provided herein are configured to draw and/or return a portion of cell culture media to the bioreactor without introducing undesired microorganisms (e.g., bacterial or fungal cells) to the culture.

In some embodiments, the system comprises a gas filter configured to permit gas flow from a separate gas source and/or external environment while barring the passage of cells. The gas filter may be used to maintain the sterility of the system. In some embodiments, the gas filter is disposed between a relief valve and an external atmosphere, e.g., to prevent cells, particulates, and/or other contamination in the external atmosphere from entering the portion of cell culture media in the fluid conduit. A gas filter used in this way may, in some embodiments, double as an effusion barrier and thereby may act as a relief valve in the manner discussed above. However, in some embodiments, the gas filter may not maintain a significant pressure differential for gas on opposite sides of the gas filter, and a separate relief valve may be used under such circumstances.

In some embodiments, it may also be desirable to prevent potential contamination from a pressure source and/or pressure sink to the bioreactor. Thus, depending on the embodiment, a gas filter may be used to separate the pressure source and/or the pressure sink from the portion of cell culture media, e.g., so that the gas filter prevents entry of cells from the pressure source and/or pressure sink into the cell culture. However, depending on the configuration of the pressure source and/or the pressure sink, a gas filter might not be used in every embodiment. For example, in some embodiments the pressure source is a reversable pump configured to physically isolate fluid in the system from fluid outside the system. For example, the pressure source could be a diaphragm pump comprising a diaphragm that mechanically separates fluid in the system from an external atmosphere, rendering use of a gas filter to isolate the pump from the cell culture media unnecessary. In some embodiments, it is advantageous to include a gas filter that separates the fluid conduit from an external atmosphere (e.g., by acting as or covering a relief valve) without filtering fluid flowing between the fluid conduit and the pressure source (or pressure sink). Various configurations of pressure sources, pressure sinks, relief valves, and gas filters are described in greater detail with reference to the figures below.

Any of a variety of gas filters may be used, depending on the embodiment, and the disclosure is not limited to any particular type of gas filter. In some embodiments, the gas filter is a membrane. For example, the gas filter may be a hollow fiber membrane, which may, advantageously, be used to filter gas flow between the fluid conduit and a relief valve without filtering flow between the fluid conduit and the pressure source (and/or pressure sink). Where a hollow fiber membrane is used as a gas filter, it may be advantageous, according to some embodiments, to use a plurality of hollow fiber membranes to parallelize gas flow and increase overall flow rate of fluid through the fluid conduit.

The gas filter may be configured such that gas passes through the gas filter during at least some stages of the system’s operation. However, in some embodiments, it is advantageous to prevent contact between the gas filter and the cell culture media, e.g., to prevent loss of cell culture media via transport of cell culture media through the gas filter. Any of a variety of methods may be used to control the flow of the cell culture media (e.g., to prevent contact between the cell culture media and the gas filter). For example, in some embodiments, the system comprises a volume displacement control configured to limit the volume of cell culture media flowed into the fluid conduit. The volume displacement control may be configured to limit the volume of a portion of cell culture media drawn through the port to ensure that the volume of the portion remains below a volume that would result in contact between the gas filter and the cell culture media. The volume displacement control can be implemented using any of a variety of appropriate methods, including but not limited to, pump encoders, timers, variable frequency drives for pumps, and/or proportional valves. In some embodiments, the system does not comprise a volume displacement control (e.g., does not comprise one or more of a pump encoder, a timer, a variable frequency drive for pump, or proportional valve).

While volume displacement control can be used in some embodiments, in some embodiments it may be advantageous to use active feedback controls. Active feedback controls may be advantageous as an alternative to (or in combination with) volume displacement controls, at least because volume displacement controls can sometimes lose calibration. By controlling fluid flow based, at least in part, on active sensing the position of fluid (e.g., within the conduit or within the bioreactor), the system can be configured to prevent contact between the cell culture media and the gas filter without recalibration. Active feedback controls may be configured to operate using a sensor. According to some embodiments, the sensor is configured to use the one or more properties sensed by the sensor to control the flow of cell culture media in the system. Any of a variety of appropriate sensors may be used. For example, in some embodiments, the sensor is configured to differentiate between gas and liquid, according to some embodiments. For example, in some embodiments the sample is a bubble sensor. As another example, in some embodiments, the sensor is a level sensor configured to measure a level of liquid in the bioreactor. The level sensor may be used to detect changes in the volume associated with withdrawal from and/or return of liquid to the bioreactor via the port.

The sensor may be operatively coupled to a controller configured to control the flow of the cell culture media through the fluid conduit by appropriately controlling the operation and/or fluid connection of the pressure source and/or pressure sink fluidly coupled to the fluid conduit. Thus, according to some embodiments the pressure source or pressure sink may be configured to draw cell culture media from the bioreactor into the fluid conduit until the sensor detects a fluid front of the portion of the cell culture media drawn into the fluid conduit is detected at a desired location by the sensor. Once the cell culture media has been detected by the sensor, the pressure source and/or pressure sink may be actuated to stop the drawing of cell culture media into the fluid conduit using the controller. Optionally, the controller can rely on the same signal from the detector to reverse the flow of cell culture media within the fluid conduit, pressurizing the fluid conduit to return the cell culture media to the bioreactor. Any of a variety of sensor types other than bubble sensors or level sensors may be used, as the disclosure is not limited to any particular sensor arrangements. To provide a few non-limiting examples, in some embodiments the sensor is a spectroscopic sensor, a volumetric sensor, a pH sensor, a temperature sensor, a pressure sensor, a flow sensor, an electrical and/or optical based meniscus sensor configured to differentiate gas from the cell culture media, and/or any other appropriate sensor configured to detect the presence of a fluid front of the cell culture media flowing within the fluid conduit. In some embodiments, the system does not comprise a bubble sensor or level sensor.

One or more controllers including associated one or more processors and non-volatile computer readable memory with corresponding processor executable instructions may be configured to control the various components of a bioreactor disclosed herein to control the disclosed agitation process, according to some embodiments. For example, in some embodiments, the one or more controllers may be operatively coupled to the pressure source, the pressure sink (if present), the relief valve, and/or the sensor(s) of the system, depending on the embodiment. In some embodiments, the one or more controllers are configured to control the actuation process, e.g., by automatically controlling the agitation of cells near the port of the bioreactor. Any of a variety of suitable controllers may be used (e.g., the Repligen ® ATF2 controller or ATF6 controller), as described in greater detail elsewhere herein. The gas filter, pressure source and/or pressure sink, and relief valve may each, if present, be arranged in any of a variety of appropriate configurations. However, it has been recognized herein that, according to some embodiments, a tangential flow filtration (TFF) system such as an alternating tangential flow filtration (ATF) system may provide an advantageous arrangement of these components for use in agitating cells near the port of the bioreactor. A TFF system or an ATF system configured for this purpose is herein referred to as an “agitation TFF” system, to distinguish it from a TFF system or an ATF system that is configured to remove cell culture media from the cell culture during a perfusion process. An agitation TFF system may comprise arrangements of components and membranes as discussed elsewhere herein in the context of TFF systems for filtering spent cell culture media. For example, an agitation TFF system connected to a bioreactor may have a membrane (e.g., a hollow fiber membrane) substantially similar to a hollow fiber membrane of another TFF system connected to the bioreactor and configured for filtering spent cell culture media. However, the agitation TFF system, unlike the TFF system for filtering spent cell culture media, may be configured such that cell culture media never contacts its membrane(s). Rather, the cell culture media may, in some embodiments, be retained within the fluid conduit while a pressure source of the agitation TFF system is configured to control the pressure in the fluid conduit and the one or more hollow fiber membranes of the TFF system are used as one or more gas filters. Depending on the embodiment, the hollow fiber membranes may serve as a relief valve. In some embodiments, the relief valve (if present) is disposed at a permeate outlet of the agitation TFF system so that gas can flow between an external atmosphere and the fluid conduit through the one or more gas filters and the relief valve.

The use of an agitation TFF system may provide a number of advantages, depending on the embodiment. For example, in some embodiments, the agitation TFF system can be chosen to mirror the filtration properties of a TFF system used for cell culture media exchange (e.g., as part of a perfusion process). The agitation TFF system may be controlled, at least in part, using the same controller and/or control software as the TFF system used for cell culture media exchange, simplifying process design. Furthermore, the agitation TFF system may be available as a commercial TFF system, reducing the likelihood of component incompatibility and/or component failure during use of the cell culture system. Finally, the use of an agitation TFF system for cell culture agitation may, in some embodiments, reduce the risks associated with accidental fluid contact with pressure sources and/or gas membranes, since an agitation TFF system can be cleaned and reused by a procedure similar to the procedure used to clean and reuse a TFF system used for separating spent cell culture media from the bioreactor. However, it should of course be understood that the use of an agitation TFF system is not strictly required, and that other arrangements of these system components are also contemplated.

FIGs. 2A-2C provide schematic cross-sectional views of various non-limiting cell culture systems 200, according to some embodiments. FIG. 2A shows cell culture system 200, which is similar to cell culture system 100 of FIG. 1 A. Cell culture system 200 comprises a media exchange system 250 (a perfusion TFF system) similar to media exchange system 150 of FIG. 1A. Cell culture system 200 comprises bioreactor 210 and is configured to support continuous perfusion using column 252 (represented as an ATF column) connected to bioreactor 210 via outlet channel 216 that fluidically connects the bioreactor 210 to column 252. Cell culture system 200 further comprises diaphragm pump 254, which is configured to flow cell culture media into column 252, and to flow retentate from column 252 back to bioreactor 210. Bioreactor 210, outlet channel 216, column 252, and diaphragm pump 254 are configured to operate like bioreactor 110, outlet channel 116, column 152, and diaphragm pump 154 of FIG. 1 A, and their operation is detailed above in the description of FIG. 1 A.

As illustrated in FIG. 2A, cell culture system 200 further comprises mixer 212 (which is analogous to mixer 112 described with reference to FIG. 1A). Mixer 212 is represented as an impeller and is configured to circulate cell media by causing rotation of liquid within the bioreactor. However, as schematically illustrated in FIG. 2A, bioreactor 210 contains a zone 299 that the impeller is not capable of adequately mixing, e.g., because zone 299 is directly below mixer 212 (and aligned with its axis of rotation) and/or because the presence of port 260 in the bottom of bioreactor 210 causes too much fluid drag to allow adequate circulation of fluid in zone 299. The problem may be compounded because bioreactor 210 has a conical bottom that contributes to cells settling to port 260. Without additional agitation, zone 299 would be considered a dead-zone because although fluid (and cells) may enter or exit the dead-zone, they typically would not do so with adequate frequency to replace spent media in the dead-zone. Not all bioreactors have a dead-zone. For example, bioreactor 110 of FIG. 1 A does not have a deadzone, whereas in bioreactor 210, zone 299 could be a dead zone, absent additional agitation. The existence of potential dead-zone 299 in bioreactor 210 can be problematic for cell culture because, as mentioned above, a dead zone’s mere existence in the bioreactor can result in the death of as much as 90% of the cultured cells over the course of a long cell culturing process.

Cell culture system 200 further comprises a fluid conduit 262 fluidly coupled to the interior volume of the bioreactor 210 via port 260. Flow through the fluid conduit 262 is configured to agitate the fluid in zone 299 (near port 260) by drawing a portion of a cell culture media out of bioreactor 210 and into fluid conduit 262 through port 260 in the bottom of the bioreactor. The cell culture system 200 may return the cell culture media drawn into fluid conduit 262 to bioreactor 210 via the port 260 in order to provide agitation, and the process can be iterated in order to provide continuous agitation and disbursement of the culture media and cells adjacent to the port 260 back into the bulk of the interior volume of bioreactor 210. Any of a variety of fluid conduits could be used, depending on the embodiment. For example, the fluid conduit could be a hose, a tube, a pipe, a channel, or any of a variety of other fluid conduits, depending on the embodiment.

The cell culture media can be drawn into and/or returned to the bioreactor by any of a variety of appropriate methods. For example, referring again to FIG. 2A, cell culture system 200 comprises pressure source 270, which is fluidically connected to fluid port 260 via fluid conduit 262. In the schematic of FIG. 2A, pressure source 270 is represented as a reversable-direction fluid pump (as indicated by the double-arrow representing the double-arrow drawn on the pump to indicate the possible flow directions). Pressure source 270 may be configured to pressurize fluid conduit 262 when pressure source 270 is in a first state, forcing a portion of cell culture media in fluid conduit 262 to return to bioreactor 210. In a second state, pressure source 270 may be configured to depressurize fluid conduit 262 to draw a portion of cell culture media into fluid conduit 262 from zone 299 of bioreactor 210. According to some embodiments, cell culture system 200 may be configured to change the state of pressure source 270 in order to altematingly draw a portion of cell culture media into the fluid conduit and to return the portion of cell culture media to the bioreactor, thereby providing agitation to fluid in zone 299 near port 260 of the bioreactor.

In the depicted embodiment of FIG. 2A, pressure source 270 doubles as a pressure sink. It should, of course, be understood that in other embodiments, a pressure source may be separate from a pressure sink of the cell culture system. For example, in some embodiments, the cell culture system comprises a switchable valve configured to alternate fluidic connection between the fluid conduit and the pressure source with fluidic connection between the fluid conduit and the pressure sink in order to alternate pressurization and depressurization of the fluid conduit. Thus, the operation of the pressure source is not limited to any particular configuration.

Although control of the pressure source and/or pressure sink can be handled manually, it may be advantageous to automate control of the pressure source and or pressure sink. Cell culture system 200 comprises one or more controllers 280 configured to actuate the pressure source 270 between a first state (wherein it is configured to pressurize the fluid conduit) and a second state (wherein it is configured to depressurize the fluid conduit). One or more controllers 280 comprise one or more processors 281, which may be configured to control the pressure source using processor-executable instructions for controlling various aspects of the cell culture system 200 (e.g., including pressure source 270). One or more controllers 280 further comprise non-volitile computer readable memory 283, which may be configured to store processorexecutable instructions for controlling various aspects of the cell culture system 200 (e.g., including pressure source 270) according to any of the methods disclosed herein.

Various methods for controlling cell culture system 200 are described in greater detail below, with reference to FIG. 3. One or more controllers 280 are configured to control pressure source 270 at least in part through the use of a sensor 282 of cell culture system 200. Sensor 282 may be configured to detect cell culture media, e.g., and one or more controllers 280 may be configured to actuate pressure source 270 in order to control the portion of cell culture media drawn into fluid conduit 262 based at least in part on data collected from sensor 282. Any of a variety of sensors may be used as detailed above. For example, sensor 282 could be a bubble sensor, a spectroscopic sensor (e.g., a color sensor), a volumetric sensor, a pH sensor, a temperature sensor, or an electrical sensor, depending on the embodiment. And it should, of course, be understood that sensor 282 is merely intended to be representative and that a plurality of sensors could be used, depending on the embodiment, e.g., to provide more precise control of the pressure source.

As discussed above, in some embodiments it is advantageous for a cell culture system to comprise a gas filter. FIG. 2A presents an embodiment where a gas filter 264 is used to maintain the sterility of fluid in fluid conduit 262. In the depicted embodiment, gas filter 264 is disposed between pressure source 270 and fluid conduit 262. The gas filter is configured to prevent passage of microorganisms (e.g., cells from the bioreactor, bacteria, fungi, or any of a variety of other types of cells), according to some embodiments. And the disposition of the gas filter between the pressure source and the fluid conduit shown in FIG. 2A may help to maintain the sterility of the fluid conduit, e.g., where pressure source 270 is configured to pressurize the fluid cavity by pumping non-sterile fluid drawn from an external atmosphere (e.g., air) into the fluid conduit. Of course, it should be understood that sterile pressure sources can also be used (e.g., as discussed with reference to subsequent FIGs. 2B-2C); however, the use of a gas filter such as gas filter 264 between fluid conduit 262 and pressure source 270 could be advantageous in at least some embodiments. As discussed above, in some embodiments it is advantageous to prevent contact between the gas filter and the cell culture media (e.g., to prevent leakage of cell culture media). Thus, sensor 282 of cell culture system 200 is configured to detect cell culture media from the fluid conduit before the cell culture media contacts the gas filter. This may allow one or more controllers 280 to actuate pressure source 270 to pressurize fluid conduit 262 before unintended contact between the portion of cell media and the gas filter can occur, according to at least some embodiments. Although the use of a relief valve as a secondary pressure control may be advantageous for reasons described above, as cell culture system 200 of FIG. 2A illustrates, a relief valve is not necessary in every embodiment. For example, cell culture system 200 does not contain a relief valve, instead relying on pressure source 270 as the sole source and sink for the pressure of the fluid conduit.

FIG. 2B provides an example of another cell culture system 200 similar, in most respects, to cell culture system 200 of FIG. 2A. Certain details, such as sensor 282 and one or more controllers 280 shown in FIG. 2A, are not represented in FIG. 2B, though they could still be used.

As shown in FIG. 2B, cell culture system 200 comprises a relief valve 268 fluidically connected to fluid conduit 262. Relief valve 268 may be used, according to some embodiments, to prevent overshear of a portion of cell culture media as it is drawn into fluid conduit 262 and/or returned to bioreactor 210. For example, the relief valve may be configured to limit the pressure differential between the fluid conduit and an external atmosphere, e.g., by passing gas into and/or out of the fluid conduit, depending on the embodiment. Thus, relief valve 262 may be configured to help provide low-shear agitation to zone 299, to prevent undesired cell loss that could result from excessive shear stress. The relief valve itself may be actuated by any of a variety of appropriate methods. For example, in some embodiments the relief valve is actuated manually, or is controlled in response to pressure sensed in the fluid conduit. According to some embodiments, the relief valve is configured to remain closed until subjected to a threshold pressure, at which point it self-actuates to relieve excess pressure. In some embodiments, the relief valve is configured to stay open, and is configured to maintain but limit a pressure differential between the external atmosphere and the fluid conduit by acting as a fluid flow barrier. For example, in some embodiments, the relief valve may be or comprise a high- resistance flow barrier configured to limit the rate of gas flow through the relief valve, thereby maintaining a pressure differential between the external atmosphere and the fluid conduit. Other embodiments are also possible, as the disclosure is not so limited.

In FIG. 2B, no gas filter separates pressure source 270 from fluid conduit 262, since pressure source 270 is a sterile pressure source. However, cell culture system 200 comprises gas filter 264, which is disposed between relief valve 268 and fluid conduit 262 such that fluid flowing between the external atmosphere and the fluid conduit is forced through gas filter 264. Thus, gas filter 264 is configured to maintain the sterility of cell culture system 200 by preventing exchange of microorganisms between the external atmosphere and the bioreactor via the relief valve. It should be noted that although cell culture system 200 is schematically depicted as including a separate relief valve and gas membrane, in some embodiments the gas filter 264 is configured to act as a relief valve (e.g., by acting as an effusion barrier).

FIG. 2C provides still another example of a cell culture system 200, which is substantially similar to the cell culture systems shown in FIGs. 2A-2B. However, in cell culture system 200 of FIG. 2C, the pressure of fluid conduit 262 is configured to be actuated using an agitation tangential flow filtration (agitation TFF) system 290 separate from the media exchange system 250, represented as a perfusion TFF system. The use of an agitation TFF system can, in some embodiments, conveniently consolidate several of the above-described features for use in cell culture systems. For example, agitation TFF system 290 comprises pressure source 270 (in the form of a diaphragm pump), which can act as a reversable pressure source, as well as a column 266 comprising one or more hollow-fiber membranes (not shown), which can be used as gas filters analogous to gas filter 264 shown in FIG. 2B, in that they are configured to separate a relief valve 268 from fluid conduit 262 to maintain the sterility of the fluid conduit. The relief valve 268 is connected to the agitation TFF 290 at a permeate line of the ATF column, though it should, of course, be understood that in other embodiments the permeate line could be closed and/or the hollow fiber membranes of the agitation TFF system could be used as a relief valve, removing the need for a separate relief valve 268 as shown.

Agitation TFF system 290 may differ from, e.g., media exchange system 250 or another TFF system in that it is not used to separate cell culture media. Rather, agitation TFF system 290 is, in some embodiments, used as a convenient way to control pressure of the fluid conduit 262 while maintaining the sterility of gas entering or leaving the fluid conduit. Agitation TFF system 290 is not, in such a configuration, used as a backup system for media exchange when media exchange system 250 is disabled or clogged; rather, it performs the separate function of preventing dead-zone formation in the bioreactor via agitation of fluid near a port of the bioreactor. The hollow fiber membranes of agitation TFF system 290 may be similar to or different from the hollow fiber membranes of perfusion TFF system 250, depending on the embodiment. For example, in some embodiments, the hollow fiber membranes of agitation TFF system 290 and perfusion TFF system 250 have the same porosity and pore size. However, in some embodiments, it may be advantageous for the porosity and pore size of the hollow fiber membranes of agitation TFF system 290 and perfusion TFF 250 system to differ — e.g., because the hollow fiber membranes of agitation TFF system 290 are intended to double as a relief valve, and because it is therefore advantageous to change the pore structure for the sake of imposing higher flow resistance to gas flow through the membranes. Thus, according to some embodiments the agitation TFF system comprises one or more membranes with a smaller pore size and/or a lower porosity than the perfusion TFF, according to some embodiments. Other configurations are, of course, also possible, as the disclosure is not so limited.

The membrane(s) of the agitation TFF system may have any of a variety of appropriate areas, relative to the membranes of the perfusion TFF system. However, in some embodiments, it is advantageous for the perfusion TFF system to have a higher total membrane area than the agitation TFF system, e.g., because the perfusion TFF system uses the membrane(s) to filter cell culture media, whereas the agitation TFF system uses the membrane(s) to filter gas. Such a configuration may be advantageous since, without wishing to be bound by any particular theory, a membrane may have a comparably lower resistance to gas flow than to the flow of cell culture media across the membranes. Other configurations are, of course, also possible, as the disclosure is not so limited.

FIG. 3 provides a non-limiting, schematic illustration of a method 200 of agitating fluid near a port of a bioreactor, according to some embodiments. At a first step 301, the method comprises a step of drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity e.g., when a base of the bioreactor is disposed on a level surface during operation of the cell culture system. The fluid can be drawn out of the bioreactor using a pressure sink, e.g., as described above. At a second step 303, the method comprises returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port. As indicated by dashed arrow 305, the method comprises alternatingly performing steps 301 and 303 to agitate fluid near the port, e.g., by using low-shear pressure waves resulting from the withdrawal and return of the cell media into the fluid conduit. The amount of cell culture media in the portion may be determined by any of a variety of appropriate methods. For example, in some embodiments, the one or more controllers are configured to control the volume of the portion of cell culture media, e.g., by acting as a volume displacement control that limits volume displacement of the cell culture media of the bioreactor into the fluid conduit. In some embodiments, the one or more controllers are configured to determine the amount of cell culture media using a sensor. For example, the one or more controllers may be configured to control the amount of withdrawn and returned cell culture media by sensing the cell culture media, e.g., to determine whether enough cell culture media has been drawn into the fluid conduit to reach the position of the sensor.

Volume displacement and/or sensor-based methods may also be used to prevent the pressure source from pumping excess fluid into the bioreactor. For example, in some embodiments, the method comprises stopping the return of the portion of cell culture media to the bioreactor when gas passes a sensor near the drain port, in order to prevent gas from entering the bioreactor. In some embodiments, the method comprises retaining a small amount of cell culture media within the fluid conduit, e.g., so that it is more difficult for gas to bubble past cell culture media and into the bioreactor. This may, advantageously, reduce the risk of pressurization of the bioreactor, and lessen the exposure of cell culture media to high shear conditions near the drain port.

Method 300 may be performed manually, or may be performed, e.g., using one or more controllers operatively coupled to the various components of the disclosed systems. The one or more controllers may be associated with non-volatile computer readable memory storing processor-executable instructions for performing the method. Likewise, the one or more controllers may comprise one or more processors configured to execute the method.

Various embodiments below, and elsewhere herein, may refer to a tangential flow filtration (TFF) system, and in some specific embodiments, an alternating tangential flow filtration (ATF) system or other similar term. It should be understood that the various properties, and parameters (e.g., lumen size, pore size, length, material, flow rates, etc.) disclosed in use relative to these types of filtration systems may be used with both as the disclosure is not limited to a specific type of tangential flow filtration though some benefits associated with cell cluster growth and sizing have been noted relative to ATF systems disclosed herein.

As used in this application and in the claims, the term “coupled” generally means physically, mechanically, fluidically, chemically, magnetically, and/or electrically coupled or linked and does not exclude the presence of intermediate elements between the coupled or associated items absent specific contrary language.

In some examples, values, procedures, or apparatus may be referred to as “lowest,” “best,” “minimum,” or the like. It will be appreciated that such descriptions are intended to indicate that a selection among many alternatives can be made, and such selections need not be better, smaller, or otherwise preferable to other selections.

In the description, certain terms may be used such as “up,” “down,” “upper,” “lower,” “horizontal,” “vertical,” “left,” “right,” and the like. These terms are used, where applicable, to provide some clarity of description when dealing with relative relationships. But, these terms are not intended to imply absolute relationships, positions, and/or orientations. For example, with respect to an object, an “upper” surface can become a “lower” surface simply by turning the object over. Nevertheless, it is still the same object.

In addition, provided definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

In this application, the use of “or” means “and/or” unless stated otherwise. The terms “and/or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

The term “about” means plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

The term “diabetes” and its grammatical equivalents as used herein can refer to is a disease characterized by high blood sugar levels over a prolonged period. For example, the term “diabetes” and its grammatical equivalents as used herein can refer to all or any type of diabetes, including, but not limited to, type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes. In some embodiments, diabetes can be a form of hereditary diabetes. In some embodiments, diabetes can be an autoimmune form of diabetes. The term “endocrine cell(s),” if not particularly specified, can refer to hormone- producing cells present in the pancreas of an organism, such as “islet”, “islet cells”, “islet equivalent”, “islet- like cells”, “pancreatic islets” and their grammatical equivalents. In an embodiment, the endocrine cells can be differentiated from pancreatic progenitor cells or precursors. Islet cells can comprise different types of cells, including, but not limited to, pancreatic a cells, pancreatic P cells, pancreatic 5 cells, pancreatic F cells, and/or pancreatic a cells. Islet cells can also refer to a group of cells, cell clusters, or the like.

The terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. Often, progenitor cells can also have significant or very high proliferative potential. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

A “precursor thereof’ as the term related to an insulin-positive endocrine cell can refer to any cell that is capable of differentiating into an insulin-positive endocrine cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, that if cultured under suitable conditions will differentiate the precursor cell into the insulin-positive endocrine cell.

As used herein, “culturing” one or more cells with a reagent means that the viable cells in the culture are contacted with the reagent for a sufficient time for it to have a biological effect. The disclosure also describes other active steps such as administering reagents to one or more cells, or contacting one or more cells with a reagent. In each of these action steps, one or more of the referenced cells are treated/contacted with the referenced reagent for a sufficient time for it to have a biological effect. Cells that are cultured with, contacted with, treated with, or administered any of the reagents disclosed herein are done so in a liquid media (e.g., StemScale, NutriStem, TeSR-E8, StemFit, StemPro, DMEM, such as DMEM/F12). In some embodiments, the media is the E8 media described in Chen et al., 2011, Nat. Methods, 8(5):424-29. In some embodiments, the media comprises DMEM/F12. In some embodiments, the media comprises ascorbic acid. In some embodiments, the media comprises sodium selenium. In some embodiments, the media comprises a growth factor from the FGF family (e.g., keratinocyte growth factor (KGF), FGF2 (bFGF), FGF8B, FGF10 and FGF21). In some embodiments, the media comprises insulin. In some embodiments, the media comprises NaCOa. In some embodiments, the media comprises transferrin. In some embodiments, the media comprises a growth factor of the TGF-P superfamily (e.g., TGF-P 1 or NODAL). Pancreatic differentiation as disclosed herein may be carried out in a step-wise manner. In an exemplary embodiment of the step-wise progression, “Stage 1” or “SI” or “Stl” refers to the first step in the differentiation process, the differentiation of pluripotent stem cells into cells expressing markers characteristic of definitive endoderm cells (“DE”, “Stage 1 cells” or “Stl cells” or “SI cells”). In some embodiments, “Stage 2” refers to the second step, the differentiation of cells expressing markers characteristic of definitive endoderm cells into cells expressing markers characteristic of gut tube cells (“GT”, “Stage 2 cells” “St2 cells” or “S2 cells”). In some embodiments, “Stage 3” refers to the third step, the differentiation of cells expressing markers characteristic of gut tube cells into cells expressing markers characteristic of pancreatic progenitor 1 cells (“PPI”, “Stage 3 cells” or “St3 cells” or “S3 cells”). In some embodiments, “Stage 4” refers to the fourth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 1 cells into cells expressing markers characteristic of pancreatic progenitor 2 cells (“PP2”, “Stage 4 cells” or “St4 cells” or “S4 cells”). In some embodiments, “Stage 5” refers to the fifth step, the differentiation of cells expressing markers characteristic of pancreatic progenitor 2 cells (e.g., PDX.1+, NKX6.1+) into cells expressing markers characteristic of pancreatic endoderm cells and/or pancreatic endocrine progenitor cells (e.g., insulin+) (“EN”, “Stage 5 cells” or “St5 cells” or “S5 cells”). In some embodiments, “Stage 6” refers to the differentiation of cells expressing markers characteristic of pancreatic endocrine progenitor cells (e.g., insulin) into cells expressing markers characteristic of pancreatic endocrine P cells (“SC-P cells”) or pancreatic endocrine a cells (“SC-a cells”). It should be appreciated, however, that not all cells in a particular population progress through these stages at the same rate, i.e., some cells may have progressed less, or more, down the differentiation pathway than the majority of cells present in the particular population. For example, in some embodiments, SC-P cells can be identified during stage 5, at the conclusion of stage 5, at the beginning of stage 6, etc. It should also be appreciated that not all cells necessarily differentiate to a specific cell type at the completion of particular stage. For example, in some embodiments, 30-100%, 50-100%, 30-80%, 30-50%, 70-90%, 80-100%, or 80-95% of the cells differentiate into cells expressing markers characteristic of pancreatic endoderm cells and/or pancreatic endocrine progenitor cells (e.g., insulin+) following completion of stage 5. Examples of methods of making cells of any one of stages 1-6 are provided in, for example, US Patent 10,030,229; US Patent 10,443,042; US Patent No. US 11,466,256; published application US 20200332262; and published application US 20210198632, published application US 20220090020, published application US 2022-0233646; published application US 2022- 0090020; published application US 20230218676; and published application WO2022147056, each of which is incorporated by reference in its entirety. The terms “stem cell-derived P cell,” “SC-P cell,” “functional P cell,” “functional pancreatic P cell,” “mature SC-P cell,” “P-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic P cells) that display at least one marker indicative of a pancreatic P cell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucose stimulated insulin secretion (GSIS) response similar or superior to that of an endogenous mature P cell (e.g., a mature P from a healthy functioning pancreas from a healthy adult non-diabetic patient). For simplicity, SC-P cells may be referred to as simply “P cells” in this disclosure. In some embodiments, the terms “SC-P cell” and “non-native P cell” as used herein are interchangeable. In some embodiments, the “SC-P cell” expresses lower levels of MAFA than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of MAFB than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” expresses higher levels of SIX2, HOPX, IAPP and/or UCN3 than a pancreatic P cell from a healthy adult human patient. In some embodiments, the “SC-P cell” comprises a mature pancreatic cell. It is to be understood that the SC-P cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-P cells from any insulin-positive endocrine cell or precursor thereof using any cell as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells such as definitive endoderm cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the disclosure is not intended to be limited in this manner). In some embodiments, the SC-P cells exhibit a response to multiple glucose challenges (e.g., at least one, at least two, or at least three or more sequential glucose challenges). In some embodiments, the response resembles the response of endogenous islets (e.g., human islets) to multiple glucose challenges. In some embodiments, the morphology of the SC-P cell resembles the morphology of an endogenous P cell. In some embodiments, the SC- P cell exhibits an in vitro GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits an in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the SC-P cell exhibits both an in vitro and in vivo GSIS response that resembles the GSIS response of an endogenous P cell. In some embodiments, the GSIS response of the SC-P cell can be observed within two weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed within three weeks of transplantation of the SC- P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC- P cell can be observed within four weeks of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the GSIS response of the SC-P cell can be observed between one month and three months of transplantation of the SC-P cell into a host (e.g., a human or animal). In some embodiments, the SC-P cells package insulin into secretory granules. In some embodiments, the SC-P cells exhibit encapsulated crystalline insulin granules when viewed using electron microscopy. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 1.1. In some embodiments, the SC-P cells exhibit a stimulation index of greater than 2. In some embodiments, the stimulation index of the cell is characterized by the ratio of insulin secreted in response to high glucose concentrations (e.g., 15 mM) compared to low glucose concentrations (e.g., 2.5 mM).

In some embodiments, the SC-P cells exhibit cytokine-induced apoptosis in response to cytokines. In some embodiments, insulin secretion from the SC-P cells is enhanced in response to known antidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-P cells are monohormonal. In some embodiments, the SC-P cells do not abnormally co-express other hormones, such as glucagon, somatostatin or pancreatic polypeptide. In some embodiments, the SC-P cells exhibit a low rate of replication. In some embodiments, the SC-P cells increase intracellular Ca2+ in response to glucose.

The terms “stem cell-derived a cell,” “SC-a cell,” “functional a cell,” “functional pancreatic a cell,” “mature SC-a cell,” “a-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic a cells) that display at least one marker indicative of a pancreatic a cell (e.g., glucagon, expressing ISL1 but not NKX6.1), expresses glucagon, and is capable of secreting functional glucagon in response to a stimulus that induces an endogenous pancreatic a cell to secrete functional glucagon. In some embodiments, the “SC-a cell” does not express somatostatin. In some embodiments, the “SC-a cell” does not express insulin. In some embodiments, the terms “SC-a cell” and “non-native a cell” as used herein are interchangeable. In some embodiments, the “SC-a cell” comprises a mature pancreatic cell. For short, these cells may be referred to as simply “a cells” in this disclosure.

The terms “stem cell-derived 5 cell,” “SC-5 cell,” “functional 5 cell,” “functional pancreatic 5 cell,” “mature SC-5 cell,” “5-like cell” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic 5 cells) that display at least one marker indicative of a pancreatic 5 cell (e.g., somatostatin), expresses and is capable of secreting somatostatin in response to a stimulus that induces an endogenous pancreatic 5 cell to secrete functional glucagon. For simplicity, SC- 5 cells may be referred to as simply “5 cells” in this disclosure. In some embodiments, “SC-5 cell” does not express glucagon. In some embodiments, “SC-5 cell” does not express insulin. In some embodiments, the terms “SC-5 cell” and “non-native 5 cell” as used herein are interchangeable. In some embodiments, the “SC-5 cell” comprises a mature pancreatic cell.

The terms “stem cell-derived enterochromaffin (EC) cell,” “SC-EC cell,” and their grammatical equivalents can refer to cells (e.g., non-native pancreatic EC cells) that display at least one marker indicative of a pancreatic EC cell (e.g., VMAT1 (vesicular monoamine transporter 1), expressing NKX6.1 but not ISL1). In some embodiments, the terms “SC-EC cell” and “non-native EC cell” as used herein are interchangeable.

Similar to SC-P cells, it is to be understood that the SC-a, SC-5 cells, and SC-EC cells need not be derived (e.g., directly) from stem cells, as the methods of the disclosure are capable of deriving SC-a cells from other precursor cells generated during in vitro differentiation of SC-P cells as a starting point (e.g., one can use embryonic stem cells, induced-pluripotent stem cells, progenitor cells, partially reprogrammed somatic cells (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an induced pluripotent stem cell and the somatic cell from which it was derived), multipotent cells, totipotent cells, a transdifferentiated version of any of the foregoing cells, etc., as the disclosure is not intended to be limited in this manner).

As used herein, the term “insulin producing cell” and its grammatical equivalent refer to a cell differentiated from a pancreatic progenitor, or precursor thereof, which secretes insulin. An insulin-producing cell can include pancreatic P cell as that term is described herein, as well as pancreatic P-like cells (e.g., insulin-positive, endocrine cells) that synthesize (e.g., transcribe the insulin gene, translate the proinsulin mRNA, and modify the proinsulin mRNA into the insulin protein), express (e.g., manifest the phenotypic trait carried by the insulin gene), or secrete (release insulin into the extracellular space) insulin in a constitutive or inducible manner. A population of insulin producing cells e.g., produced by differentiating insulin-positive endocrine cells or a precursor thereof into SC-P cells according to the methods of the present disclosure can be pancreatic P cells or P-like cells (e.g., cells that have at least one, or at least two least characteristics of an endogenous P cell and exhibit a glucose stimulated insulin secretion (GSIS) response that resembles an endogenous adult P cell). The population of insulin-producing cells, e.g., produced by the methods as disclosed herein can comprise mature pancreatic P cell or SC-P cells, and can also contain non-insulin-producing cells (e.g., cells of cell like phenotype with the exception they do not produce or secrete insulin).

The terms “insulin-positive P-like cell,” “insulin-positive endocrine cell,” and their grammatical equivalents can refer to cells (e.g., pancreatic endocrine cells) that display at least one marker indicative of a pancreatic P cell and also expresses insulin but, unless specified otherwise, lack a glucose stimulated insulin secretion (GSIS) response characteristic of an endogenous P cell. Exemplary markers of “insulin-positive endocrine cell” include, but are not limited to, NKX6.1 (NK6 homeobox 1), ISL1 (Isletl), and insulin.

The term “P cell marker” refers to, without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analyte which are expressed or present in pancreatic P cells. Exemplary P cell markers include, but are not limited to, pancreatic and duodenal homeobox 1 (PDX1) polypeptide, insulin, c-peptide, amylin, E-cadherin, Hnf3p, PCV3, B2, Nkx2.2, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Infib, Hnf-6, Hnf-3beta, VMAT2, NKX6.1, and MafA, and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). In some embodiments, the P cell marker is a nuclear P-cell marker. In some embodiments, the P cell marker is PDX1 or PH3.

The term “pancreatic endocrine marker” can refer to without limitation, proteins, peptides, nucleic acids, polymorphism of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes which are expressed or present in pancreatic endocrine cells. Exemplary pancreatic endocrine cell markers include, but are not limited to, Ngn-3, NeuroD and Islet- 1.

The term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” “pancreatic endocrine precursor” and their grammatical equivalents are used interchangeably herein and can refer to a stem cell which is capable of becoming a pancreatic hormone expressing cell capable of forming pancreatic endocrine cells, pancreatic exocrine cells or pancreatic duct cells. These cells are committed to differentiating towards at least one type of pancreatic cell, e.g. P cells that produce insulin; a cells that produce glucagon; 5 cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “PDX1 -positive pancreatic progenitor” as used herein can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into SC-P cells, such as pancreatic P cells. A PDXl-positive pancreatic progenitor expresses the marker PDX1. Other markers include, but are not limited to Cdcpl, or Ptfla, or HNF6 or NRx2.2. The expression of PDX1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-PDXl antibody or quantitative RT-PCR. In some embodiments, a PDXl-positive pancreatic progenitor cell lacks expression of NKX6.1. In some embodiments, a PDXl-positive pancreatic progenitor cell can also be referred to as PDXl-positive, NKX6.1 -negative pancreatic progenitor cell due to its lack of expression of NKX6.1. In some embodiments, the PDXl- positive pancreatic progenitor cells can also be termed as “pancreatic foregut endoderm cells.” The terms “PDX1 -positive, NKX6.1 -positive pancreatic progenitor,” and “NKX6.1- positive pancreatic progenitor” are used interchangeably herein and can refer to a cell which is a pancreatic endoderm (PE) cell which has the capacity to differentiate into insulin-producing cells, such as pancreatic P cells. A PDX1 -positive, NKX6.1 -positive pancreatic progenitor expresses the markers PDX1 and NKX6-1. Other markers may include, but are not limited to Cdcpl, or Ptfla, or HNF6 or NRx2.2. The expression of NKX6-1 may be assessed by any method known by the skilled person such as immunochemistry using an anti-NKX6-l antibody or quantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” are equivalent and interchangeable. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells can also be termed as “pancreatic foregut precursor cells.”

The terms “NeuroD” and “NeuroDl” are used interchangeably and identify a protein expressed in pancreatic endocrine progenitor cells and the gene encoding it.

The term “differentiated cell” or its grammatical equivalents means any primary cell that is not, in its native form, pluripotent as that term is defined herein. Stated another way, the term “differentiated cell” can refer to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., a stem cell such as an induced pluripotent stem cell) in a cellular differentiation process. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell may lead to the pancreatic pathway, where -98% of the cells become exocrine, ductular, or matrix cells, and -2% become endocrine cells. Early endocrine cells are islet progenitors, which can then differentiate further into insulin-producing cells (e.g. functional endocrine cells) which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc.

As used herein, the term “somatic cell” can refer to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as “gametes”) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body - apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells - is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a “non- embryonic somatic cell”, by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an “adult somatic cell”, by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro. Unless otherwise indicated the methods for converting at least one insulin-positive endocrine cell or precursor thereof to an insulin-producing, glucose responsive cell can be performed both in vivo and in vitro (where in vivo is practiced when at least one insulin-positive endocrine cell or precursor thereof are present within a subject, and where in vitro is practiced using an isolated at least one insulin-positive endocrine cell or precursor thereof maintained in culture).

As used herein, the term “adult cell” can refer to a cell found throughout the body after embryonic development.

The term “endoderm cell” as used herein can refer to a cell which is from one of the three primary germ cell layers in the very early embryo (the other two germ cell layers are the mesoderm and ectoderm). The endoderm is the innermost of the three layers. An endoderm cell differentiates to give rise first to the embryonic gut and then to the linings of the respiratory and digestive tracts (e.g., the intestine), the liver and the pancreas.

The term “a cell of endoderm origin” as used herein can refer to any cell which has developed or differentiated from an endoderm cell. For example, a cell of endoderm origin includes cells of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. Without wishing to be bound by theory, liver and pancreas progenitors (also referred to as pancreatic progenitors) are developed from endoderm cells in the embryonic foregut. Shortly after their specification, liver and pancreas progenitors rapidly acquire markedly different cellular functions and regenerative capacities. These changes are elicited by inductive signals and genetic regulatory factors that are highly conserved among vertebrates. Interest in the development and regeneration of the organs has been fueled by the intense need for hepatocytes and pancreatic P cells in the therapeutic treatment of liver failure and type I diabetes. Studies in diverse model organisms and humans have revealed evolutionarily conserved inductive signals and transcription factor networks that elicit the differentiation of liver and pancreatic cells and provide guidance for how to promote hepatocyte and P cell differentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell (e.g., a pancreatic P cell). A definitive endoderm cell expresses the marker Sox 17. Other markers characteristic of definitive endoderm cells may include, but are not limited to MIXL2, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, 0TX2, goosecoid, C-Kit, CD99, CMK0R1 and CRIP1. In particular, definitive endoderm cells herein express Soxl7 and in some embodiments Soxl7 and HNF3B, and do not express significant levels of GATA4, SPARC, APF or DAB. Definitive endoderm cells are not positive for the marker PDX1 (e.g. they are PDX1 -negative). Definitive endoderm cells have the capacity to differentiate into cells including those of the liver, lung, pancreas, thymus, intestine, stomach and thyroid. The expression of Soxl7 and other markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-Soxl7 antibody, or quantitative RT-PCR.

The term “pancreatic endoderm” can refer to a cell of endoderm origin which is capable of differentiating into multiple pancreatic lineages, including pancreatic P cells, but no longer has the capacity to differentiate into non-pancreatic lineages.

The term “pancreatic islet cells” refers to a population of cells that include different types of pancreatic endocrine cells (P-cells, a-cells, 5-cells, s-cells) and enterochromaffin (EC) cells, e.g., as described in Xavier et al. (J Clin Med. 2018 Mar; 7(3): 54), incorporated herein by reference.

The term “primitive gut tube cell” or “gut tube cell” as used herein can refer to a cell differentiated from an endoderm cell and which can be differentiated into a SC-P cell (e.g., a pancreatic P cell). A primitive gut tube cell expresses at least one of the following markers: HNP1-P, HNF3-P or HNF4-a. In some embodiments, a primitive gut tube cell is FOXA2- positive and SOX2-positive, i.e., expresses both FOXA2 (also known as HNF3-P) and SOX2. In some embodiments, a primitive gut tube cell is FOXA2-positive and PDX1 -negative, i.e., expresses FOXA2 but not PDX1. Primitive gut tube cells have the capacity to differentiate into cells including those of the lung, liver, pancreas, stomach, and intestine. The expression of HNF1-P and other markers of primitive gut tube may be assessed by any method known by the skilled person such as immunochemistry, e.g., using an anti-HNFl-P antibody.

The term “phenotype” can refer to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.

The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. The “non-human animals” and “non-human mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like. “Patient in need thereof’ or “subject in need thereof’ is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, for instance, but not restricted to diabetes.

“Administering” as used herein can refer to providing one or more compositions described herein to a patient or a subject. By way of example and not limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration can be by the oral route. Additionally, administration can also be by surgical deposition of a bolus or pellet of cells, or positioning of a medical device. In an embodiment, a composition of the present disclosure can comprise engineered cells or host cells expressing nucleic acid sequences described herein, or a vector comprising at least one nucleic acid sequence described herein, in an amount that is effective to treat or prevent proliferative disorders. A pharmaceutical composition can comprise the cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and any individual numbers present in this range.

All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

Overview

Disclosed herein are methods of culturing cells. The methods can include culturing a cell culture in a bioreactor, wherein the cell culture includes a liquid media and a plurality of cell clusters, transporting a portion of the cell culture from the bioreactor into a TFF system, removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system, returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor, and/or replacing the removed portion of the liquid media with a new portion of liquid media.

The disclosed methods relate, in various embodiments, to methods particularly advantageous for the culturing of cell clusters with desired size ranges that may improve a viability of the cells and/or efficacy of an associate treatment. Cell clusters can have a variety of useful biomedical applications, particularly when the cell clusters comprise a variety of distinct differentiated cell types that can cooperatively perform a function. Culturing cell clusters can be a challenging task, complicated both by the generic difficulties recognized for all cell culturing and by problems specific to the culturing of cell clusters (rather than, e.g., freestanding cells). For example, in some embodiments, cell clusters may exhibit improved functionality if they fall within a narrowly tailored size range. Without wishing to be bound by any particular theory, overly small clusters, according to some embodiments, may be insufficiently large or differentiated to perform a desired function. In contrast, according to some embodiments, excessively large clusters may comprise a core of cells too physically isolated from an exterior environment of the cluster to receive sufficient oxygen and nutrients which may result in death of the interior cells and, in some cases, death of the clusters as a whole.

In view of the above, it has been recognized that it is desirable to provide cell clusters within predetermined size ranges. However, a particular challenge of culturing cell clusters relates to scale-up of the cluster formation processes. For example, discontinuous processes (e.g., as represented in FIG. ID, described above) for replacing spent cell culture media in a bioreactor may be difficult to perform at an industrial scale, e.g., because they are too slow, wasteful or cumbersome to produce cell clusters in useful quantities and qualities. The present disclosure relates, in various embodiments, to processes and systems for continuously replacing cell culture media that can improve the scalability of cell cluster culturing. As a particular example, in some embodiments the disclosure relates to continuous processes for removing and replacing cell culture media while providing cell clusters within a desired size range.

During typical cell culturing processes, shear stresses applied to the cell clusters are typically minimized as excessive shear stresses result in clusters breaking apart and/or otherwise resulting in cell death. However, it has been recognized that the shear stresses applied to the cell clusters can be adjusted to control the resulting size of the cell clusters produced during a cell culturing process. More specifically, it has been recognized that the application of shear stresses within an appropriate range to cell clusters within a cell culture media in combination with appropriate filtration of cell debris and cell clusters below a desired size threshold may be used to produce cell clusters within a desired size range. In some embodiments, the filtration used in such methods and systems may correspond to tangential flow filtration systems. In other embodiments, the filtration used in such methods and systems may be alternating flow filtration systems. Appropriate physical constructions, combinations of fluid flow parameters, and other appropriate process parameters disclosed herein may be used to apply the appropriate combination of shear stresses and growth parameters to produce cell clusters of the type and size needed for a desired application as elaborated on further below. In some embodiments the plurality of cell clusters includes stem cells. In some embodiments least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells. In some embodiments the stem cells are embryonic stem cells. In some embodiments the stem cells are induced pluripotent stem cells.

In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are OCT4-negative and/or SOX17- positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are FOXA2-positive, and/or PDX1 -negative. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDXl-positive and/or NKX6.1-negative. In some embodiments at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDXl-positive and/or NKX6.1 -positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are ISLl-positive. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are chromogranin-positive. In some embodiments 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600 pm, 75-500 pm, 75- 400 pm, 75-300 pm in diameter. In some embodiments, combinations of these features are present.

In some embodiments the plurality of cell clusters are generated from a plurality of dissociated cells. In some embodiments, prior to culturing a cell culture in a bioreactor the method includes seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters. In some embodiments the dissociated cells are cultured until 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600 pm, 75-500 pm, 75-400 pm, or 75-300 pm in diameter. In some embodiments, an average maximum transverse dimension (e.g., a diameter) of the cell clusters may be greater than or equal to 75 pm. In some embodiments, the average maximum transverse dimension (e.g., a diameter) of the cell clusters may also be less than or equal to 600 pm.

In some embodiments the culturing step is 12-72 hours, 12-60 hours, 12-50 hours, 12-36 hours, 12-36 hours, 18-60 hours, 18-50 hours, 18-36 hours, 18-26 hours, 26-60 hours, 26-50 hours, 26-36 hours, 36-60 hours, 36-50 hours, or 44-52 hours in length before the step of transporting the portion of the cell culture from the bioreactor into the TFF. In some embodiments the TFF system is an ATF system. In some embodiments 0.3-1, 0.3-0.8, 1-5, 1-4, 1-3, 2-5, 2-4, 2-3, 2.5-3.0, or 2.5-3.5 volumes of media are exchanged in a 24-hour period. In some embodiments the cell culture is transported from the bioreactor through the TFF system at a shear rate of 400-800 sec'¹, 400-3500 sec'¹, 400-3000 sec'¹, 400-2500 sec'¹, 400-2000 sec'¹, 400-1500 sec'¹, 1000-3500 sec'¹, 1000-3000 sec'¹, 1000-2000 sec'¹, 2000-3500 sec'¹, 2000-3000 sec'¹, 1200-1800 sec'¹, 1400-1600 sec'¹, or 1450-1550 sec'¹.

In some embodiments the TFF system includes one or more filters, wherein the one or more filters include a plurality of pores, wherein from the pores are 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments the TFF system includes a filter made of polyethersulfone (PES).

In some embodiments a shear protectant is present in the cell culture in the TFF. In some embodiments the shear protectant is polaxamer, polyvinyl alcohol (PVA) or pluronic. In some embodiments the shear protectant is PVA, and the PVA is PVA80 or PVA87-89. In some embodiments the shear protectant is Pluronic, and the Pluronic is P188 or PF68.

Generally, tangential flow filtration is performed using a cassette housing a tangential flow filter and configured to permit a flow of fluid to contact and pass tangent to the filter. Accordingly, in some embodiments, tangential flow filtration comprises contacting one or more mixtures (e.g., eluents, retentates, or other mixtures) with a tangential flow filter to form a retentate and/or a permeate (e.g., a retentate and/or a permeate comprising an analyte from the mixture). The tangential flow filter may be a membrane (e.g., a porous membrane), according to some embodiments. In some embodiments, the tangential flow filter is configured to retain or permit permeation of species based on their size (e.g., by allowing smaller species to pass through pores of the filter while retaining larger species that cannot pass through the pores in the tangential flow of fluid). After filtration, fluid retained in the flow that passed tangentially to the filter is the retentate, while fluid that passed through the filter is the permeate. The methods provided herein may comprise further purification of a tangential flow filter retentate (e.g., using additional filtration or chromatography steps. In some embodiments, the tangential flow filtration is used for viral filtration.

Any of a variety of suitable tangential flow filter materials may be used. For example, in some embodiments, the tangential flow filter comprises a polymer membrane. The polymer membrane may be hydrophilic, in some embodiments. According to some embodiments, for example, the polymer membrane comprises polyethersulfone (PES). For example, the polymer membrane may be a PALL OMEGA™ PES membrane or a generic equivalent thereof. In some embodiments, the polymer membrane is hydrophobic. According to some embodiments, for example, the polymer membrane comprises poly vinylidene fluoride (PVDF). For example, the polymer membrane may be a PLANOVA™ membrane (e.g., a PLANOVA™ 35N membrane) or a generic equivalent thereof. Different flow conditions (e.g., different pressures, loadings, and flow-rates may be suitable for different tangential flow filters, e.g., depending on the hydrophilicity or hydrophobicity of the tangential flow filter.

A tangential flow filter may have any of a variety of suitable areas, depending on the embodiment. In some embodiments, a tangential flow filter has an area of greater than or equal to 0.1 m², greater than or equal to 0.2 m², greater than or equal to 0.5 m², greater than or equal to 1 m², greater than or equal to 2 m², greater than or equal to 3 m², greater than or equal to 4 m², greater than or equal to 5 m², greater than or equal to 6 m², greater than or equal to 7 m², greater than or equal to 8 m², or greater than or equal to 9 m². In some embodiments, a tangential flow filter has an area of less than or equal to 10 m², less than or equal to 9 m², less than or equal to 8 m², less than or equal to 7 m², less than or equal to 6 m², less than or equal to 5 m², less than or equal to 4 m², less than or equal to 3 m², less than or equal to 2 m², less than or equal to 1 m², less than or equal to 0.5 m², or less than or equal to 0.2 m². Combinations of these ranges are also possible (e.g., greater than or equal to 0.1 m² and less than or equal to 10 m², greater than or equal to 1 m² and less than or equal to 8 m², or greater than or equal to 0.1 m² and less than or equal to 0.5 m²). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

In some embodiments, tangential flow filtration is performed with a flow rate of greater than or equal to 5 L/hr, greater than or equal to 10 L/hr, greater than or equal to 50 L/hr, greater than or equal to 100 L/hr, greater than or equal to 200 L/hr, greater than or equal to 300 L/hr, greater than or equal to 400 L/hr, greater than or equal to 500 L/hr, greater than or equal to 600 L/hr, greater than or equal to 700 L/hr, greater than or equal to 800 L/hr, greater than or equal to 900 L/hr, greater than or equal to 1000 L/hr, greater than or equal to 1100 L/hr, greater than or equal to 1200 L/hr, greater than or equal to 1300 L/hr, or greater than or equal to 1400 L/hr. In some embodiments, tangential flow filtration is performed with a flow rate of less than or equal to 1500 L/hr, less than or equal to 1400 L/hr, less than or equal to 1300 L/hr, less than or equal to 1200 L/hr, less than or equal to 1100 L/hr, less than or equal to 1000 L/hr, less than or equal to 900 L/hr, less than or equal to 800 L/hr, less than or equal to 700 L/hr, less than or equal to 600 L/hr, less than or equal to 500 L/hr, less than or equal to 400 L/hr, less than or equal to 300 L/hr, less than or equal to 200 L/hr, less than or equal to 100 L/hr, or less than or equal to 50 L/hr. Combinations of these ranges are also possible (e.g., greater than or equal to 100 L/hr and less than or equal to 1500 L/hr, greater than or equal to 1000 L/hr and less than or equal to 1500 L/hr, greater than or equal to 400 L/hr and less than or equal to 700 L/hr, greater than or equal to 100 L/hr and less than or equal to 200 L/hr, or greater than or equal to 5 L/hr and less than or equal to 100 L/hr). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. In some embodiments, the TFF (e.g., ATF) is performed with a flow rate of greater than any of 0.2 liters per minute, 0.3 liters per minute, 0.4 liters per minute, 0.5 liters per minute, 0.6 liters per minute, or 0.7 liters per minute. In some embodiments, the TFF (e.g., ATF) is performed with a flow rate of less than any of 10 liters per minute, 8 liters per minute, 6 liters per minute, 4 liters per minute, 2 liters per minute, 1 liters per minute, or 0.8 liters per minute. In particular embodiments, the TFF (e.g., ATF) is performed with a flow rate of any of 1-10 liters per minute, 1-5 liters per minute, 1-3 liters per minute, 0.1-0.8 liters per minute, 0.2-0.7 liters per minute, 0.3-0.6 liters per minute, 0.4-0.6 liters per minute, 0.5-0.8 liters per minute, 0.6-0.8 liters per minute, or 0.8- 1.0 liters per minute.

In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed at a filtrate flux rate of 10-25 liters/m²/hour (LMH), 25-50 LMH, 50-70 LMH, 0.5-75 LMH, 0.5-50 LMH, 0.5-25 LMH, 0.5-10 LMH, 0.5-6 LMH, 0.5-4 LMH, 0.5-2 LMH, 1-2 LMH, or 1.5-2 LMH. In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed at a filtrate flux rate of 1-2 LMH or 1.5-2 LMH or 1.6- 1.8 LMH.

In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed with a membrane residence time of 60-300 seconds, 60-240 seconds, 60- 180 seconds, 60-120 seconds, 1-60 seconds, 1-45 seconds, 1-30 seconds, 1-20 seconds, 1-15 seconds, 5-15, 5-10 seconds, or 10-15 seconds. In some embodiments, any of the TFF methods (e.g., any of the ATF methods) disclosed herein are performed with a membrane residence time of 30-60 seconds, 30-45 seconds, 45-60 seconds, or 20-30 seconds.

In some embodiments, a filter used with any of the cell culturing systems disclosed herein, including the ATF and/or TFF systems disclosed herein, may include one or more cassette membranes. In some embodiments, the disclosed filters, including the disclosed ATF and TFF systems, may include one or more hollow fiber membranes. In some embodiments, the disclosed ATF and TFF systems may include a plurality of hollow fiber membranes. A hollow fiber membrane as described herein may refer to a structure including a porous membrane formed into an elongated structure where the porous membrane extends around the perimeter of an internal lumen that extends through an axial length of the hollow fiber membrane to form a thin tube with a porous side wall. In some embodiments a plurality of substantially parallel hollow fiber membranes may be arranged such that the lumens of the plurality of hollow fiber membranes are in fluid communication with an upstream inlet of the filtration system and an exterior surface of the hollow fiber membranes opposite from the associated lumens may be in fluid communication with a waste outlet of the filtration system. In embodiments where fluid flows through the filtration system during operation, the lumens of the plurality of hollow fiber membranes may also be in fluid communication with a downstream outlet of the filtration system. Depending on the embodiment, the filtration system may be any TFF system including, in some instances, an ATF system.

Depending on the embodiment, the lumens of one or more hollow fiber membranes may have any of a variety of appropriate average transverse dimensions (e.g., a radius or other appropriate dimension perpendicular to a longitudinal axis of the hollow fiber membrane), depending on the desired flow characteristics. In some embodiments, the lumens of the one or more hollow fiber membranes may have an average radius of greater than or equal to 0.1 mm, greater than or equal to 0.2 mm, greater than or equal to 0.3 mm, greater than or equal to 0.4 mm, greater than or equal to 0.5 mm, greater than or equal to 0.6 mm, greater than or equal to 0.7 mm, greater than or equal to 0.8 mm, greater than or equal to 0.9 mm, greater than or equal to 1 mm, greater than or equal to 1.1 mm, greater than or equal to 1.2 mm, greater than or equal to 1.3 mm, greater than or equal to 1.4 mm, greater than or equal to 1.5 mm, greater than or equal to 1.6 mm, greater than or equal to 1.7 mm, greater than or equal to 1.8 mm, greater than or equal to 1.9 mm, greater than or equal to 2 mm, greater than or equal to 3 mm, greater than or equal to 5 mm, or greater than or equal to 7 mm. In some embodiments, the lumens of the one or more hollow fiber membranes have an average radius of less than or equal to 10 mm, less than or equal to 7 mm, less than or equal to 5 mm, less than or equal to 3 mm, less than or equal to 2 mm, less than or equal to 1.9 mm, less than or equal to 1.8 mm, less than or equal to 1.7 mm, less than or equal to 1.6 mm, less than or equal to 1.5 mm, less than or equal to 1.4 mm, less than or equal to 1.3 mm, less than or equal to 1.2 mm, less than or equal to 1.1 mm, less than or equal to 1 mm, less than or equal to 0.9 mm, less than or equal to 0.8 mm, less than or equal to 0.7 mm, less than or equal to 0.6 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, or less than or equal to 0.2 mm. Combinations of these ranges are also possible. For example, in some embodiments, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.1 mm and less than or equal to 10 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.1 mm and less than or equal to 2 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membranes may be greater than or equal to 0.5 mm and less than or equal to 2 mm. In another embodiment, an average radius of the lumens of the one or more a hollow fiber membranes may be greater than or equal to 0.5 mm and less than or equal to 1.2 mm. In another embodiment, an average radius of the lumens of the one or more hollow fiber membrane may be greater than or equal to 0.8 mm and less than or equal to 1.2 mm). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. In some embodiments, the lumens of the one or more hollow fiber membranes may have an average a radius between or equal to 0.5-10 mm, 5-10 mm, 2-5 mm, 0.5-7 mm, 0.5-5 mm, 0.5-3 mm, 0.5-2 mm, 0.5- 1.2 mm, 0.8- 1.2 mm, or 0.9- 1.1 mm.

To provide an appropriate balance of applied shear forces and cell cluster size, in some embodiments the disclosed hollow fiber membrane(s) may have an average lumen radius, or other appropriate average transverse dimensions, which is larger than a target radius of cultured cell clusters by an appropriate ratio. Without wishing to be bound by any particular theory, in some embodiments the appropriate sizing of the lumens of a hollow fiber membrane may help to size cell clusters during continuous perfusion, e.g., by mechanically limiting the maximum transverse dimension of cell clusters. For example, in some embodiments, the ratio of the target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes is greater than or equal to 0.1, greater than or equal to 0.2, greater than or equal to 0.3, greater than or equal to 0.4, greater than or equal to 0.5, greater than or equal to 0.6, greater than or equal to 0.7, greater than or equal to 0.8, or greater than or equal to 0.9. In some embodiments, the ratio of the target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes is less than 1, less than or equal to 0.9, less than or equal to 0.8, less than or equal to 0.7, less than or equal to 0.6, less than or equal to 0.5, less than or equal to 0.4, less than or equal to 0.3, or less than or equal to 0.2. Combinations of these ranges are also possible (e.g., greater than or equal to 0.1 and less than or equal to 1, greater than or equal to 0 and less than or equal to 2, or greater than or equal to 0.8 and less than or equal to 0.4). In one embodiment, a target average maximum transverse dimension of a plurality of cell clusters to the average radius of the lumens of the one or more hollow fiber membranes may be between or equal to 0.1 and 1. While the above ranges may be beneficial for applying the desired ranges of shear stresses, other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

A TFF system (e.g., the ATF system or other appropriate type of TFF system) may include one or more hollow fiber membranes having any of a variety of suitable lengths. In some embodiments, a TFF system includes one or more hollow fiber membranes having a length of greater than or equal to 1 cm, greater than or equal to 2 cm, greater than or equal to 3 cm, greater than or equal to 4 cm, greater than or equal to 5 cm, greater than or equal to 6 cm, greater than or equal to 7 cm, greater than or equal to 8 cm, greater than or equal to 9 cm, greater than or equal to 10 cm, greater than or equal to 20 cm, greater than or equal to 50 cm, or greater than or equal to 80 cm. In some embodiments, a TFF system includes one or more hollow fiber membranes having a length of less than or equal to 100 cm, less than or equal to 80 cm, less than or equal to 50 cm, less than or equal to 20 cm, less than or equal to 10 cm, less than or equal to 9 cm, less than or equal to 8 cm, less than or equal to 7 cm, less than or equal to 6 cm, less than or equal to 5 cm, less than or equal to 4 cm, less than or equal to 3 cm, or less than or equal to 2 cm. Combinations of these ranges are also possible (e.g., greater than or equal to 1 cm and less than or equal to 100 cm, or greater than or equal to 5 cm and less than or equal to 50 cm). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

As noted above, the one or more hollow fiber membranes of a TFF system may each comprise a porous membrane that forms the porous sidewall of the corresponding hollow fiber membrane extending circumferentially around the lumen and along a length of the hollow fiber membrane. For example, the hollow fiber membrane may comprise a plurality of separate and/or interconnected pores extending through the wall of the hollow fiber membrane. These pores may be sized, shaped, and/or otherwise configured to permit transmission of a permeate such as liquid cell culture media, cell debris, cells, and/or cell clusters below a threshold size from the internal lumens of the individual hollow fiber membranes to a waste outlet of the TFF system through the porous walls of the hollow fiber membranes. The pores may also have an appropriate average size and size distribution to retain the desired cell clusters above a threshold size. Depending on the size of the desired clusters, in some embodiments, the hollow fiber membrane includes a plurality of pores, wherein an average pore size of the porous hollow fiber membranes is between or equal to 0.15-0.2 microns, 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. Of course, different pore size ranges may also be used depending on the desired target size of the cell clusters.

It should be understood that the above size parameters may be determined using any appropriate measurement technique typically used for measuring parameters generally associated with filters and membranes. For example, in some embodiments, the pore and lumen sizes of the hollow fiber membranes may be measured using an optical measurement device, and where appropriate applicable equations related to pore size determination where interconnected nonlinear pores are used in the hollow fiber membranes.

A hollow fiber membrane may be comprised of any of a variety of suitable materials. In some embodiments, the hollow fiber membrane includes PES.

In some embodiments, the method includes the steps of seeding the bioreactor with 0.01 x 10⁶-10 x 10⁶ viable cells/ml, 0.01 x 10⁶-5 x 10⁶ viable cells/ml, 0.01 x 10⁶-l x 10⁶ viable cells/ml, 0.01 x 10⁶-0.5 x 10⁶ viable cells/ml, 0.01 x 10⁶-0.05 x 10⁶ viable cells/ml, 0.1 x 10⁶-l x 10⁶ viable cells/ml, or 0.3 x 10⁶-0.8 x 10⁶ viable cells/ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the viable cells are dissociated cells. In some embodiments 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the viable cells are dissociated cells. In some embodiments, the method is repeatedly performed over a period of 1-20 days, 1-15 days, 1-10 days, 1-7 days, 1-5 days, 1-3 days, 2-12 days, 8-12 days, 3-8 days, 4-7 days, or 4-6 days. In some embodiments, after repeatedly performing the method of a period of days, at the end of the period, the cell clusters are dissociated. In some embodiments, the cell clusters are dissociated by treating the cell clusters with a one or more proteolytic and collagenolytic enzymes. In some embodiments, the one or more proteolytic and collagenolytic enzymes include any one or more of trypsin, collagenase, Trypsin-like protease XIV, or thermolysin. In some embodiments, the cell clusters are dissociated by treating the cell clusters with ACCUTASE™. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed.

In some embodiments, the bioreactor holds a volume of 1-250 liters, 1-200 liters, 1-150 liters, 1-100 liters, 1-50 liters, 1-25 liters, 1-10 liters, 1-5 liters, 200-250 liters, 150-200 liters, 100-150 liters, 50-100 liters, 45-55 liters, or 190-210 liters of media. In some embodiments, the bioreactor is a stirred tank reactor. In some embodiments, the bioreactor includes stem cells and a stem cell media.

In some embodiments, the bioreactor includes a (Rho-associated, coiled-coil containing protein kinase) ROCK inhibitor. In some embodiments, the ROCK inhibitor is thiazovivin, Fasudil, Y-27632, and/or HA1077. In some embodiments the bioreactor includes basic fibroblast growth factor (bFGF).

In some embodiments, a portion of the cell culture is transported from the bioreactor into the TFF system by means of a pump. In some embodiments, the pump is in the TFF system. In some embodiments the pump is a diaphragm pump (e.g., a 4-piston diaphragm pump), a peristaltic pump, or a magnetic levitation pump. In some embodiments, the cell culture is agitated in the bioreactor to prevent settling of the cell clusters in the bioreactor. In some embodiments, the agitation is performed using a wave reactor, a continuous stirred tank reactor or vertical wheel reactor.

In some embodiments, the method includes replacing a removed portion of liquid media with a new portion of liquid media. In some embodiments the new portion of liquid media includes one or more cell differentiation and/or survival factors. In some embodiments the one or more cell differentiation or survival factors include any one or more of: a ROCK inhibitor (e.g., Y-27632 or thiazovivin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a F0X01 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

Also disclosed herein is a TFF system including a cell culture, wherein the cell culture includes a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor. In some embodiments the plurality of cell clusters includes stem cells. In some embodiments at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells.

In some embodiments, the TFF system is an ATF system. In some embodiments the cell culture flows through the TFF system at a shear rate of 400-800 sec'¹, 400-3500 sec'¹, 400-3000 sec'¹, 400-2500 sec'¹, 400-2000 sec'¹, 400-1500 sec'¹, 1000-3500 sec'¹, 1000-3000 sec'¹, 1000- 2000 sec'¹, 2000-3500 sec'¹, 2000-3000 sec'¹, 1200-1800 sec'¹, 1400-1600 sec'¹, or 1450-1550 sec'¹. In some embodiments, the TFF system includes one or more filters. In some embodiments, the one or more filters include a plurality of pores. In some embodiments the pores are 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2- 5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments the TFF system includes a filter made of PES.

In some embodiments, a shear protectant is present in the cell culture. In some embodiments, the shear protectant is PVA or pluronic. In some embodiments, the shear protectant is PVA, and the PVA is PVA80 or PVA87-89. In some embodiments, the shear protectant is Pluronic, and the Pluronic is P188 or PF68.

In some embodiments, the TFF system includes one or more cassette membranes. In some embodiments, the TFF system includes one or more hollow fiber membrane. In some embodiments, the hollow fiber membrane has a radius of at 0.5-10 mm, 5-10 mm, 2-5 mm, 0.5-7 mm, 0.5-5 mm, 0.5-3 mm, 0.5-2 mm, 0.5-1.2 mm, 0.8-1.2 mm, or 0.9-1.1 mm. In some embodiments, the TFF system includes a plurality of hollow fiber membranes. In some embodiments, the hollow fiber membrane includes a plurality of pores. In some embodiments, the pore sizes are 0.15-0.2 microns, 0.2-100 microns, 0.2-75 microns, 0.2-50 microns, 0.2-25 microns, 0.2-10 microns, 0.2-5 microns, 0.2-1 microns, 1-10 microns, 5-10 microns, 25-50 microns, 50-75 microns, or 75-100 microns. In some embodiments, the hollow fiber membrane includes PES.

In some embodiments, the cell culture includes a ROCK inhibitor. In some embodiments the ROCK inhibitor is selected from the group consisting of thiazovivin, Fasudil, Y-27632, and HA1077. In some embodiments the bioreactor includes Basic fibroblast growth factor (bFGF).

In some embodiments the TFF system includes a pump. In some embodiments the pump is a diaphragm pump (e.g., a 4-piston diaphragm pump), a peristaltic pump, or a magnetic levitation pump.

In some embodiments the cell culture includes one or more cell differentiation or survival factors. In some embodiments the one or more cell differentiation or survival factors include any one or more of: a ROCK inhibitor (e.g., Y-27632 or thiazovivin), a TGF-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a BMP inhibitor (e.g., DMH1, EDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

Cell culture systems, compositions, and methods for cell culture

In some embodiments, the present disclosure provides compositions for cell culture, methods of cell culture, cell culture systems, and methods of using the same. In some embodiments, the compositions, methods and systems can provide continuous perfusion cell culture and/or dynamic cell culture. In some embodiments, the compositions, methods, and systems facilitate the production of a pancreatic islet cell, for example from a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell. In some embodiments the compositions, methods, and systems facilitate the formation of cell clusters. In some embodiments, the compositions, methods and systems facilitate the formation of cell clusters from single cells. In some embodiments, the cells are differentiated in vitro using the compositions, methods and systems described herein. In some embodiments, the cells described herein can be used to form a composition to treat diseases or can be used in a method of treating diseases (e.g., diabetes). In further embodiments, the methods for producing the cell clusters in suspension described herein are amenable to large scale manufacturing.

In embodiments, the cell clusters are stem cell clusters, e.g., embryonic stem cell clusters or iPSC cell clusters. The systems disclosed herein can be used from the propagation of stem cells, or for the differentiation of stem cells into pancreatic cell populations.

In some embodiments the present disclosure provides for a method of continuous perfusion. The method can include the step of culturing a cell culture in a bioreactor. The cell culture can include a liquid media and/or a plurality of cell clusters. A portion of the cell culture can be transported from the bioreactor into a TFF system, such as an ATF system. A portion of the liquid media from the cell culture can be removed in the TFF system. A portion of the liquid media and/or cell clusters can be retained in the TFF system. A retained portion of the liquid media and/or cell clusters can be returned from the TFF system to the bioreactor. A removed portion of the liquid media can be replaced, such as with a new portion of liquid media.

In some embodiments the present disclosure provides for a TFF system, such as an ATF system. The TFF and/or ATF system can include a cell culture, such a cell culture including a liquid media and/or a plurality of cell clusters. The TFF and/or ATF system can be in fluid communication with a bioreactor.

Continuous perfusion or dynamic cell culture

The disclosed systems can include a bioreactor. The disclosed compositions can be included in a bioreactor. The disclosed methods can utilize a bioreactor. In some embodiments, the system uses TFF, such as ATF. The TFF and ATF systems described herein can facilitate the formation and/or maintenance of cell clusters. In some embodiments, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9% of the cells in a cell culture are found in cell clusters, or a range between any two of the preceding values. In a specific example, greater than 6% of the total cells, such as the total viable cells, are found in cell clusters. In another specific example, greater than 10% of the total cells, such as the total viable cells, are found in cell clusters.

The bioreactor can be a vessel of any shape which permits the introduction of nutrients and oxygen and which facilitates the growth of cells, such as those described herein. In some embodiments, the bioreactor is used for an in vitro culture. In some embodiments the bioreactor facilitates perfusion of a cell culture within the bioreactor, such as continuous perfusion. Perfusion or continuous perfusion can entail the passage of culture media through the bioreactor. In some embodiments the perfusion or continuous perfusion can supply one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collageno lytic enzymes, or other agents. In some embodiments the perfusion or continuous perfusion can remove waste products. In some embodiments the bioreactor can facilitate agitation, such as where the cells experience intentional active motion. In some embodiments, this agitation can allow propagation of cell clusters in the bioreactor. Agitation can be achieved by mechanisms such as a wave reactor, a stirred tank reactor, a continuous stirred tank reactor or vertical wheel reactor. In some embodiments, a cell culture within the bioreactor is agitated in the bioreactor using tangential flow. In some embodiments, this agitation can prevent settling of the cell clusters in the bioreactor. In some embodiments the bioreactor can facilitate cell settling, such as by deactivating a mechanism which induces deactivating a mechanism which agitates the cell culture. Exemplary bioreactors are described in U.S. Pat. Nos. 5,320,963, 5,605,822, and 5,155,035, each of which is incorporated by reference herein.

A tangential flow filtration (TFF) system can be used in the compositions, methods, and systems described herein. “Tangential flow filtration” refers to flow tangential to a filtration element, such as to a membrane surface of the filtration element. In some embodiments, TFF is used for the filtration of cells. In some embodiments, media including cells, and/or cell clusters are fed into the TFF system, and pass tangentially across the filtration element. In some embodiments, the filtration element separates a portion of the media from the cells (or cell clusters). In specific embodiments, tangential passage across the filtration element removes a portion of liquid media from a cell culture. In some embodiments, the TFF system includes an inlet or outlet for the input of liquid media, cells (and/or cell clusters) and another inlet or outlet for the removal of the portion of the liquid media. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the TFF system. An exemplary TFF system is described in U.S. Pat. No. 5,102,545.

In some embodiments, the TFF system includes, or is an alternating tangential flow filtration (ATF) system. “Alternating tangential flow filtration” refers to a flow tangential to a filtration element, followed by the flow being reversed. In some embodiments, media including cells and/or cell clusters are fed into an ATF system and pass tangentially across the filtration element, subsequently, when the flow is reversed, the media including the cells and/or the cell clusters again pass tangentially across the filtration element, but in a substantially reversed direction. In some embodiments, the ATF system includes a single inlet or outlet, and a filtration element, for input of the media, cells and/or cell clusters for input when the flow is in the direction toward bioreactor, and for removal when the flow is in the direction away from the bioreactor. An ATF system can include a diaphragm pump. In some embodiments, the air chamber of the diaphragm pump can become pressurized, pushing medium and cells tangentially across the filter element, subsequently the air chamber of the diaphragm pump can empty, pulling medium and cells tangentially across the filter element again, and pulling medium and cells into a diaphragm liquid chamber. In some embodiments the ATF system includes a 4- piston diaphragm pump, a peristaltic pump, and/or a magnetic levitation pump. In specific embodiments, tangential passage across the filtration element removes a portion of liquid media from a cell culture.

The TFF system, such as the ATF system, includes a filtration element. In some embodiments, the filtration element separates a portion of the media from the cells. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the ATF system. In specific embodiments, tangential passage across the filtration element retains a portion of the liquid media and cell clusters in the ATF system. An exemplary ATF system is described in U.S. Pat. No. US 8,206,981, which discloses ATF systems are beneficial because they diminish the aggregation of cells during the process of cell culture.

In some embodiments, TFF and/or ATF can operate continuously or intermittently to filter media, such as media received from a cell culture in a bioreactor. In some embodiments, TFF and/or ATF can separate one or more of cells, media, cell waste products, differentiation factors, and/or other agents. In a specific embodiment, the TFF and/or ATF can separate the cell waste products, media, and/or differentiation factors from cells, such as pluripotent stem cells, definitive endoderm cells, primitive gut tube cells, pancreatic progenitor cells, endocrine progenitor cells, and/or clusters of any of the aforementioned examples of cells.

In some embodiments, a TFF system and/or ATF system can include one or more filters and/or membranes. The one or more filters and/or membranes can contribute to the separation of the cells, cell clusters, media, cell waste products, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collagenolytic enzymes, and/or other agents. Characteristics of the filters and/or membranes such as porosity and material can affect the speed of filtration. The one or more filters and/or membranes can include a plurality of pores. The pores can be about 0.1 microns, about 0.15 microns, about 0.2 microns, about 1 micron, about 5 microns, about 10 microns, about 25 microns, about 50 microns, about 75 microns, about 100, microns, or a range between any of the preceding values such as about 0.1-100 microns, about 0.1-75 microns, about 0.1-50 microns, about 0.1-25 microns, about 0.1-10 microns, about 0.1-5 microns, about 0.1-1 microns, about 0.15-100 microns, about 0.15-75 microns, about 0.15-50 microns, about 0.15-25 microns, about 0.15-10 microns, about 0.15-5 microns, about 0.15-1 microns, about 0.2-100 microns, about 0.2- 75 microns, about 0.2-50 microns, about 0.2-25 microns, about 0.2-10 microns, about 0.2-5 microns, about 0.2-1 microns, about 1-10 microns, about 5-10 microns, about 25-50 microns, about 50-75 microns, or about 75-100 microns. In some embodiments, the TFF and/or ATF system includes a filter which includes PES. In a specific example, the TFF and/or ATF system includes a filter made of PES.

In some embodiments, a TFF system and/or ATF system can include one or more cassette membranes. In some embodiments, a cassette membrane can be contained within a housing element to form a cassette. In some embodiments, media can pass through the cassette, and pass tangentially across a filtration element, such as a membrane element, within the cassette. In some embodiments, the cassette membrane can separate a portion of media from cells. In some embodiments, the cassette membrane can be exchangeable/replaceable, for example if the membrane element is fouled. In some embodiments, the TFF and/or ATF system can form a fluid-tight seal with the housing element and/or with the cassette membrane. In some embodiments the housing of the cassette membrane can have an inlet configured for liquid intake. In some examples, the housing of the cassette membrane can have an outlet configured for liquid discharge. The inlet and/or the outlet can be fluidly coupled to other embodiments of a TFF system and/or ATF system. An exemplary cassette which can be used in a TFF system is described in U.S. Pat. No. 6,312,591.

In some embodiments, a TFF system and/or ATF system can include one or more hollow fiber membranes. In a specific example, the TFF and/or ATF system includes a plurality of hollow filter membranes. In some embodiments, a hollow filter membrane can have a body, which defines a lumen through which a liquid can pass. In some embodiments, the lumen of the hollow filter membrane has a diameter of 0.1-10 mm, 0.1-5 mm, 0.1-2 mm, 0.1-1.5 mm, 0.1-1.1 mm, 0.1-0.8 mm, 0.1-0.4 mm, 0.1-0.2 mm, 0.8- 1.2 mm, or 0.9- 1.1 mm, or about 1 mm. The body of the hollow filter membrane can include pores or is porous. In some embodiments, the porous body allows for filtration. In a specific example, the hollow fiber membrane includes a plurality of pores. In some embodiments, the pore sizes are about 0.1 microns, about 0.15 microns, about 0.2 microns, about 1 microns, about 5 microns, about 10 microns, about 25 microns, about 50 microns, about 75 microns, about 100 microns, such as about 0.1-100 microns, about 0.1-75 microns, about 0.1-50 microns, about 0.1-25 microns, about 0.1-10 microns, about 0.1-5 microns, about 0.1-1 microns, about 0.15-100 microns, about 0.15-75 microns, about 0.15-50 microns, about 0.15-25 microns, about 0.15-10 microns, about 0.15-5 microns, about 0.15-1 microns, about 0.2-100 microns, about 0.2-75 microns, about 0.2-50 microns, about 0.2-25 microns, about 0.2-10 microns, about 0.2-5 microns, about 0.2-1 microns, about 1-10 microns, about 5-10 microns, about 25-50 microns, about 50-75 microns, or about 75-100 microns. In some embodiments, the hollow fiber membrane has a radius of about 0.5 mm, about 0.8 mm, about 0.9 mm, about 1.0 mm, about 1.1 mm, about 1.2 mm, about 2 mm, about 3 mm, about 5 mm, about 7 mm, or about 10 mm, or a range between any of the preceding values such as about 0.5-10 mm, about 5-10 mm, about 2-5 mm, about 0.5-7 mm, about 0.5-5 mm, about 0.5-3 mm, about 0.5-2 mm, about 0.5- 1.2 mm, about 0.8- 1.2 mm, or about 0.9-1.1 mm. In some embodiments the hollow fiber membrane includes PES. In a specific example, the hollow fiber membrane is made of PES. In some embodiments, the one or more hollow fiber membranes can be bundled together within a housing element. In some embodiments, a plurality of hollow fiber membranes can be disposed in parallel, or substantially parallel in relation to each other. In some embodiments the hollow fiber membrane can have an inlet configured for liquid intake. In some embodiments, the hollow fiber membrane can have an outlet configured for liquid discharge. The inlet and/or the outlet can be fluidly coupled to other embodiments of a TFF system and/or ATF system. In some embodiments, the hollow fiber membranes used in a TFF system are those described in U.S. Pat. No. 10,166,511.

In some embodiments a TFF system and/or ATF system can include one or more pumps. In a specific embodiment the pump is in the TFF system and/or ATF system. In a specific embodiment the pump is external to the TFF system and/or ATF system. In some embodiments, one or more pumps can be in fluid communication with a bioreactor, a TFF system, an ATF system, a media source, and/or a permeate reservoir. In some embodiments a pump is configured for unidirectional flow. In some embodiments a pump is configured for bidirectional flow. In some embodiments the pump is a diaphragm pump, such as a 4-piston diaphragm pump. In some embodiments the pump is a peristaltic pump. In some embodiments the pump is a magnetic levitation pump. In some embodiments a pump is configured to operate continuously. In some embodiments a pump is configured to operate intermittently. In some embodiments a pump is configured to reverse the direction of its flow after a set period of time, such as about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minutes, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 12 hours, about 24 hours, or a range between any two of the preceding values.

In some embodiments a pump transports a portion of a cell culture from a bioreactor into a TFF (e.g., ATF) system. In some embodiments a pump removes a portion of liquid media from the cell culture. In some embodiments a portion of the liquid media and cells and/or cell clusters are retained in the TFF system. In some embodiments a pump replaces a removed portion of liquid media from the cells and/or cell clusters with a new portion of liquid media. In some embodiments a pump causes cells, cell aggregates, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and collagenolytic enzymes, other agents, and/or waste products to pass tangentially across a filtration element. In some embodiments, the filtration element separates a portion of the media from the cells and/or cell clusters. In some embodiments a pump removes permeate from a TFF (e.g., ATF) system. In some embodiments a pump provides liquid media to a bioreactor. In some embodiments a pump provides agitation of a bioreactor.

In some embodiments the bioreactor can hold a volume of about 1 liter, about 5 liters, about 10 liters, about 25 liters, about 45 liters, about 50 liters, about 55 liters, about 100 liters, about 150 liters, about 190 liters, about 200 liters, about 210 liters, about 250 liters of media, or a range between any two of the preceding values such as about 1-250 liters, about 1-200 liters, about 1-150 liters, about 1-100 liters, about 1-50 liters, about 1-25 liters, about 1-10 liters, about 1-5 liters, about 200-250 liters, about 150-200 liters, about 100-150 liters, about 50-100 liters, about 45-55 liters, about 190-210 liters of media. In some embodiments, the bioreactor can hold a volume of about 250-1000, about 250-750, about 250-500, 500-1000, 500-750, or 750-1000 liters of media. In some embodiments the total cell culture has a higher volume of media than can be held in the bioreactor, for example if some portion of the volume of the cell culture is present in a TFF system.

In some embodiments, a volume of media is exchanged by removing a portion of liquid media, retaining a portion of liquid media, returning a retained portion of liquid media, and/or replacing a removed portion of liquid media, such as with a new portion of liquid media. A “volume of media” is in relation to the original volume of media in the bioreactor. In some embodiments the original volume of media is about 1 liter, about 5 liters, about 10 liters, about 25 liters, about 45 liters, about 50 liters, about 55 liters, about 100 liters, about 150 liters, about 190 liters, about 200 liters, about 210 liters, or about 250 liters of media. In some embodiments, the original volume of media is about 1-250 liters, about 1-200 liters, about 1-150 liters, about 1- 100 liters, about 1-50 liters, about 1-25 liters, about 1-10 liters, about 1-5 liters, about 200-250 liters, about 150-200 liters, about 100-150 liters, about 50-100 liters, about 45-55 liters, about 190-210 liters of media. In some embodiments, the original volume of media is about 250-1000, about 250-750 liters, about 250-500 liters, 500-1000 liters, 500-750 liters, or 750-1000 liters of media. A set number of volumes of media can be exchanged in a set period of time. For example, a set number of volumes of media can be exchanged in about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, or a range between any two of the preceding values. In a specific example, a set number of volumes of media are exchanged in a 24-hour period. When the exchange is over a 24-hour period, it can be referred to as an exchange of vessel volumes per day (VVD), such as about 2.7 vessel volumes per day. In one example, about 0.3, about 0.8, about 1, about 2, about 2.5, about 2.7, about 3, about 3.5, about 4, about 5, or a range between any two of the preceding values, such as about 0.3-1, about 0.3-0.8, about 1-5, about 1-4, about 1-3, about 2-5, about 2-4, about 2-3, about 2.5-3.0, about 2.6-2.8, or about 2.5-3.5 volumes of media can are exchanged in a set period of time (e.g., 24 hours). In one example, about 2.5-3.0 volumes are exchanged, such as within a 24-hour period.

Any of the cell cultures disclosed herein comprises one or more cells (e.g., a plurality of any of the cell clusters disclosed herein) and a liquid media. A cell culture can include one or more of cells, cell clusters, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and/or collagenolytic enzymes, other agents, and/or waste products. In some embodiments, a liquid media comprises one or more of cells, cell clusters, nutrients, oxygen, culture media, one or more differentiation factors, one or more growth factors, one or more survival factors, one or more proteolytic and/or collagenolytic enzymes, other agents, and/or waste products. Cell culturing can be performed in a bioreactor, such as a bioreactor in fluid communication with a TFF and/or ATF system. In some embodiments, the liquid media is substantially free of cells and/or cell clusters. In some examples, the liquid media has less than about 1.00e-5 cells per liter and/or cell clusters per liter, less than about 1.00e-4 cells per liter and/or cell clusters per liter, less than about 1.00e-3 cells per liter and/or cell clusters per liter, less than about 1.00e-2 cells per liter and/or cell clusters per liter, less than about 1.00e-l cells per liter and/or cell clusters per liter, l.OOel cells per liter and/or cell clusters per liter, less than about 1.00e2 cells per liter and/or cell clusters per liter, less than about 1.00e3 cells per liter and/or cell clusters per liter, less than about 1.00e4 cells per liter and/or cell clusters per liter, less than about 1.00e5 cells per liter and/or cell clusters per liter, less than about 1.00e6 cells per liter and/or cell clusters per liter, or less than 1.00e7 cells per liter and/or cell clusters per liter.

A density of cells or cell clusters can be found in the cell culture, in the liquid media, and/or in the bioreactor. In some embodiments, the density of cells or cell clusters in the cell culture and/or in the liquid media is different than the density in the bioreactor, for example if a portion of the culture within a TFF system has a different density than in the bioreactor. In some embodiments, about 1.00e4 cells/mL, about 2.00e4 cells/mL, about 3.00e4 cells/mL, about 4.00e4 cells/mL, about 5.00e4 cells/mL, about 6.00e4 cells/mL, about 7.00e4 cells/mL, about 8.00e4 cells/mL, about 9.00e4 cells/mL, about 1.00e5 cells/mL, about 2.00e5 cells/mL, about 3.00e5 cells/mL, about 4.00e5 cells/mL, about 5.00e5 cells/mL, about 6.00e5 cells/mL, about 7.00e5 cells/mL, about 8.00e5 cells/mL, about 9.00e5 cells/mL, about 1.00e6 cells/mL, about 2.00e6 cells/mL, about 3.00e6 cells/mL, about 4.00e6 cells/mL, about 5.00e6 cells/mL, about 6.00e6 cells/mL, about 7.00e6 cells/mL, about 8.00e6 cells/mL, about 9.00e6 cells/mL, about 1.00e7 cells/mL, about 1.10e7 cells/mL, about 2.00e7 cells/mL, about 3.00e7 cells/mL, about 4.00e7 cells/mL, about 5.00e7 cells/mL, about 6.00e7 cells/mL, about 7.00e7 cells/mL, about 8.00e7 cells/mL, about 9.00e7 cells/mL, about 1.00e8 cells/mL, about 2.00e8 cells/mL, about 3.00e8 cells/mL, about 4.00e8 cells/mL, about 5.00e8 cells/mL, about 6.00e8 cells/mL, about 7.00e8 cells/mL, about 8.00e8 cells/mL, about 9.00e8 cells/mL, or a range between any two of the preceding values such as about 3.00e5-4.00e5 cells/mL, about 7.00e5-8.00e5 cells/mL, or about 9.00e6-1.10e7 cells/mL can be present in the cell culture, in the liquid media and/or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and/or into the bioreactor, or can be present in the cell culture, present in the liquid media, and/or present in the bioreactor after a defined period of time. In some embodiments, the cell density reflects the density of viable cells.

In some embodiments, about 1.00e4 clusters/mL, about 2.00e4 clusters/mL, about 3.00e4 clusters/mL, about 4.00e4 clusters/mL, about 5.00e4 clusters/mL, about 6.00e4 clusters/mL, about 7.00e4 clusters/mL, about 8.00e4 clusters/mL, about 9.00e4 clusters/mL, about 1.00e5 clusters/mL, about 2.00e5 clusters/mL, about 3.00e5 clusters/mL, about 4.00e5 clusters/mL, about 5.00e5 clusters/mL, about 6.00e5 clusters/mL, about 7.00e5 clusters/mL, about 8.00e5 clusters/mL, about 9.00e5 clusters/mL, about 1.00e6 clusters/mL, about 2.00e6 clusters/mL, about 3.00e6 clusters/mL, about 4.00e6 clusters/mL, about 5.00e6 clusters/mL, about 6.00e6 clusters/mL, about 7.00e6 clusters/mL, about 8.00e6 clusters/mL, about 9.00e6 clusters/mL, about 1.00e7 clusters/mL, about 1.10e7 clusters/mL, about 2.00e7 clusters/mL, about 3.00e7 clusters/mL, about 4.00e7 clusters/mL, about 5.00e7 clusters/mL, about 6.00e7 clusters/mL, about 7.00e7 clusters/mL, about 8.00e7 clusters/mL, about 9.00e7 clusters/mL, about 1.00e8 clusters/mL, about 2.00e8 clusters/mL, about 3.00e8 clusters/mL, about 4.00e8 clusters/mL, about 5.00e8 clusters/mL, about 6.00e8 clusters/mL, about 7.00e8 clusters/mL, about 8.00e8 clusters/mL, about 9.00e8 clusters/mL, or a range between any two of the preceding values can be present in the cell culture, in the liquid media and/or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and/or into the bioreactor, or can be present in the cell culture, present in the liquid media, and/or present in the bioreactor after a defined period of time. In some embodiments, the cluster density reflects the density of viable cell clusters.

In some embodiments, about 0.05-3 viable cells (VCs)/ml are present in the cell culture, in the liquid media and/or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and/or into the bioreactor, or can be present in the cell culture, present in the liquid media, and/or present in the bioreactor after a defined period of time. In some embodiments, 0.05-0.1 VCs/ml, 0.1-2 VCs/ml, 0.1-1 VCs/ml, 0.2-0.9 VCs/ml, 0.3-0.7 VCs/ml, 0.4-0.6 VCs/ml, or about 0.5 VCs/ml are present in the cell culture, in the liquid media and/or in the bioreactor, can be inoculated into the cell culture, into the liquid media, and/or into the bioreactor, or can be present in the cell culture, present in the liquid media, and/or present in the bioreactor after a defined period of time.

In some embodiments, the cells or cell clusters are pluripotent stem cells, and are cultured in stem cell media. Exemplary stem cell medias include STEMSCALE™, NUTRISTEM®, TESR™, STEMSPAN™, STEMDIFF™, and STEMPRO™-34.

A particular challenge of culturing cell clusters in bioreactors relates to the sensitivity of cell clusters to fluid shear. Excessive shear rates can subject cell clusters to excessively high shear stresses which tear cell clusters apart. It is this challenge that has typically led practitioners to apply shear stresses that are as low as possible to cell clusters during growth. Unexpectedly, however, it has been recognized in the context of the present disclosure that intermittently subjecting cell clusters to occasional, high shear rates in combination with filtration (e.g., in a system or method provided herein) can favorably improve a size distribution of the resulting cell clusters improving both the yield and efficiency of the process. For example, subjecting cell clusters to relatively high shear rates can, in some embodiments, reduce the size of cell clusters greater than a desired size range. Cell clusters, debris, and other waste less than the desired size range may also be filtered out of the cell culture media using appropriate filtration methods as disclosed herein to further improve the selectivity of the desire cell cluster size formation. Accordingly, certain aspects of the present disclosure relate to the control of shear rates to which cell clusters and/or cell media are subjected. In some embodiments, the liquid media includes a shear protectant, one or more differentiation factors, one or more survival factors, one or more growth factors, one or more proteolytic and/or collagenolytic enzymes, nutrients, oxygen and/or other agents. In some embodiments the liquid media includes cells, cell aggregates, and/or waste products. In some embodiments, a new portion of liquid media is added (e.g., to replace any removed liquid media), such as a new portion including one or more differentiation or survival factors. In some embodiments, a new portion of liquid media is added, such as a new portion including one or more shear protectants.

In some embodiments, cells and/or cell clusters are transported from the bioreactor through a TFF and/or ATF system at a shear rate. In some embodiments, cells within said cell culture experience shear stress. Both shear rate and shear stress can be used to define the fluid shear within a system, such as within a TFF and/or ATF system.

The shear rate applied to a fluid, and materials within the fluid (e.g., cell clusters) may be determined by any of a variety of suitable measurements. Without wishing to be bound by any particular theory, in some embodiments, the shear rate may be calculated using a pipe flow model by assuming that the fluid is a Newtonian fluid subject to laminar flow. For example, in some embodiments (e.g., where fluid flows through a cylindrical tube such as a hollow fiber of a hollow fiber membrane), the shear rate (units: sec'¹) may be determined based on the rate of volumetric flow (Q, units: m³/s) and the radius (r, units: m) of the pipe, or other appropriate fluid path, using the equation:

4Q

Shear Rate = — - (1) nr³

The above parameters may either be commanded during operation and design of the system and/or may be measured dynamically. It should be understood that the above shear rates are determined for laminar flows. Therefore, other appropriate methods, such as computational fluid dynamic simulations may be performed to determine the shear rate applied to the fluids and cell clusters contained therein.

The liquid media comprising the cells and/or cell clusters has a fluid viscosity. Fluid viscosity can be measured in poise (1 poise=l dyne sec/cm²=100 centipoise (cp) = 0.1 Pa*s). The viscosity of water is 1 cp. The viscosity of an exemplary suspension of cells (or cell clusters) in media can be between 1.0 and 1.1 cp at 25°C. More generally, suspensions of cells may have any of a variety of suitable viscosities. In some embodiments, a suspension of cells has a viscosity of greater than or equal to 1 cp, greater than or equal to 1.1 cp, greater than or equal to 1.5 cp, greater than or equal to 2 cp, greater than or equal to 5 cp, greater than or equal to 10 cp, greater than or equal to 20 cp, greater than or equal to 30 cp, greater than or equal to 40 cp, greater than or equal to 50 cp, greater than or equal to 60 cp, greater than or equal to 70 cp, greater than or equal to 80 cp, greater than or equal to 90 cp, greater than or equal to 100 cp, greater than or equal to 110 cp, greater than or equal to 120 cp, greater than or equal to 130 cp, greater than or equal to 140 cp, greater than or equal to 150 cp, greater than or equal to 160 cp, greater than or equal to 170 cp, greater than or equal to 180 cp, or greater than or equal to 190 cp.

In some embodiments, a suspension of cells has a viscosity of less than or equal to 200 cp, less than or equal to 190 cp, less than or equal to 180 cp, less than or equal to 170 cp, less than or equal to 160 cp, less than or equal to 150 cp, less than or equal to 140 cp, less than or equal to 130 cp, less than or equal to 120 cp, less than or equal to 110 cp, less than or equal to 100 cp, less than or equal to 90 cp, less than or equal to 80 cp, less than or equal to 70 cp, less than or equal to 60 cp, less than or equal to 50 cp, less than or equal to 40 cp, less than or equal to 30 cp, less than or equal to 20 cp, less than or equal to 10 cp, less than or equal to 5 cp, less than or equal to 2 cp, less than or equal to 1.5 cp, less than or equal to 1.2 cp, or less than or equal to 1.1 cp. Combinations of these ranges are also possible (e.g., greater than or equal to 1 cp and less than or equal to 200 cp, greater than or equal to 1 cp and less than or equal to 10 cp, greater than or equal to 1 cp and less than or equal to 2 cp, or greater than or equal to 1 cp and less than or equal to 1.1 cp). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited. Density and temperature can affect fluid viscosity. The concentration of cells or cell media components can also affect fluid viscosity. Therefore, it should be understood that the above noted viscosities may be measured at a desired operational temperature during culturing and cell cluster formation. The viscosities associated with any of the embodiments disclosed herein may be measured at an operational temperature of the fluid during operation of the system and may be measured with any appropriate type of viscometer for the viscosity range of the liquid media.

The cell culture and/or the liquid media can be transported from the bioreactor through the TFF system. In some embodiments, the cell culture and/or liquid media can include cells and/or cell clusters. In some embodiments the cell culture and/or liquid media is transported from the bioreactor through the TFF system at a shear rate of about 400 sec'¹, about 500 sec'¹, about 525 sec'¹, about 550 sec'¹, about 600 sec'¹, about 800 sec'¹, about 1000 sec'¹, about 1200 sec'¹, about 1400 sec'¹, about 1450 sec'¹, about 1500 sec'¹, about 1550 sec'¹, about 1600 sec'¹, about 1800 sec'¹, about 2000 sec'¹, about 2500 sec'¹, about 3000 sec'¹, about 3500 sec'¹, or a range between any two of the preceding values, such as about 400-800 sec'¹, about 400-3500 sec' about 400-3000 sec'¹, about 400-2500 sec'¹, about 400-2000 sec'¹, about 400-1500 sec'¹, about 1000-3500 sec'¹, about 1000-3000 sec'¹, about 1000-2000 sec'¹, about 2000-3500 sec'¹, about 2000-3000 sec'¹, about 1200-1800 sec'¹, about 1400-1600 sec'¹, or about 1450-1550 sec'¹. In a specific example, cell culture and/or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 525-3000 sec'¹. In a further specific example, cell culture and/or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 525-1600 sec'¹. In a further specific example, cell culture and/or the liquid media is transported from the bioreactor through the TFF system at a shear rate of about 1400-1600 sec'¹. In some embodiments, cell culture and/or the liquid media is transported from the bioreactor through the TFF system at a shear rate of greater than 500 sec'¹.

As discussed above, certain advantages have been recognized to using comparatively high shear rates for the purpose of controlling cell cluster size. The shear rate may be chosen to fall within a range particularly suitable for a desired cell cluster size. In some embodiments, the cell culture and/or liquid media is transported from the bioreactor through the TFF system at a shear rate of greater than or equal to 400 sec'¹, greater than or equal to 600 sec'¹, greater than or equal to 800 sec'¹, greater than or equal to 1000 sec'¹, greater than or equal to 1200 sec'¹, greater than or equal to 1400 sec'¹, greater than or equal to 1600 sec'¹, or greater than or equal to 1800 sec'¹. In some embodiments, the cell culture and/or liquid media is transported from the bioreactor through the TFF system at a shear rate of less than or equal to 2000 sec'¹, less than or equal to 1800 sec'¹, less than or equal to 1600 sec'¹, less than or equal to 1400 sec'¹, less than or equal to 1200 sec'¹, less than or equal to 1000 sec'¹, less than or equal to 800 sec'¹, or less than or equal to 600 sec'¹. Combinations of these ranges are also possible (e.g., greater than or equal to 400 sec'¹ and less than or equal to 2000 sec'¹, or greater than or equal to 600 sec'¹ and less than or equal to 1800 sec'¹). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

Fluid velocity can affect whether a fluid flow will be laminar or turbulent. Laminar flow is seen when viscous forces are dominant whereas turbulent flow is seen when high velocity and inertial forces are dominant. Laminar flow is characterized by smooth and/or even streamlines at low velocity. Turbulent flow is characterized by eddies, vortices, and chaotic fluctuations.

As discussed above, controlling the shear rate to which a fluid is subjected can have important implications for controlling the viability and size of cell clusters cultured in a bioreactor. Whereas shear rate is consistent in fluids under laminar flow (e.g., as described in the case of pipe-flow by equation (1) above), turbulent flow introduces local fluctuations in shear stress that can expose cell clusters to chronically extreme shear forces. Accordingly, controlling whether cell media flows laminarly or turbulently in a system or method provided herein can have a significant effect on average cluster size of cells. It should, of course, be understood that laminar and/or turbulent flow may be used to culture cells of a desired size range, depending on the embodiment. However, the ability of laminar flow to provide relatively consistent shear rates, rather than fluctuating shear rates, may have advantages for homogenizing the size of cell clusters and/or for sizing them appropriately for a desired application. Therefore, in some embodiments, the various bioreactors and filtration systems disclosed herein may be operated in a laminar flow regime with a Reynold’s number (Re) less than 2300 as elaborated on further below.

Reynold's number (Re) can be used to quantify the presence of laminar or turbulent flow. Reynold's number is the ratio of inertial to viscous forces, quantitated as (density*velocity*length scale)/(viscosity). Laminar flow dominates when Re<2300. Turbulent flow dominates when Re>4000. Re is directly proportional to the shear rate and shear stress experienced by cells in a cell suspension. The Reynold’s number is a dimensionless quantity that, without wishing to be bound by any particular theory, can be calculated using a pipe flow model by assuming that the fluid is a Newtonian fluid. For example, in some embodiments (e.g., where fluid flows through a cylindrical tube such as a hollow fiber of a hollow fiber membrane), the Reynold’s number (Re, dimensionless) may be determined based on the rate of volumetric flow (Q, units: m³/s), the hydraulic diameter (DH, units: m) of the pipe (equivalent to the pipe diameter for a cylindrical pipe or fiber), the cross sectional area of the pipe, (A, units: m²), the mass density of the fluid (p, units: kg/m³), and the dynamic viscosity of the fluid (p, units: Pa*s) using the equation:

PQDH

Re = pA

Which simplifies, in the case of a cylindrical pipe or fiber, to:

2pQ

Re pnr where r is the pipe radius (units: m).

A cell culture may be transported from the bioreactor through the TFF system with any of a variety of appropriate Reynold’s numbers. In some embodiments, according to some embodiments, a cell culture is transported from the bioreactor through the TFF system with a Reynold’s numbers of greater than 0, greater than or equal to 50, greater than or equal to 100, greater than or equal to 200, greater than or equal to 300, greater than or equal to 400, greater than or equal to 500, greater than or equal to 600, greater than or equal to 700, greater than or equal to 800, greater than or equal to 900, greater than or equal to 1000, greater than or equal to 1250, greater than or equal to 1500, or greater than or equal to 1750. In some embodiments, a cell culture is transported from the bioreactor through the TFF system with a Reynold’s numbers of less than or equal to 2000, less than or equal to 1750, less than or equal to 1500, less than or equal to 1250, less than or equal to 1000, less than or equal to 900, less than or equal to 800, less than or equal to 700, less than or equal to 600, less than or equal to 500, less than or equal to 400, less than or equal to 300, less than or equal to 200, or less than or equal to 100.

Combinations of these ranges are also possible (e.g., greater than 0 and less than or equal to 2000, greater than or equal to 50 and less than or equal to 300, or greater than or equal to 100 and less than or equal to 300). Other ranges, both higher and lower than those described above, are also possible, as the disclosure is not so limited.

There is an initial tendency for liquid to resist movement. Without being bound by theory, fluid closest to a solid surface experiences attractive forces. This generates a boundary layer and/or a region of no-flow immediately adjacent to the surface. Thus, fluid velocity will form a gradient between the region immediately adjacent to the surface and the region at the center of the fluid flow. The gradient’s steepness is affected by the speed of the liquid and the distance from the boundary to the region of highest velocity. As the flow rate accelerates, the velocity of the flow overcomes viscosity of the liquid and laminar flow breaks down resulting in turbulent flow. Cell lysis can occur under turbulent conditions, particularly in regions of high local shear stress and/or energy dissipation, which can be found near the region immediately adjacent to the surface. Thus, specific shear stress can be used to maintain clusters in a culture, or to maintain the disassociation of single cells, or some combination thereof.

In some embodiments a shear protectant is present in cell culture, such as in cell culture in a TFF and/or ATF. A shear protectant can protect cells in the cell culture from shear stress. In some embodiments the shear protectant includes polaxamer, PVA (such as PVA80 and/or PVA87-89) and/or pluronic (such as P188 or PF68). In some embodiments, a cell culture within a bioreactor comprises the shear protectant. In some embodiments a cell culture within a bioreactor does not comprise a shear protectant. In some embodiments a cell culture within a TFF system comprises the shear protectant. In some embodiments a cell culture within an ATF system comprises the shear protectant. In some embodiments, a new portion of liquid media is added to a bioreactor, and the new portion of liquid media comprises a shear protectant. In some embodiments a shear protectant is added to a cell culture with a TFF and/or ATF.

The bioreactor, TFF system, ATF system, and/or pumps described herein can be in fluid communication. In some embodiments the bioreactor, TFF system, ATF system, and/or pumps can be fluidly connected to additional elements such as a reservoir (such as a reservoir which provides a new portion of liquid media, or such as a reservoir which accepts permeate). A fluid connection can be unidirectional, or bidirectional. In a specific example, the bioreactor is in fluid communication with the TFF system. Fluid communication can be achieved by tubing, piping, or other methods known to one of ordinary skill in the relevant art. In some embodiments, a cell culture and/or liquid media is present in the bioreactor, and contacts a bioreactor, a TFF system, and the bioreactor, in that order. In some embodiments, a cell culture and/or liquid media is present in the bioreactor, and contacts the bioreactor, and a TFF system, in that order, at which point a portion of the liquid media from the cell culture is removed. The removed portion of the liquid media can contact the TFF system and a permeate outlet, in that order. In some embodiments, the cell culture includes cell clusters.

In some embodiments, the cell culture and/or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged using rapid media exchange. In some embodiments, the cell culture and/or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged within 1- 30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7- 8 hours. In some embodiments, the cell culture and/or liquid media (which may or may not comprise one or more cell differentiation or survival factors, such as any of the cell differentiation or survival factors disclosed herein) within the bioreactor is exchanged using centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, the disclosure provides for a method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; e) replacing the removed portion of the liquid media with a new portion of liquid media; and f) removing and replacing a portion of the liquid media from the cell culture in the bioreactor by means of rapid media exchange. In some embodiments, steps a)-e) are repeated continuously for a period of time (e.g., 12 hours- 1 day, 1- 14 days, 1-10 days, 1-7 days, 1-5 days, 3-14 days, 3-10 days, 3-7 days, or 1-2 days or 1-3 days) before step f) is performed. In particular embodiments, step f) is performed over a shorter period of time as compared to steps a)-e). In some embodiments, the rapid media exchange is completed within 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In particular embodiments, the rapid media exchange is completed within 30 minutes and 2.5 hours. In some embodiments, the rapid media exchange is performed using centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, the disclosure provides for a method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; e) replacing the removed portion of the liquid media with a new portion of liquid media; and wherein the method further comprises step f), wherein step f) comprises removing and replacing media in the cell culture, wherein step f) is performed for 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4- 5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In some embodiments, the method comprises performing steps a)-e) for 12 hours to 9 days, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 3 days, 12 hours to 1 day, 2-4 days, 4-6 days, or 7-9 days before step f) is performed. In particular embodiments, step f) is performed for 30 minutes to 2.5 hours. In some embodiments, during the media exchange step f), the replacement media comprises one or more differentiation or survival factors. In some embodiments, 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90- 95%, 95-99%, 95-100%, 95-98%, 95-97%, 91-96%, or 92-95% of the media in the reactor is removed and replaced in step f). In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the replacement media in step f) does not comprise one or more differentiation or survival factors that were present in the media removed during step f). In some embodiments, the rapid media exchange is completed within 1-120 minutes, 60-120 minutes, 1-60 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes or 1-5 minutes. In some embodiments, dead or dying cells are removed during step f). In some embodiments, steps a)-e) are repeated following the completion of step f). In some embodiments, the replacement media from steps a)-e) comprise one or more cell differentiation or survival factors. In some embodiments, step f) is performed to remove one or more cell differentiation or survival factors from the cell culture. In some embodiments, step f) is performed to remove one or more cell differentiation or survival factors from the media previously used in steps a)-e). For example, in some embodiments, media used in steps a)-e) comprises a Wnt activator (e.g., CHIR99021), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a Wnt activator (e.g., CHIR99021). In some embodiments, step f) is performed to add one or more new cell differentiation or survival factors to the cell culture. For example, in some embodiments, media used in steps a)-e) does not comprise a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 95-100%, 90-95%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208). In some embodiments, step f) is performed to add one or more new cell differentiation or survival factors to the cell culture and also to remove one or more cell differentiation or survival factors. For example, in some embodiments, media used in steps a)-e) comprises a protein kinase C activator (e.g., PDBU or TPPB) but does not comprise a thyroid receptor activator (e.g., T3 or GC-1), and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a protein kinase C activator (e.g., PDBU or TPPB) but comprises a thyroid receptor activator (e.g., T3 or GC-1). In some embodiments, the one or more cell differentiation or survival factors comprise a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, the one or more cell differentiation or survival factors comprise a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11). In some embodiments, the one or more cell differentiation or survival factors comprise a Wnt activator (e.g., CHIR99021). In some embodiments, the one or more cell differentiation or survival factors comprise a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, the one or more cell differentiation or survival factors comprise a retinoic acid receptor activator (e.g., retinoic acid). In some embodiments, the one or more cell differentiation or survival factors comprise a sonic hedgehog inhibitor (e.g., Santl). In some embodiments, the one or more cell differentiation or survival factors comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin). In some embodiments, the one or more cell differentiation or survival factors comprise a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, the one or more cell differentiation or survival factors comprise a FOXO1 inhibitor (e.g., AS 1842856). In some embodiments, the one or more cell differentiation or survival factors comprise a gamma- secretase inhibitor (e.g., XX, XXI or DAPT). In some embodiments, the one or more cell differentiation or survival factors comprise a thyroid receptor activator (e.g., T3 or GC-1). In some embodiments, the one or more cell differentiation or survival factors comprise a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208). In some embodiments, the one or more cell differentiation or survival factors comprise an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin). In some embodiments, the one or more cell differentiation or survival factors comprise a protein kinase inhibitor (e.g., staurosporine). In some embodiments, the one or more cell differentiation or survival factors comprise an epigenetic modifying compound (e.g., DZNEP). In some embodiments, the one or more cell differentiation or survival factors comprise a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the media removed in step f) comprises one or more of bFGF or Y27632, and the replacement media comprises one or more of Activin A, CHIR99021, or PVA80, and optionally lacks one or more of bFGF or Y27632. In some embodiments, the media removed in step f) comprises one or more of Activin A, CHIR99021, or PVA80, and the replacement media comprises one or more of KGF and PVA80, and the replacement media optionally lacks one or more of Activin A or CHIR99021. In some embodiments, the media removed in step f) comprises one or more of KGF and PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, DMH-1, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, DMH-1, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, wherein the replacement media lacks DMH-1. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, PDBU, Thiazovivin, Activin A, Vitamin C or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, or PVA80, wherein the replacement media optionally lacks PDBU. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, or PVA80, and the replacement media comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, PVA80, AS 1842856, XXI, or PDBU. In some embodiments, the media removed in step f) comprises one or more of KGF, Retinoic Acid, Santl, Thiazovivin, Activin A, Vitamin C, PVA80, AS 1842856, XXI, or PDBU, and the replacement media comprises one or more of Retinoic Acid, GC-1, XXI, Alk5i, Santl, Betacellulin, EDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media optionally lacks one or more of Activin A, KGF, PVA80, or AS 1842856. In some embodiments, the media removed in step f) comprises one or more of Retinoic Acid, GC- 1, XXI, Alk5i, Santl, Betacellulin, EDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media comprises one or more of GC-1, XXI, Alk5i, Santl, LDN-193189, Staurosporine, DZNEP, Thiazovivin, PVA89, NVPTNKS656, Vitamin C, Glutamine, Formate, Taurine, Acetate, P-hydroxybutyrate, or Biotin, and the replacement media lacks one or more of retinoic acid, Betacellulin, or Santl.

In some embodiments, the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). In some embodiments, the media removed in step f) comprises one or more of ITS-X, Activin A, WNT3A, Y-27632, CHIR99021, or LDN193189, and the replacement media comprises one or more of ITS-X, KGF or Vitamin C, and the replacement media optionally lacks one or more of Activin A, WNT3A, Y-27632, CHIR99021, or LDN193189. In some embodiments, the media removed in step f) comprises one or more of ITS-X, Vitamin C, or KGF, and the replacement media comprises one or more of DMH-1, retinoic acid, SANT-1, KGF, Vitamin C, B27, TPPB or PDBU, Activin A, Y-27632 or thiazovivin, or IWR-I-Endo, and the replacement media optionally lacks ITS-X. In some embodiments, the media removed in step f) comprises one or more of DMH-1, retinoic acid, SANT-1, KGF, Vitamin C, B27, TPPB or PDBU, Activin A, Y- 27632 or thiazovivin, or IWR-I-Endo, and the replacement media comprises one or more of EGF, SANT-1, Nicotinamide, KGF, TPPB or PDBU, Ascorbic Acid, B27, retinoic acid, Y- 27632, IWR-I-Endo or WIKI4, and the replacement media optionally lacks DMH-1, TPPB or PDBU. In some embodiments, the media removed in step f) comprises one or more of EGF, SANT-1, Nicotinamide, KGF, TPPB, Ascorbic Acid, B27, retinoic acid, Y-27632, IWR-I-Endo or WIKI4 and the replacement media comprises one or more of galactose, LDN193189, T3 or GC-1, Heparin, ALK5iII, GSI-XX, UNC0321 or ZnSO4, and the replacement media optionally lacks one or more of EGF, Nicotinamide, KGF, TPPB or PDBU, retinoic acid, IWR-I-Endo, or WIKI4. In some embodiments, the media removed in step f) comprises one or more of galactose, SANT-1, LDN193189, Y-27632, Vitamin C, T3 or GC-1, Heparin, ALK5iII, GSI- XX, UNC0321 or ZnSO4, and the replacement media comprises one or more of Alk5i II, LDN193189, Heparin, T3 or GC-1, Vitamin C, N-acetylcysteine, B27, UNC0321, SANT-1, or DNase I, and the replacement media optionally lacks one or more of galactose, Y-27632, or GSI- XX. In some embodiments, the media removed in step f) comprises one or more of Alk5i II, LDN193189, Heparin, T3 or GC-1, Vitamin C, N-acetylcysteine, B27, UNC0321, SANT-1, or DNase I, and the replacement media comprises one or more of CD Lipid, Heparin, CuSO4, ZnSO4, Selenite, Ferric citrate, MnS04, Na2SiO3, Molydbic acid, NH4VO3, NiSO4, SnCI2, Trolox, Carnitine, T3 or GC1, Vitamin C, N-acetylcysteine, LDN193189, or UNC0321, and the replacement media optionally lacks one or more of ALK5i II, B27, SANT-1, or DNase I. In some embodiments, the media removed in step f) comprises one or more of CHIR99021 or CP21R7 or Activin A, and the replacement media comprises one or more of Revitacell, LDN193189 or DMH-1, or AGN193109 and optionally lacks one or more of CHIR99021 or CP21R7 or Activin A. In some embodiments, the media removed in step f) comprises one or more of Revitacell, LDN193189 or DMH-1, or AGN193109 and the replacement media comprises one or more of Revitacell, KGF, LDN193189 or DMH-1, or AGN193109. In some embodiments, the media removed in step f) comprises one or more of Revitacell, KGF, LDN193189 or DMH-1, or AGN193109 and the replacement media comprises one or more of Revitacell, KGF, or AGN193109 and the replacement media optionally lacks LDN193189 or DMH-1. In some embodiments, the media removed in step f) comprises one or more of Revitacell, KGF, or AGN193109 and the replacement media comprises one or more of Glutamax, HAS, ITS-X, NaHCO3, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Betacellulin or EGF, Heparin, Staurosporine, Forskolin, TCS-JNK60, or Linifanib, and the replacement media optionally lacks one or more of Revitacell, KGF, or AGN193109. In some embodiments, the media removed in step f) comprises one or more of Glutamax, HAS, ITS-X, NaHCO3, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Betacellulin or EGF, Heparin, Staurosporine, Forskolin, TCS-JNK60, or Linifanib, and the replacement media comprises one or more of Glutamax, HAS, ITS-X, NaHC03, Vitamin C, ZnSO4, gamma secretase inhibitor XX or XXI, T3 or GC-1, thiazovivin or Y27632, LDN193189, Heparin, Staurosporine, TCS-JNK60, or Linifanib, but the replacement media optionally lacks one or more of Betacellulin or EGF, Forskolin, or TCS-JNK60.

In some embodiments, media used in steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) and/or a Wnt activator (e.g., CHIR99021); and step f) is performed to remove this media (e.g., to remove 70-90%, 80- 90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a Wnt activator (e.g., CHIR99021). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12-72 hours. In some embodiments, replacement media from steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) and/or a Wnt activator (e.g., CHIR99021); and replacement media from step f) comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a Wnt activator (e.g., CHIR99021).

In some embodiments, media used in steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a fibroblast growth factor (e.g., KGF or FGF10); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that does not comprise a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but comprises a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, steps a)-e) are performed 1-5, 1-3, 2-3, or 2-4 days. In some embodiments, replacement media from steps a)-e) comprises a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but does not comprise a fibroblast growth factor (e.g., KGF or FGF10) and replacement media from step f) does not comprise a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11) but comprises a fibroblast growth factor (e.g., KGF or FGF10).

In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10) but does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92- 95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, steps a)-e) are performed 1-5, 1-3, 2-3, 2-4, or 3-5 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10) but does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12-72 hours. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a bone morphogenic protein (BMP) inhibitor (e.g., DMH1, LDN193189, or dorsomorphin).

In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, steps a)-e) are performed for 12-24 hours, 12-36 hours, 12-48 hours, or 12- 72 hours. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a protein kinase C activator (e.g., PDBU or TPPB), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin) and replacement media from step f) comprises fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB).

In some embodiments, steps a)-e) are performed for 1-6, 1-8, 5-8, 4-5, 3-4, 3-5, 2-4, or 4- 7 days, wherein the media comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that not comprise a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), and/or a gamma-secretase inhibitor (e.g., XX, XXI or DAPT); and steps a)-e) are then performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days, wherein the media comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y- 27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a F0X01 inhibitor (e.g., AS1842856), and/or a gamma- secretase inhibitor (e.g., XX, XXI or DAPT); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92- 95% of the media) and replace it with media that comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, steps a)- e) are performed for 1-6, 1-8, 5-8, 3-5, 2-4, or 4-7 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), and/or a gamma-secretase inhibitor (e.g., XX, XXI or DAPT) and replacement media from step f) comprises fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin).

In some embodiments, media used in steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-1- Endo or WIKI4); and step f) is performed to remove this media (e.g., to remove 70-90%, 80- 90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a protein kinase C activator (e.g., PDBU or TPPB), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4), but that does not comprise a FOXO1 inhibitor (e.g., AS 1842856), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a fibroblast growth factor (e.g., KGF or FGF10). In some embodiments, steps a)-e) are performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days. In some embodiments, replacement media from steps a)-e) comprises a fibroblast growth factor (e.g., KGF or FGF10), a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), and/or a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), but that does not comprise a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4) and replacement media from step f) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a protein kinase C activator (e.g., PDBU or TPPB), a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, EY364947, EY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4), but that does not comprise a FOXO1 inhibitor (e.g., AS 1842856), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), and/or a fibroblast growth factor (e.g., KGF or FGF10).

In some embodiments, media used in steps a)-e) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4); and step f) is performed to remove this media (e.g., to remove 70-90%, 80-90%, 70-100%, 80-100%, 90- 100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media) and replace it with media that comprises a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83- 01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), a protein kinase inhibitor (e.g., staurosporine), and/or an epigenetic modifying compound (e.g., DZNEP), but that does not comprise a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR- l-Endo or WIKI4), and/or a protein kinase C activator (e.g., PDBU or TPPB). In some embodiments, steps a)-e) are performed for 1-3, 1-4, 2-4, 2-3, or 1-2 days. In some embodiments, replacement media from steps a)-e) comprises a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), a transforming growth factor (TGF)-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), a protein kinase C activator (e.g., PDBU or TPPB), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP) and/or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4) and replacement media from step f) comprises a gamma-secretase inhibitor (e.g., XX, XXI or DAPT), a Rho- associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), a protein kinase inhibitor (e.g., staurosporine), and/or an epigenetic modifying compound (e.g., DZNEP), but that does not comprise a sonic hedgehog inhibitor (e.g., Santl), a retinoic acid receptor activator (e.g., retinoic acid), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR- 1-Endo or WIKI4), and/or a protein kinase C activator (e.g., PDBU or TPPB).

It should be noted that, because media is removed and replaced in steps a)-e), references to “media used in steps a)-e)” and the like contemplates that older media may be continually removed and replaced with newer media during these steps (i.e., the media used in the steps a)-e) is not static and is not the identical media used throughout those steps). In some embodiments, the older removed media may comprise single cells, dead or dying cells, waste products, and/or lower concentrations of reagents (e.g., glucose or one or more cell differentiation or survival reagents) than the newer replacement media. The replacement media used any point during the specified steps a)-e) may be the same type of media or substantially the same media (i.e., the same or substantially the same reagents and concentrations of reagents in the media) as the replacement media used any other point during the specified steps a)-e). It should also be noted that “media” and “medium” may be used interchangeably herein, unless the context clearly specifies otherwise.

As used herein, the terms “rapid media exchange” or “rapidly exchanging media” mean removal and replacement of media in a vessel (e.g., a bioreactor) by means other than continuous perfusion media exchange. Continuous perfusion media exchange is a process that continuously exchanges culture medium in a cell culture in a vessel (e.g., a bioreactor), while retaining cells and/or cell clusters in the cell culture (e.g., retaining 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-100%, 70-90%, 70-80%, 80-90%, 90-95%, 95-99%, of the cells and/or cell clusters in cell culture) over a period of time. Continuous media exchange may, in some embodiments, preserve the total volume of cell culture in the vessel while exchanging spent cell culture media.

Rapid media exchange may comprise reducing the volume of cell culture within the vessel, e.g., by removing spent culture media and adding new cell culture media. In particular embodiments, rapid media exchange is completed within 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours. In particular embodiments, rapid media exchange is completed with 30 minutes and 2.5 hours. In some embodiments, rapid media exchange removes 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media from the vessel (e.g., bioreactor). In some embodiments, rapid media exchange is performed by means of centrifugation, rapid tangential flow filtration (TFF), rapid alternating tangential flow filtration (ATF) system exchange, or settling. In some embodiments, agitation is performed in a bioreactor (e.g., by an impeller) such that the cells or cell clusters do not settle on a surface of the bioreactor. In particular embodiments, agitation (e.g., by an impeller) is not halted in the bioreactor when performing steps a)-e). In particular embodiments, agitation (e.g., by an impeller) is not halted in the bioreactor when performing continuous perfusion media exchange. In particular embodiments, agitation (e.g., by an impeller) in a bioreactor is significantly reduced (e.g., by 10-100%, 50-100%, 80-100%, 10-20%, 20-40%, 40-60%, 60-80%, 80-90%, or 90- 100%) or halted while performing rapid media exchange. In some embodiments, agitation (e.g., by an impeller) in a bioreactor is maintained when using continuous perfusion media exchange.

Rapid media exchange may, in some embodiments, be partially continuous. For example in some embodiments, rapid media exchange is performed by two or more steps of rapid media exchange (e.g., removing 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95-98%, 95-97%, 91-96%, or 92-95% of the media from the vessel (e.g., bioreactor)), while continuous perfusion is performed between at least two steps of rapid media exchange (e.g., so that media exchange continues during the period between a first step of rapid media exchange and a second step of rapid media exchange).

In some embodiments, the cell culture and/or liquid media within the bioreactor is settled. In some embodiments, settling is achieved by reducing or eliminating agitation in a bioreactor, resulting in the cells and/or clusters to settle to the bottom of the bioreactor. In some embodiments, once the cells and/or clusters have settled to the bottom of the bioreactor, the media (e.g., 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-100%, 95-99%, 95- 98%, 95-97%, 91-96%, or 92-95% of the media) is removed. In some embodiments, the removed media is replaced with new media. In some embodiments, the settling can be part of a settling media exchange, for example, the settling can assist in removing a portion of the liquid media from the cell culture while retaining a portion of the liquid media and the cells and/or cell clusters, and the removed portion of the liquid media can be replaced with a new portion of liquid media. In some embodiments the cell culture and/or liquid media within the bioreactor is not allowed to settle. In some embodiments, any of the methods disclosed herein does not comprise a settling step.

In some embodiments, the cell culture and/or liquid media within the bioreactor is centrifuged. The centrifugation can be part of a centrifugation media exchange, for example, the cell culture and/or liquid media can be transported from the bioreactor into a centrifugation system, the centrifugation system can assist in removing a portion of the liquid media (e.g., 70- 90%, 80-90%, 80-97%, 70-100%, 80-100%, 90-100%, 90-97%, 90-95%, 95-100%, 95-99%, 95- 98%, 95-97%, 91-96%, or 92-95% of the media) from the cell culture and/or liquid media while retaining a portion of the liquid media and cells and/or cell clusters. The retained portion of liquid media and the cells and/or cell clusters can be returned to the bioreactor, and the removed portion of the liquid media can be replaced with a new portion of liquid media. In some embodiments the cell culture and/or liquid media within the bioreactor is not centrifuged. In some embodiments, any of the methods disclosed herein does not comprise a centrifugation step.

In some embodiments, the disclosure provides for methods of removing and/or replacing liquid media from a cell culture using any combination of the methods disclosed herein. For example, in some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the liquid media in the cell culture is exchanged by rapid media exchange (e.g., settling, centrifugation, rapid TFF, and/or rapid ATF). For example, in some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the cell culture and/or liquid media are centrifuged. In some embodiments, the disclosure provides for a method of media exchange in a cell culture by perfusion for a period of time (e.g., 12 hours-7 days, 12 hours-4 days, 12 hours-2 days, 1-3 days, 1.5-2.5 days) before the cell culture and/or liquid media are settled.

In some embodiments, dissociated cells are cultured for a period of time in a bioreactor to allow them to “adapt” to form one or more 3-dimensional cell clusters. In some embodiments, the adaptation period is 1-6 hours, 6-96 hours, 6-72 hours, 6-48 hours, 6-24 hours, 6-12 hours, 12-24 hours, 12-36 hours, 24-36 hours, 36-48 hours, or 36-60 hours.

In some embodiments, cells and/or cell clusters are cultured for a defined period of time after adapting the cells and/or cell clusters from a 2D culture, or for a defined period of time after an in- vessel-passage. In some embodiments, the cells and/or cell aggregates are cultured for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or a range between any two of the preceding values, such about 1-20 days, about 1-15 days, about 1-10 days, about 1-7 days, about 1-5 days, about 1-3 days, about 2-12 days, about 8-12 days, about 3- 8 days, about 4-7 days, about 4-6 days, about 2-4 days, about 5-7 days, about 1-3 days, or about 1-6 days.

Compositions and methods for culturing pluripotent stem cells or stem cell clusters

Disclosed herein are methods for culturing, expanding and differentiating stem cells. A stem cell can, under suitable conditions, differentiate into a diverse range of specialized cell types, while under other suitable conditions it can self-renew and remain in an essentially undifferentiated pluripotent state. “Stem cell” refers to a cell (e.g., plant stem cell, vertebrate stem cell) that has the ability both to self-renew and to generate a differentiated cell type (Morrison et al. (1997) Cell 88:287-298). In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term. The term stem cell also encompasses a pluripotent stem cell, multipotent stem cell, precursor cell and progenitor cell. Stem cells can be characterized by both the presence of specific markers (e.g., proteins, RNAs, etc.) and the absence of specific markers. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays relating to the ability of stem cells to give rise to multiple differentiated progeny. In an embodiment, the host cell is an adult stem cell, a somatic stem cell, a non- embryonic stem cell, an embryonic stem cell, hematopoietic stem cell, an include pluripotent stem cells, and a trophoblast stem cell. In preferred embodiments, the compositions and methods of the disclosure do not comprise a cancer cell.

Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from genital tissue of a fetus. Stem cells can be any cells derived from any kind of tissue (for example embryonic tissue such as fetal or pre-fetal tissue, or adult tissue), which stem cells have the characteristic of being capable under appropriate conditions of producing progeny of different cell types, e.g., derivatives of all of at least one of the 3 germinal layers (endoderm, mesoderm, and ectoderm). These cell types may be provided in the form of an established cell line, or they may be obtained directly from primary embryonic tissue and used immediately for differentiation. Included are cells listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-1, FISF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically-induced differentiation into mature, insulin positive cells did not involve destroying a human embryo.

The term “pluripotent stem cell” or “PSC” is used herein to mean a stem cell capable of producing all cell types of the organism. Therefore, a PSC can give rise to cells of all germ layers of the organism (e.g., the endoderm, mesoderm, and ectoderm of a vertebrate). Pluripotent cells are capable of forming teratomas and of contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.

Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by the expression of certain transcription factors associated with pluripotency; these cells can be called induced pluripotent stem cells or iPSCs. iPSCs can be generated using fetal, postnatal, newborn, juvenile, or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells to pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3/4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors to reprogram a somatic cell to a pluripotent stem cell. An embryonic stem (ES) cell can be an undifferentiated pluripotent cell which is obtained from an embryo in an early stage, such as the inner cell mass at the blastocyst stage, or produced by artificial means (e.g., nuclear transfer) and can give rise to any differentiated cell type in an embryo or an adult, including germ cells (e.g., sperm and eggs). Embryonic stem cell lines are commercially available. In some embodiments, an ES cell is produced without the destruction of an embryo, such as a human embryo. The “plasticity” of a cell refers to a cell’s ability to differentiate into a particular cell type found in tissues or organs from an embryo, fetus or developed organism. The “more plastic” a cell, the more tissues into which the cell may be able to differentiate.

In some embodiments, the pluripotent stem cells can be modified, such as to express an exogenous gene, increase expression of an endogenous gene, increase copy number of a gene, to correct a gene mutation, or to silence the expression of a mutant gene. In some specific nonlimiting examples, a mutation or a deletion in an endogenous gene is corrected. Methods for performing gene editing in stem cells are disclosed, for example, in Hockenmeyer and Jaenisch, “Induced Pluripotent Stem Cell Meets Genome Editing,” Cell Stem Cell 18: 573-586, 2016, incorporated herein by reference in its entirety. Any of the methods disclosed therein are of use. The method can include the use of a viral vector, such as an adeno-associated viral vector or a lentiviral vector ending a transgene of interest. The method can include the use of CRISPR/Cas9, TALEN nuclease, Zinc-finger nuclease, lentiviral mediated correction, adeno- associated virus mediated correction, shRNA, siRNA, or F-prime editing.

In some embodiments, the pluripotent stem cell can be modified to express exogenous nucleic acids, such as to include a promoter and a nucleic acid sequence encoding a protein of interest, such as, but not limited to, a marker. Suitable promoters include, but are not limited to, any promoter expressed in endocrine cells including the insulin, glucagon, and somatostatin promoter. The construct can also include other elements, such as a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. Generally, it is advantageous to transfect cells with the construct. Suitable vectors for stable transfection include, but are not limited to retroviral vectors, lentiviral vectors and Sendai virus.

Plasmids can achieve regulated high copy number and are compatible with use in mammalian cells, including human cells. In some examples, plasmids, they are suitable for maintenance and fermentation in E. coli, so that large amounts of DNA can be produced and purified. Plasmids can be safe and suitable for use in human patients and animals. High copy number plasmids can be selected for and stably maintained relatively easily during bacterial fermentation. Elements such as selectable markers and other coding sequences can be included in a plasmid. In some embodiments plasmids that encode a marker include: (1) a high copy number replication origin, (2) a selectable marker, such as, but not limited to, the neo gene for antibiotic selection with kanamycin, (3) transcription termination sequences, including the tyrosinase enhancer and (4) a multicloning site for incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors that are known in the art for inducing a nucleic acid encoding a protein, such as the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425; and U.S. Patent No. 6,416,998, which are incorporated herein by reference in their entireties.

A viral gene delivery system can be an RNA-based or DNA-based viral vector. An episomal gene delivery system can be a plasmid, an Epstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.

In some embodiments, the cells are transfected with a nucleic acid molecule encoding a marker. Markers include, but are not limited to, fluorescence proteins (for example, green fluorescent protein or red fluorescent protein), enzymes (for example, horse radish peroxidase or alkaline phosphatase or firefly /renilla luciferase or nanoluc), or other proteins. A marker may be a protein (including secreted, cell surface, or internal proteins; either synthesized or taken up by the cell); a nucleic acid (such as an mRNA, or enzymatically active nucleic acid molecule) or a polysaccharide. Included are determinants of any such cell components that are detectable by antibody, lectin, probe or nucleic acid amplification reaction that are specific for the marker of the cell type of interest. The markers can also be identified by a biochemical or enzyme assay or biological response that depends on the function of the gene product.

In some embodiments, any of the cells disclosed herein comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said at least one gene sequence is the ABO sequence, such that the disruption results in the cell being blood type O.

In one embodiment, human pluripotent stem cells are utilized that lack some or all classic HLA-Class I cell surface protein expression and NK activating ligand expression. In one embodiment, a cell derived from a human pluripotent stem cell, such as a pancreatic cell, is provided that lack some or all classic HLA-Class I cell surface protein expression and NK activating ligand expression. In one embodiment, a cell derived from a human pluripotent stem cell, such as a pancreatic cell, is provided that lack some or all classic HLA-Class I cell surface protein expression and/or NK activating ligand expression. In some embodiments, wherein the function of at least one major histocompatibility complex (MHC)-Class I gene and at least one Natural killer (NK) cell activating ligand is disrupted or inhibited in the pluripotent stem cells.

MHC-Class I molecules are one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC-Class II). Their function is to display peptide fragments of non- self proteins from within the cell to cytotoxic T cells; this will trigger an immediate response from the immune system against a particular non-self antigen displayed with the help of an MHC-Class I protein. In humans, the HLAs corresponding to MHC-Class I are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. The human HLA-E, HLA-F, and HLA-G are non-classical MHC class I molecules characterized by limited polymorphism and a lower cell surface expression than the classical paralogues (HLA-A, -B and -C). All MHC class I proteins must associate with p2-microglobulin (B2M) to produce a functional heterodimer MHC Class I protein complex prior to functional expression on the cell surface. MHC-Class I molecules can also serve as an inhibitory ligand for NK cells. In some embodiments, the NK cell activating ligand is ICAM1, CD58, CD155, PVR, CEACAM1, CADM1, MICA, MICB, or a combination thereof. In more embodiments, the NK cell activating ligand is: CD58 and ICAM1; or CD58, ICAM1, and CD155; or CD58 and CADM1; or CD58 and CD155; or CD58, ICAM1, CD 155, and CADM1; or ICAM1, CADM1, and CD 155. Hypoimmune pluripotent stem cells are disclosed, for example, in PCT Publication No. WO 2019/014351, incorporated herein by reference.

In some embodiments, said at least one gene sequence encodes an MHC-Class I gene. In some embodiments, said MHC-Class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, said at least one gene sequence encodes CIITA. In some embodiments, the cells comprise a genomic disruption in the genes encoding HLA-A and HLA-B, but do not comprise a genomic disruption in the gene encoding HLA-C. In some embodiments, the cells comprise a genomic disruption in the gene encoding CXCL10. In some embodiments, the cells comprise a genomic disruption in the gene encoding renalase. In some embodiments, said cells comprise a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, said natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD 155, carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1), MHC-Class I polypeptide-related sequence A (MICA), or MHC-Class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR, relative to stem cells that are not genetically modified. In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, CTLA, PDL2, HLA-C, HLA-E, HLA-G, Cl-inhibitor, IL- 35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2-M3, CD200, FASLG, MFGE8, and/or SERPINB9 relative to cells that are not genetically modified.

In some embodiments, the genomic disruption is induced by use of a gene editing system, e.g., CRISPR Cas technology. In some embodiments, the cells comprises a disruption (e.g., deletion, insertion, translocation, inversion, or substitution of one or more nucleotides) in any one or more of the genes encoding: B2M, CIITA, CXCL10, renalase, HLA-A, HLA-B, HLA-C, RFX-ANK, NFY-A, NLRC5, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAPI, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, 0X40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, IDO1, TRAC, TRB, NFY-A, CCR5, F3, CD142, MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D, PDGFRa, OLIG2, and/or GFAP. In some embodiments, a cell comprises disrupted expression of B2M, CXCL10, renalase, tissue factor, and/or ABO, and/or comprises increased expression or activity of CD47. In some embodiments, disruption of a gene results in an at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in expression of the gene as compared to the expression of the gene in the same type of cell without the disruption. In some embodiments, the gene is disrupted using CRISPR/Cas, piggybac transposon, TALEN, and/or zinc finger technology. In some embodiments, the stem cell is any of the genetically engineered cells disclosed in WO2024097697, which is herein incorporated by reference in its entirety.

In some embodiments, the stem cell is a multipotent cell and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https://doi.org/10.1101/2023.10.20.563345.

1. Embryonic Stem Cells

By “embryonic stem cell” (ES) is meant a PSC that was isolated from an embryo, typically from the inner cell mass of the blastocyst. Exemplary ES lines are listed in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-Ol, hESBGN-02, hESBGN-03, hESBGN- 04 (BresaGen, Inc ); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz- hESl (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and Hl, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). Stem cells of interest also include embryonic stem cells from other primates, such as Rhesus stem cells and marmoset stem cells. The stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al. (1998) Science 282: 1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod 55:254; Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). In culture, ESCs typically grow as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nucleoli. In addition, ESCs express SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1 . Examples of methods of generating and characterizing ESCs may be found in, for example, U.S. Pat. No. 7,029,913, U.S. Pat. No. 5,843,780, and U.S. Pat. No. 6,200,806, each of which is incorporated herein by its entirety. Methods for proliferating hESCs in the undifferentiated form are described in WO 99/20741, WO 01/51616, and WO 03/020920, each of which is incorporated herein by its entirety.

Human embryonic stem (hES) cells, described by Thomson et al, (1998) Science 282: 1145; embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998) can be used in the disclosed methods and systems. The stem cells may be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mice, rats, hamster, primate, etc. In some embodiments, the cells are human. In some embodiments, a human embryo was not destroyed for the source of pluripotent cell used on the methods and compositions as disclosed herein.

ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. The replated cells can continue to proliferate and produce new colonies of ES cells which can be removed, dissociated, replated again and allowed to grow. This process of “subculturing” undifferentiated ES cells can be repeated a number of times to produce cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). ES cells have the potential to proliferate while maintaining their pluripotency. For example, ES cells are useful in research on cells and on genes which control cell differentiation. The pluripotency of ES cells combined with genetic manipulation and selection can be used for gene analysis studies in vivo via the generation of transgenic, chimeric, and knockout mice.

Human ES cells can be produced or derived from a zygote or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, pathogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell by previously described methods. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass which is cultured on a feeder layer of mouse embryonic fibroblasts. Further, clumps of cells derived from the inner cell mass are chemically or mechanically dissociated, replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor. In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGEL® or laminin in the presence of conditioned medium containing basic fibroblast growth factor. Human ES cell lines are available. In some embodiments, a human ES cell did not require destruction of a human embryo. These include the use of established ES cell lines.

ES cells can also be derived from other organisms including rhesus monkey and marmoset by previously described methods, as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30. As a further example, mouse ES cell lines that have been established include the CGR8 cell line established from the inner cell mass of the mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without feeder layers.

ES stem cells can be detected by protein markers including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REXI.

Pluripotent stem cells also can be prepared through the method of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from skin fibroblasts of a primate are introduced into the cytoplasm of spindle-free, mature metaphase II primate ooctyes by electrofusion. The fused oocytes are activated by exposure to ionomycin, and then incubated until the blastocyst stage. The inner cell mass of selected blastocysts are then cultured to produce embryonic stem cell lines. The embryonic stem cell lines show normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo. Embryos are not destroyed in the production of these ES cells.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EG cell,” it is meant a PSC that is derived from germ cells and/or germ cell progenitors, e.g., primordial germ cells, i.e. those that can become sperm and eggs. Embryonic germ cells (EG cells) are thought to have properties similar to embryonic stem cells as described above. Examples of methods of generating and characterizing EG cells may be found in, for example, U.S. Pat. No. 7, 153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 13726; and Koshimizu, U., et al. (1996) Development, 122: 1235, each of which are incorporated herein by its entirety.

2. Induced Pluripotent Stem Cells

By “induced pluripotent stem cell” or “iPSC,” it is meant a PSC that is derived from a cell that is not a PSC (i.e., from a cell this is differentiated relative to a PSC). iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an ES cell-like morphology, growing as flat colonies with large nucleo -cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26al, TERT, and zfp42. Examples of methods of generating and characterizing iPSCs can be found in, for example, Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, each of which are incorporated herein by its entirety. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art to reprogram the somatic cells to become pluripotent stem cells.

The induction of pluripotency was achieved in 2006 using mouse cells by Yamanaka et al., and in 2007 using human cells by reprogramming of somatic cells via the introduction of transcription factors that are linked to pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and are capable of differentiating into almost any cell type. The use of iPSCs circumvents most of the ethical and practical problems associated with large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive treatments to prevent graft rejection.

With the exception of germ cells, any cell can be used as a starting point for iPSCs. For example, cell types could be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or stomach cells. The cells can be a multipotent cells, such as but not limited to a hematopoietic stem cell, such as, but no limited to, CD34+ cells. In some embodiments, the stem cell is a multipotent cell and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https://doi.org/10.1101/2023.10.20.563345. T cells may also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). There is no limitation on the degree of cell differentiation or the age of an animal from which cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself an endocrine cell such as a human endocrine cell. However, other cell types are also of use, such as, but not limited to, fibroblasts and muscle cells. The cell can be an adult or a fetal cell. iPSCs can be grown under conditions that are known to differentiate human ES cells into specific cell types, and express human ES cell markers including: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

Somatic cells and pluripotent stem cells can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to one of skill in the art. One of skill in the art can readily produce induced pluripotent stem cells, see for example, Published U.S. Patent Application No. 20090246875, Published U.S. Patent Application No. 2010/0210014; Published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,278,620; PCT Publication No. WO 2007/069666 Al, and U.S. Patent No. 8,268,620, which are incorporated herein by reference in its entirety. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least three, or at least four, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3/4, Sox2, c-Myc and Klf4 are utilized.

The cells are treated with a nuclear reprogramming substance, which is generally one or more factor(s) capable of inducing an iPSC from a somatic cell or a nucleic acid that encodes these substances (including forms integrated in a vector). The nuclear reprogramming substances generally include at least Oct3/4, Klf4 and Sox2 or nucleic acids that encode these molecules. A functional inhibitor of p53, L-myc or a nucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acid that encodes Lin28 or Lin28b, can be utilized as additional nuclear reprogramming substances. Nanog can also be utilized for nuclear reprogramming. As disclosed in published U.S. Patent Application No. 20120196360, exemplary reprogramming factors for the production of iPSCs include (1) Oct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klfl, Klf2 or Klf5); (2) Oct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen (SV40LT); (3) Oct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus (HPV)16 E6; (4) Oct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3/4, Klf4, Sox2, L- Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3/4, Klf4, Sox2, L-Myc, TERT, Bmil; (7) Oct3/4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3/4, Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3/4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT; (10) Oct3/4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3/4, Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3/4, Klf4, Sox2; (13) Oct3/4, Klf4, Sox2, TERT, SV40LT; (14) Oct3/4, Klf4, Sox2, TERT, HP VI 6 E6; (15) Oct3/4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3/4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3/4, Klf4, Sox2, TERT, Bmil; (18) Oct3/4, Klf4, Sox2, Lin28 (19) Oct3/4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3/4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3/4, Klf4, Sox2, SV40LT; or (22) Oct3/4, Esrrb, Sox2 (Esrrb is replaceable with Esrrg). In one nonlimiting example, Oct3/4, Klf4, Sox2, and c-Myc are utilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized, see for example, U.S. Patent No. 7,682,828, which is incorporated herein by reference in its entirety. These factors include, but are not limited to, Oct3/4, Klf4 and Sox2. In other examples, the factors include, but are not limited to Oct 3/4, Klf4 and Myc. In some non-limiting examples, Oct3/4, Klf4, c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3/4, Klf4, Sox2 and Sal 4 are utilized. Factors like Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, an integrating vector such as the EBV element-based system can be used (U.S. Patent No. 8,546,140). In a further embodiment, reprogramming proteins could be introduced directly into somatic cells by protein transduction. Reprogramming may further comprise contacting the cells with one or more signaling receptors including glycogen synthase kinase 3 (GSK-3) inhibitor, a mitogen-activated protein kinase (MEK) inhibitor, a TGF-P receptor inhibitor or signaling inhibitor, leukemia inhibitory factor (LIF), a p53 inhibitor, an NF- kappa B inhibitor, or a combination thereof. Those regulators may include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is anticipated that virtually any iPS cells or cell lines may be used.

In some embodiments, the induced pluripotent stem cells are generated from mesenchymal stromal cells. In some embodiments, the induced pluripotent stem cells are generated from adipose-derived mesenchymal stromal cells (ADSCs) isolated. In some embodiments, the induced pluripotent stem cells are chemically-induced pluripotent stem cells (see, e.g., Guan et al., 2022, Nature, 605:325-331; Wang et al., 2024, Cell, 187, 1-13). In some embodiments, the induced pluripotent stem cells are derived from cells taken from a subject (e.g., a diabetic subject), and the induced pluripotent stem cells are then differentiated using any of the methods disclosed herein in order to make SC-islet cells or precursors thereof that may be administered back to the patient, i.e., the induced pluripotent stem cells are autologous cells to the subject (see, e.g., Wang et al., 2024, Cell, 187, 1-13).

Mouse and human cDNA sequences of these nuclear reprogramming substances are available with reference to the NCBI accession numbers recited in PCT Publication No. WO 2007/069666, which is incorporated herein by reference in its entirety. Methods for introducing one or more reprogramming substances, or nucleic acids encoding these reprogramming substances, are known and disclosed for example, in U.S. Patent Publication No. 2012/0196360 and U.S. Patent No. 8,071,369, which both are incorporated herein by reference in its entirety.

Once derived, iPSCs can be cultured in a medium sufficient to maintain pluripotency. The iPSCs may be used with various media and techniques developed to culture pluripotent stem cells, more specifically, embryonic stem cells, as described in U.S. Patent No. 7,442,548 and U.S. Patent Pub. No. 2003/0211603. In the case of mouse cells, the culture is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppression factor to an ordinary medium. In the case of human cells, it is desirable that basic fibroblast growth factor (bFGF) be added in place of LIF. Other methods for the culture and maintenance of iPSCs, may be used.

In certain embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. In some embodiments, the cell is cultured in the co-presence of embryonic fibroblasts treated with radiation or an antibiotic to terminate the cell division, as feeder cells. Alternately, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using a defined, feeder-independent culture system, such as a TESR™ medium or E8™ medium. In some embodiments, the media is the E8 media described in Chen et al., 2011, Nat. Methods, 8(5):424-29. In some embodiments, the media comprises DMEM/F12. In some embodiments, the media comprises ascorbic acid. In some embodiments, the ascorbic acid is in the form of L-ascorbic acid-2-phosphate magnesium. In some embodiments, the media comprises 1-500 mg/L, 1-250 mg/L, 1-100 mg/L, 1-50 mg/L, or 50-100 mg/L of ascorbic acid. In some embodiments, the media comprises sodium selenium. In some embodiments, the media comprises 0.5-100 pg/L, 0.5-50 pg/L, 0.5-25 pg/L, 1-25 pg/L, 5-20 pg/L or 18-22 pg/L of sodium selenium. In some embodiments, the media comprises a growth factor from the LGL family (e.g., keratinocyte growth factor (KGL), LGL2 (bLGL), LGL8B, LGL10 and LGL21). In some embodiments, the media comprises 1-1000 pg/L, 1-500 pg/L, 1-250 pg/L, 50-1000 pg/L, 50-500 pg/L, 50-250 pg/L, or 75-125 pg/L of growth factor from the LGL family (e.g., keratinocyte growth factor (KGL), LGL2 (bLGL), LGL8B, LGL10 and LGL21). In some embodiments, the media comprises insulin. In some embodiments, the media comprises 0.5-100 mg/L, 0.5-50 mg/L, 0.5-25 mg/L, 10-100 mg/L, 10-50 mg/L, 10-25 mg/L or 15-25 mg/L of insulin. In some embodiments, the media comprises NaCOa. In some embodiments, the media comprises 100-3000 mg/L, 100-1000 mg/L, 250-1000 mg/L, 250-750 mg/L, or 500-600 mg/L NaCOa. In some embodiments, the media comprises transferrin. In some embodiments, the media comprises 1-100 mg/L, 1-50 mg/L, 1-25 mg/L, 5-50 mg/L, 5-25 mg/L, or 8-12 mg/L transferrin. In some embodiments, the media comprises a growth factor of the TGL-P superfamily e.g., TGL-pi or NODAL). In some embodiments, the media comprises 0.1- 10 pg/L, 0.1-5 pg/L, 0.1-2.5 pg/L, 1-10 pg/L, 1-5 pg/L, 1-3 pg/L of the growth factor of TGL-pi. In more particular embodiments, the media comprises 1.5-2.5 pg/1 of TGL-P 1. In some embodiments, the media comprises 1-1000 pg/L, 1-500 pg/L, 1-250 pg/L, 50-1000 pg/L, 50-500 pg/L, 50-250 pg/L, or 80-120 pg/L of NODAL. In some embodiments, the culture is feeder- free. In other embodiments, the culture is matrix free. a. MHC Haplotype Matching

Major Histocompatibility Complex is the main cause of immune-rejection of allogeneic organ transplants. There are three major class I MHC haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA loci are highly polymorphic and are distributed over 4 Mb on chromosome 6. The ability to haplotype the HLA genes within the region is clinically important since this region is associated with autoimmune and infectious diseases and the compatibility of HLA haplotypes between donor and recipient can influence the clinical outcomes of transplantation. HLAs corresponding to MHC class I present peptides from inside the cell and HLAs corresponding to MHC class II present antigens from outside of the cell to T-lymphocytes. Incompatibility of MHC haplotypes between the graft and the host triggers an immune response against the graft and leads to its rejection. Thus, a subject can be treated with an immunosuppressant to prevent rejection. HLA-matched pluripotent stem cell lines can be used to overcome the risk of immune rejection.

Because of the importance of HLA in transplantation, the HLA loci are usually typed by serology and PCR for identifying favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be accomplished using a complement mediated lymphocytotoxicity test with purified T or B lymphocytes. This procedure is predominantly used for matching HLA- A and -B loci. Molecular-based tissue typing can often be more accurate than serologic testing. Low resolution molecular methods such as SSOP (sequence specific oligonucleotide probes) methods, in which PCR products are tested against a series of oligonucleotide probes, can be used to identify HLA antigens, and currently these methods are the most common methods used for Class ILHLA typing. High resolution techniques such as SSP (sequence specific primer) methods which utilize allele specific primers for PCR amplification can identify specific MHC alleles.

MHC compatibility between a donor and a recipient increases significantly if the donor cells are HLA homozygous, i.e. contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can serve as super donors and grafts generated from their cells can be transplanted in all individuals that are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype found in high frequency in a population, these cells may have application in transplantation therapies for a large number of individuals.

Accordingly, iPSCs can be produced from cells of the subject to be treated, or another subject with the same or substantially the same HLA type as that of the patient. In one case, the major HLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR) of the donor are identical to the major HLAs of the recipient. In some cases, the somatic cell donor may be a super donor; thus, iPSCs derived from a MHC homozygous super donor may be used to generate, e.g., SC-islet cells. Thus, the iPSCs derived from a super donor may be transplanted in subjects that are either homozygous or heterozygous for that haplotype. For example, the iPSCs can be homozygous at two HLA alleles such as HLA-A and HLA-B. As such, iPSCs produced from super donors can be used in the methods disclosed herein, to produce cells that can potentially “match” a large number of potential recipients. b. Episomal Vectors

In certain embodiments, reprogramming factors are expressed from expression cassettes comprised in one or more exogenous episomal genetic elements (see U.S. Patent Publication 2010/0003757, incorporated herein by reference in its entirety). Thus, iPSCs can be essentially free of exogenous genetic elements, such as from retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extra-chromosomally replicating vectors (i.e., episomal vectors), which are vectors capable of replicating episomally to make iPSCs essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, incorporated herein by reference in its entirety; Yu et al., 2009). A number of DNA viruses, such as adenoviruses, simian virus 40 (SV40) or bovine papilloma virus (BPV), or budding yeast ARS (Autonomously Replicating Sequences)-containing plasmids replicate extra-chromosomally or episomally in mammalian cells. These episomal plasmids are intrinsically free from all these disadvantages (Bode et al., 2001) associated with integrating vectors. For example, a lymphotrophic herpes virus-based including or Epstein Barr Virus (EBV) as defined above may replicate extra- chromosomally and help deliver reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. One advantage of episomal vectors is that the exogenous elements will be lost with time after being introduced into cells, leading to self-sustained iPSCs essentially free of these elements.

Other extra-chromosomal vectors include other lymphotrophic herpes virus-based vectors. Lymphotrophic herpes virus is a herpes virus that replicates in a lymphoblast (e.g., a human B lymphoblast) and becomes a plasmid for a part of its natural life-cycle. Herpes simplex virus (HSV) is not a "lymphotrophic" herpes virus. Exemplary lymphotrophic herpes viruses include, but are not limited to EBV, Kaposi's sarcoma herpes virus (KSHV); herpes virus saimiri (HS) and Marek's disease virus (MDV). Additional sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, SV40, or BPV.

3. Single cell suspensions, cell aggregates and media

Stem cell clusters, and differentiating cell clusters, can be propagated using the methods disclosed herein. Although such methods can be used to produce cell clusters, the disclosed cell clusters and culture conditions disclosed herein can be used to produce pluripotent stem cell clusters, propagate clusters of pluripotent stem cells, and to achieve synchronous directed- differentiation.

In some embodiments, the plurality of cell clusters are generated from a plurality of disassociated cells. These single cell suspensions can be seeded into a bioreactor to form clusters and be propagated as clusters. In some embodiments, the disclosed methods include the steps of seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters.

In some embodiments, a bioreactor is seeded with dissociated cells, such as a single cell suspension of pluripotent stem cells to form pluripotent stem cell clusters. As used herein, “single cell suspension” or equivalents thereof refers to a single cell (e.g., hES single cell) suspension or a single cell suspension by any mechanical or chemical means. Several methods exist for dissociating cell clusters to form single cell suspensions from primary tissues, attached cells in culture, and aggregates, e.g., physical forces (mechanical dissociation such as cell scraper, trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Acutase and the like), or a combination of both. Further, methods and culture media conditions capable of supporting single-cell dissociation of cells (e.g., hES cells) are useful for expansion, cell sorting, and defined seeding for multi-well plate assays and enable automatization of culture procedures and clonal expansion. Methods for generating a stable single-cell enzymatic dissociation stem cell stem cell-derived culture systems are disclosed, for example, in U.S. Published Application No. 2021/0324329-Al, incorporated herein by reference. In some embodiments, the method comprises the steps of seeding the bioreactor with about 0.01 x 10⁶ to about 10 x 10⁶ viable cells/ml, about 0.01 x 10⁶-5 x 10⁶ viable cells/ml, about 0.01 x 10⁶- 1 x 10⁶ viable cells/ml, about 0.01 x 10⁶ to about 0.5 x 10⁶ viable cells/ml, about 0.01 x 10⁶ to about 0.05 x 10⁶ viable cells/ml, about 0.1 x 10⁶ to about 1 x 10⁶ viable cells/ml, or about 0.3 x 10⁶ to about 0.8 x 10⁶ viable cells/ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the method comprises the steps of seeding the bioreactor with about 0.05-0.1 viable cells/ml, 0.1-2 viable cells/ml, 0.1-1 viable cells/ml, 0.2-0.9 viable cells/ml, 0.3-0.7 viable cells/ml, 0.4-0.6 viable cells/ml, or about 0.5 viable cells/ml and culturing the viable cells to generate the plurality of cell clusters. In some embodiments, the viable cells are disassociated cells, such as disassociated pluripotent stem cells. In some embodiments, about 50 to about 100%, about 50 to about 90%, about 50 to about 75%, about 70 to about 100%, about 70 to about 85%, about 80 to about 100%, about 80 to about 90%, about 95% to about 98%, about 93% to about 97%, about 90 to about 100%, or about 90 to about 95% of the viable cells are dissociated cells.

In some embodiments, in contrast to cell clusters produced by static culture and enzymatic degradation of adherent cultures that may vary in both size and shape, the cell clusters and methods described herein may utilize clusters of a specified size and shape distribution, such that the cell aggregates are substantially uniform in size and/or shape. The size uniformity of the cell aggregates may affect differentiation performance and the culture homogeneity. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells, such as pluripotent stem cells. The stem cells can be embryonic stem cells. The stem cells can be induced pluripotent stem cells. In some embodiments, 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, of 90-95% of the clusters have a diameter between about 75 pm to about 600 pm, about 75 pm to about 500 pm, about 75 pm to about 400 pm, or about 75 pm to about 300 pm.

In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are OCT4-negative and SOX17-positive. In other embodiments, about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are FOXA2-positive, PDX1 -negative. In more embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1- positive, NKX6.1 -negative. In further embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1 -positive, NKX6.1-positive. In other embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are IS LI -positive. In more embodiments, at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are chromogranin-positive.

In some embodiments, seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell cluster includes culturing for about 12 to about 72 hours, about 12 to about 60 hours, about 12 to about 50 hours, about 12 to about 36 hours, about 12 to about 36 hours, about 18 to about 60 hours, about 18 to about 50 hours, about 18 to about 36 hours, about 18 to about 26 hours, about 26 to about 60 hours, about 26 to about 50 hours, about 26 to about 36 hours, about 36 to about 60 hours, about 36 to about 50 hours, or about 44 to about 52 hours in length before transporting a portion of the cell culture from the bioreactor into the tangential flow filtration system.

In some embodiments, these steps of the method are repeatedly performed over a period of about 1 to about 20 days, about 1 to about 15 days, about 1 to about 10 days, about 1 to about 7 days, about 1 to about 5 days, about 1 to about 3 days, about 2 to about 12 days, about 8 to about 12 days, about 3 to about 8 days, about 4 to about 7 days, or about 4 to about 6 days. In some embodiments, at the end of the period, the cell clusters are dissociated. In further embodiments, cell clusters are dissociated by treating the cell clusters with a one or more proteolytic and collageno lytic enzymes. In further embodiments, the one or more proteolytic and collagenolytic enzymes include any one or more of trypsin, collagenase, Trypsin-like protease XIV, or thermolysin. In embodiments, the cell clusters are dissociated by treating the cell clusters with Accutase. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed. In some embodiments, the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed, and fresh media lacking the proteolytic or collagenolytic enzymes is added to the cell cluster composition.

The cells and cell clusters described herein can be suspended in any physiologically acceptable medium in a bioreactor. The tissue culture media may comprise, for example, basic nutrients such as sugars and amino acids, growth factors, antibiotics (to minimize contamination) and the like. In another embodiment, the differentiable cells are cultured in suspension, using the cell media described herein. The term “suspension” as used in the context of cell culturing where the cells or cell aggregates do not adhere to a surface of the bioreactor of a component thereof. As used herein, cells are “in motion” if they are moving, or if their immediate environment is moving relative to the cells.

Generally, the cells and/or cell clusters are cultured in growth media including a carbon source, a nitrogen source and a buffer to maintain pH. The medium can also contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, pyruvic acid, buffering agents, and inorganic salts. In some embodiments, a growth medium contains a minimal essential media, such as Dulbecco’s Modified Eagle’s medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimal Essential Medium Eagle (MEM) Alpha medium, Dulbecco’s modified Eagle medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. Additionally, the minimal essential media may be supplemented with additives such as horse, calf or fetal bovine serum. Alternatively, the medium can be serum free. The alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. WO 98/30679, for example. In other cases, the growth media may contain “knockout serum replacement,” referred to herein as a serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cell, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application No. 2002/0076747, which is incorporated herein by reference in its entirety. In some embodiments, chemically-defined Lipid concentrated (Gibco), and GLUTAMAX™ (Gibco) can be used. In some embodiments, the pluripotent stem cells are cultured in a fully defined and feeder free media. Other culture medias include STEMSCALE™, NUTRISTEM®, TESR™, STEMSPAN™, STEMDIFF™, and STEMPRO™-34.

Other culturing conditions can be appropriately defined. For example, the culturing temperature can be about 30 to 40°C, for example, at least or about 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C but particularly not limited to them. In one embodiment, the cells are cultured at 36-38°C, 36.5-37.5°C, or about 37°C. The CO2 concentration can be about 1 to 10%, for example, about 2 to 5%, or any range derivable therein. The oxygen tension can be at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

In some embodiments, the medium in the bioreactor includes a ROCK inhibitor. Specific non-limiting examples of a ROCK inhibitor are thiazovivin, Fasudil, Y-27632, and HA1077. In more embodiments, the medium in the includes Basic fibroblast growth factor (bFGF). In more embodiments, the medium in the bioreactor includes a cell differentiation or survival factor. These include one or more of: a Rho-associated protein kinase (ROCK) inhibitor (e.g., Y-27632 or thiazovivin), a TGF-beta receptor agonist/ligand (e.g., activin A, GDF8, or GDF11), Wnt activator (e.g., CHIR99021), a fibroblast growth factor (e.g., KGF or FGF10), a retinoic acid receptor activator (e.g., retinoic acid), a sonic hedgehog inhibitor (e.g., Santl), a BMP inhibitor (e.g., DMH1, LDN193189, or dorsomorphin), a protein kinase C activator (e.g., PDBU or TPPB), a FOXO1 inhibitor (e.g., AS 1842856), a gamma- secretase inhibitor (e.g., XX, XXI or DAPT), a thyroid receptor activator (e.g., T3 or GC-1), a TGF-P signaling pathway inhibitor (e.g., Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, and SD-208), an epidermal growth factor (EGF) family member (e.g., EGF or betacellulin), a protein kinase inhibitor (e.g., staurosporine), an epigenetic modifying compound (e.g., DZNEP), or a Wnt inhibitor (e.g., NVPTNKS656 or XAV-939 or IWR-l-Endo or WIKI4).

In some embodiments, cells (e.g., stem cells) can be propagated using the methods disclosed herein. In some embodiments, stem cells can be differentiated using the methods disclosed herein. In certain examples, the stem cells can be undifferentiated (e.g., a cell not committed to a specific lineage) prior to exposure to at least one cell maturation factor according to the methods as disclosed herein, whereas in other examples it may be desirable to differentiate the stem cells to one or more intermediate cell types prior to exposure of the at least one cell maturation factor (s) described herein. For example, the stem cells may display morphological, biological or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryo or adult origin. In some examples, undifferentiated cells may appear in the two dimensions of a microscopic view in colonies of cells with high nuclear/cytoplasmic ratios and prominent nucleoli. The stem cells may be themselves (for example, without substantially any undifferentiated cells being present) or may be used in the presence of differentiated cells. In certain examples, the stem cells may be cultured in the presence of suitable nutrients and optionally other cells such that the stem cells can grow and optionally differentiate.

TFF Systems, compositions and methods for producing pancreatic cells

In embodiments, the present disclosure provides compositions and methods of differentiating pancreatic cells from pluripotent stem cells (e.g., differentiating from stem cells such as human embryonic stem cells, human pluripotent stem cells, or clusters thereof). The compositions, TFF systems, and methods provided herein can, in some embodiments, offer pancreatic SC-islet cells, cell populations, or cell clusters containing pancreatic SC-P cells and pancreatic SC-a cells. In some embodiments, such pancreatic SC-islet cells, cell populations or cell clusters exhibit, high insulin content, superior glucose-dependent insulin secretion response, as well as a percentage of pancreatic SC-a, SC-P, and SC-5 cells and enterochromaffin (EC) cells, which can resemble native pancreatic islets both structurally and functionally. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein comprises about 30%-45% pancreatic SC-P cells, 40%-50% pancreatic a cells, 3-10% pancreatic SC-5 cells, and/or less than 20% SC-EC cells. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has improved glucose- stimulated insulin secretion (GSIS) response as compared to cell compositions generated according to conventional methods. In some embodiments, a population of pancreatic islet cells (e.g., stem cell derived pancreatic islet cells) produced using the compositions and methods described herein has dynamic GSIS response similar to native pancreatic islets (e.g., pancreatic islets from a healthy functioning pancreas from a healthy adult non-diabetic subject).

In some embodiments, a method of producing pancreatic islet cells (e.g., SC-beta cells, SC-alpha cells, SC-delta cells) described herein comprises contacting pancreatic progenitor cells (e.g., pancreatic progenitor cells that are PDXl-positive and NKX6.1 -negative) with a medium comprising a comprising a Forkhead Box 01 (FoxOl) inhibitor and/or a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor) in the bioreactor and/or TFF system. In some embodiments, a method described herein comprises contacting pancreatic progenitor cells in a culture with a FOXO1 inhibitor and/or a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor) in the bioreactor and/or TFF system, wherein the culture comprises pancreatic progenitor cells that are PDXl-positive and NKX6.1 -negative and pancreatic progenitor cells that are PDXl-positive and NKX6.1 -positive. In some embodiments, a method described herein comprises contacting pancreatic progenitor cells (e.g., pancreatic progenitor cells that are PDXl- positive and NKX6.1 -positive) with a medium comprising a comprising a Wnt signaling pathway inhibitor and/or a PKC activator in the bioreactor and/or TFF system. In some embodiments, a method described herein comprises contacting pancreatic progenitor cells (e.g., pancreatic progenitor cells that are PDXl-positive and NKX6.1 -negative) with a medium comprising a comprising a Forkhead Box 01 (FoxOl) inhibitor and/or a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor), and contacting resulting cells with a medium comprising a comprising a Wnt signaling pathway inhibitor and/or a PKC activator in the bioreactor and/or TFF system. In some embodiments, the method does not comprise the step of contacting cells with a Wnt signaling pathway inhibitor and a FoxOl inhibitor at the same time in the bioreactor and/or TFF system.

TFF Systems and compositions comprising FoxOl inhibitor and optionally a Notch signaling inhibitor

In some embodiments, the present disclosure provides in vitro compositions, bioreactors, and TFF systems comprising a population of pancreatic progenitor cells and a medium comprising a Forkhead Box 01 (FoxOl) inhibitor. In some embodiments, the medium further comprises a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor). In some embodiments, the medium further comprises a PKC activator. In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, 4, 5) agents selected from: a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a TGF-P ligand. In some embodiments, the medium further comprises a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a TGF-P ligand.

In some embodiments, the medium comprises a FoxOl inhibitor, a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor), a PKC activator, a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, and a TGF-P ligand. In some embodiments, the medium comprises FoxOl inhibitor AS1842856 (e.g., Catalog #344355 as Sigma-Aldrich), XXI, PdBu, keratinocyte growth factor (KGF), SANT-1, RA, triazovivin, and activin A. In some embodiments, the medium does not comprise a Wnt signaling pathway inhibitor. In some embodiments, a FoxOl inhibitor (e.g., AS1842856) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a FoxOl inhibitor (AS 1842856) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM- 2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a FoxOl inhibitor (e.g., AS 1842856) is present in the medium at a concentration of 0.5 pM-5 pM (e.g., 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a FoxOl inhibitor (e.g., AS1842856) is present in the medium at a concentration of 0.7-1.3 pM, 0.8-1.2 pM, or 0.9-1.1 pM. In some embodiments, a FoxOl inhibitor (e.g., AS1842856) is present in the medium at a concentration of 1 pM.

In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM- 2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a notch signaling pathway inhibitor (e.g., y- secretase inhibitor such as XXI) is present in the medium at a concentration of 0.5 pM-5 pM (e.g., 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 1.5 pM-2.5 pM or 1.8 pM-2.2 pM. In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 2 pM.

In some embodiments, a PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.2 pM-1 pM (e.g., 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM , 0.7 pM, 0.8 pM, 0.9 pM, or 1 pM). In some embodiments, a PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.3 pM-0.7 pM or 0.4 pM-0.6 pM. In some embodiments, a PKC activator (e.g., PdBu) is present in the medium at a concentration of 0.5 pM.

In some embodiments, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT- 1) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM- 10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM- 7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.1 pM-0.5 pM (e.g., 0.1 pM, 0.15 pM, 0.2 pM, 0.25 pM, 0.3 pM, 0.35 pM, 0.4 pM, 0.45 pM, or 0.5 pM). In some embodiments, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) is present in the medium at a concentration of 0.25 pM.

In some embodiments, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM-10 pM. In some embodiments, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM- 5 pM (e.g., 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 2.5 pM.

In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 pM-0.5 pM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 pM-0.5 pM, 0.1 pM-0.5 pM, 0.15 pM-0.5 pM, 0.2 pM-0.5 pM, 0.25 pM-0.5 pM, 0.3 pM- 0.5 pM, 0.35 pM-0.5 pM, 0.4 pM-0.5 pM, 0.45 pM-0.5 pM, 0.05 pM-0.4 pM, 0.1 pM-0.4 pM, 0.15 pM-0.4 pM, 0.2 pM-0.4 pM, 0.25 pM-0.4 pM, 0.3 pM-0.4 pM, 0.35 pM-0.4 pM, 0.05 pM-0.3 pM, 0.1 pM-0.3 pM, 0.15 pM-0.3 pM, 0.2 pM-0.3 pM, 0.25 pM-0.3 pM, 0.05 pM-0.2 pM, 0.1 pM-0.2 pM, 0.15 pM-0.2 pM, or 0.05 pM-0.1 pM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 pM-0.2 pM (e.g., 0.05 pM, 0.1 pM, 0.15 pM, or 0.2 pM). In some embodiments, retinoic acid is present in the medium at a concentration of 0.1 pM.

In some embodiments, a TGF-P ligand (e.g., activin A) is present in the medium at a concentration of 1 ng/ml-10 ng/ml. In some embodiments, a TGF-P ligand (e.g., activin A) is present in the medium at a concentration of 1 ng/ml-10 ng/ml, 1 ng/ml-9 ng/ml, 1 ng/ml-8 ng/ml, 1 ng/ml-7 ng/ml, 1 ng/ml-6 ng/ml, 1 ng/ml-5 ng/ml, 1 ng/ml-4 ng/ml, 1 ng/ml-3 ng/ml, 1 ng/ml-2 ng/ml, 2 ng/ml-10 ng/ml, 2 ng/ml-9 ng/ml, 2 ng/ml-8 ng/ml, 2 ng/ml-7 ng/ml, 2 ng/ml-6 ng/ml, 2 ng/ml-5 ng/ml, 2 ng/ml-4 ng/ml, 2 ng/ml-3 ng/ml, 3 ng/ml-10 ng/ml, 3 ng/ml-9 ng/ml, 3 ng/ml-8 ng/ml, 3 ng/ml-7 ng/ml, 3 ng/ml-6 ng/ml, 3 ng/ml-5 ng/ml, 3 ng/ml-4 ng/ml, 4 ng/ml-10 ng/ml, 4 ng/ml-9 ng/ml, 4 ng/ml-8 ng/ml, 4 ng/ml-7 ng/ml, 4 ng/ml-6 ng/ml, 4 ng/ml-5 ng/ml, 5 ng/ml-10 ng/ml, 5 ng/ml-9 ng/ml, 5 ng/ml-8 ng/ml, 5 ng/ml-7 ng/ml, 5 ng/ml-6 ng/ml, 6 ng/ml-10 ng/ml, 6 ng/ml-9 ng/ml, 6 ng/ml-8 ng/ml, 6 ng/ml-7 ng/ml, 7 ng/ml-10 ng/ml, 7 ng/ml-9 ng/ml, 7 ng/ml-8 ng/ml, 8 ng/ml-10 ng/ml, 8 ng/ml-9 ng/ml, or 9 ng/ml-10 ng/ml. In some embodiments, a TGF-P ligand (e.g., activin A) is present in the medium at a concentration of 2 ng/ml-8 ng/ml (e.g., 2 ng/ml, 3 ng/ml, 4 ng/ml, 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml). In some embodiments, a TGF-P ligand (e.g., activin A) is present in the medium at a concentration of 5 ng/ml.

In some embodiments, a fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 10 ng/ml- 100 ng/ml. In some embodiments, a fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 10 ng/ml- 100 ng/ml, 10 ng/ml-90 ng/ml, 10 ng/ml-80 ng/ml, 10 ng/ml-70 ng/ml, 10 ng/ml-60 ng/ml, 10 ng/ml-50 ng/ml, 10 ng/ml-40 ng/ml, 10 ng/ml-30 ng/ml, 10 ng/ml- 20 ng/ml, 20 ng/ml- 100 ng/ml, 20 ng/ml-90 ng/ml, 20 ng/ml-80 ng/ml, 20 ng/ml-70 ng/ml, 20 ng/ml-60 ng/ml, 20 ng/ml-50 ng/ml, 20 ng/ml-40 ng/ml, 20 ng/ml-30 ng/ml, 30 ng/ml- 100 ng/ml, 30 ng/ml-90 ng/ml, 30 ng/ml-80 ng/ml, 30 ng/ml-70 ng/ml, 30 ng/ml-60 ng/ml, 30 ng/ml- 50 ng/ml, 30 ng/ml-40 ng/ml, 40 ng/ml- 100 ng/ml, 40 ng/ml-90 ng/ml, 40 ng/ml-80 ng/ml, 40 ng/ml-70 ng/ml, 40 ng/ml-60 ng/ml, 40 ng/ml-50 ng/ml, 50 ng/ml- 100 ng/ml, 50 ng/ml-90 ng/ml, 50 ng/ml-80 ng/ml, 50 ng/ml-70 ng/ml, 50 ng/ml-60 ng/ml, 60 ng/ml- 100 ng/ml, 60 ng/ml-90 ng/ml, 60 ng/ml-80 ng/ml, 60 ng/ml-70 ng/ml, 70 ng/ml- 100 ng/ml, 70 ng/ml-90 ng/ml, 70 ng/ml-80 ng/ml, 80 ng/ml- 100 ng/ml, 80 ng/ml-90 ng/ml, or 90 ng/ml- 100 ng/ml. In some embodiments, a fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 20 ng/ml-80 ng/ml (e.g., 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml). In some embodiments, a fibroblast growth factor (e.g., keratinocyte growth factor (KGF)) is present in the medium at a concentration of 50 ng/ml.

In some embodiments, the medium comprises a FoxOl inhibitor (e.g., AS 1842856) at a concentration of 0.5 pM-5 pM, a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI) at a concentration of 0.5 pM-5 pM, a PKC activator (e.g., PdBu) at a concentration of 0.2 pM-1 pM, a fibroblast growth factor (e.g., KGF) at a concentration of 20 ng/ml-80 ng/ml, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.1 pM-0.5 pM, retinoic acid at a concentration of 0.05 pM-0.2 pM, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 1 pM- 5 pM, and a TGF-P ligand (e.g., activin A) at a concentration of 2 ng/ml-8 ng/ml.

In some embodiments, the medium comprises a FoxOl inhibitor (e.g., AS 1842856) at a concentration of 1 pM, a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI) at a concentration of 2 pM, a PKC activator (e.g., PdBu) at a concentration of 0.5 pM, a fibroblast growth factor (e.g., KGF) at a concentration of 50 ng/ml, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.25 pM, retinoic acid at a concentration of 0.1 pM, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 2.5 pM, and a TGF-P ligand (e.g., activin A) at a concentration of 5 ng/ml.

In some embodiments, an in vitro composition described herein, or the bioreactor or TFF system, further comprises a water-soluble synthetic polymer. In some embodiments, the water- soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide, optionally wherein the water-soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA). In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the culture medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w/v), 0.01% (w/v), 0.05% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), to 0.4% (w/v), 0.45% (w/v), or 0.5% (w/v) of the medium. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), and the PVA is at most 85% (e.g., 75%-80%) hydrolyzed.

In some embodiments, an in vitro composition, or bioreactor and/or the TFF system, comprises a population of pancreatic progenitor cells comprising cells that are PDX1 -positive and NKX6.1 -negative. In some embodiments, an in vitro composition, or bioreactor and/or the TFF system described herein comprises a population of pancreatic progenitor cells comprising cells that are PDXl-positive and NKX6.1 -positive. In some embodiments, an in vitro composition, or bioreactor and/or the TFF system, described herein comprises a population of pancreatic progenitor cells comprising cells that are PDXl-positive and NKX6.1 -negative and cells that are PDXl-positive and NKX6.1 -positive. In some embodiments, the PDXl-positive and NKX6.1-positive cells are insulin-negative. In some embodiments, the cells are in clusters.

In some embodiments, the population of pancreatic progenitor cells comprises more cells that are PDXl-positive and NKX6.1 -negative than cells that are PDXl-positive and NKX6.1- positive. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1 -negative. In some embodiments, no more than 50% (e.g., no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1 -positive. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%- 60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1-negative. In some embodiments, 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%- 40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1-positive. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%- 80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1 -negative and 10%-50% (e.g., 10%-50%, 20%- 50%, 30%-50%, 40%-50%, 10%-40%, 20%-40%, 30%-40%, 10%-30%, 20%-30%, or 10%- 20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1-positive. In some embodiments, at least 50% of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1 -negative, and no more than 50% of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl- positive and NKX6.1 -positive. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

In some embodiments, the population of pancreatic progenitor cells comprises more cells that are PDXl-positive and NKX6.1 -positive than cells that are PDXl-positive and NKX6.1- negative. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1-positive. In some embodiments, no more than 50% (e.g., no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1-negative. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1 -positive. In some embodiments, 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%- 40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1 -negative. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%- 80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1-positive and 10%-50% (e.g., 10%-50%, 20%- 50%, 30%-50%, 40%-50%, 10%-40%, 20%-40%, 30%-40%, 10%-30%, 20%-30%, or 10%- 20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive and NKX6.1 -negative. In some embodiments, at least 50% of the population of pancreatic progenitor cells are PDXl-positive and NKX6.1-positive, and no more than 50% of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDX1- positive and NKX6.1 -negative. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

TFF systems and composition comprising Wnt inhibitor and optionally a PKC activator The present disclosure, in other embodiments, provides in vitro compositions, or bioreactor and/or a TFF system, comprising a population of pancreatic progenitor cells comprising cells that are PDX1 -positive, NKX6.1-positive, and insulin-negative; and a medium comprising a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor). In some embodiments, the medium further comprises a protein kinase C (PKC) activator. In some embodiments, the medium does not comprise a F0X01 inhibitor. In some embodiments, the population of pancreatic progenitor cells had been previously cultured in a medium comprising a F0X01 inhibitor. In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) agents selected from: a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor), a TGFP-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid. In some embodiments, the medium further comprises a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor (e.g., a y- secretase inhibitor), a TGFP-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, and retinoic acid. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

In some embodiments, the medium further comprises one or more (e.g., 1, 2, 3, or 4) agents selected from an acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate. In some embodiments, the medium further comprises acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, and a one carbon metabolism pathway intermediate.

In some embodiments, the medium further comprises a vitamin. In some embodiments, the medium further comprises glutamine.

In some embodiments, the medium comprises a PKC activator, Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor), a sonic hedgehog (SHH) signaling pathway inhibitor, an epidermal growth factor, a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor), a TGFP-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, retinoic acid, an acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, a one carbon metabolism pathway intermediate, a vitamin, and glutamine. In some embodiments, the medium comprises PdBu, NVP-TNKS656, SANT-1, betacellulin, XXI, Alk5i, GC-1, LDN-193189, thiazovivin, staurosporine, DZNEP, retinoic acid, acetate, P-hydroxybutyrate, taurine, formate, biotin, and glutamine.

In some embodiments, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP- TNKS656) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.1 pM-0.2 pM, 0.2 pM-10 pM, 0.2 pM-9 pM, 0.2 pM-8 pM, 0.2 pM-7 pM, 0.2 pM-6 pM, 0.2 pM-5 pM, 0.2 pM-4 pM, 0.2 pM-3 pM, 0.2 pM-2 pM, 0.2 pM-1 pM, 0.2 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 0.5 pM-5 pM (e.g., 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 1.5 pM-2.5 pM or 1.8 pM-2.2 pM. In some embodiments, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) is present in the medium at a concentration of 2 pM.

In some embodiments, an epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng/ml-50 ng/ml. In some embodiments, an epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng/ml-50 ng/ml, 10 ng/ml-40 ng/ml, 10 ng/ml-30 ng/ml, 10 ng/ml-20 ng/ml, 20 ng/ml-50 ng/ml, 20 ng/ml-40 ng/ml, 20 ng/ml-30 ng/ml, 30 ng/ml-50 ng/ml, 30 ng/ml-40 ng/ml, or 40 ng/ml-50 ng/ml. In some embodiments, an epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 10 ng/ml-30 ng/ml (e.g., 10 ng/ml, 20 ng/ml, 20 ng/ml). In some embodiments, an epidermal growth factor (e.g., betacellulin) is present in the medium at a concentration of 20 ng/ml.

In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM- 2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a notch signaling pathway inhibitor (e.g., y- secretase inhibitor such as XXI) is present in the medium at a concentration of 0.5 pM-5 pM (e.g., 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) is present in the medium at a concentration of 2 pM.

In some embodiments, a TGFP-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 1 pM-50 pM. In some embodiments, a TGFP-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 1 pM-50 pM, 1 pM-40 pM, 1 pM- 30 pM, 1 pM-20 pM, 1 pM-10 pM, 10 pM-50 pM, 10 pM-40 pM, 10 pM-30 pM, 10 pM-20 pM, 20 pM-50 pM, 20 pM-40 pM, 20 pM-30 pM, 30 pM-50 pM, 30 pM-40 pM, or 40 pM-50 pM. In some embodiments, a TGFP-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 5 pM-20 pM (e.g., 5 pM, 10 pM, 15 pM, or 20 pM). In some embodiments, a TGFP-R1 kinase inhibitor (e.g., ALK5i) is present in the medium at a concentration of 10 pM.

In some embodiments, a thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.1 pM-10 pM. In some embodiments, a thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.1 pM-10 pM, 0.1 pM-9 pM, 0.1 pM-8 pM, 0.1 pM-7 pM, 0.1 pM-6 pM, 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-10 pM, 0.5 pM-9 pM, 0.5 pM-8 pM, 0.5 pM-7 pM, 0.5 pM-6 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM- 2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 0.5 pM-5 pM (e.g., 0.5 pM, 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a thyroid hormone (e.g., GC-1) is present in the medium at a concentration of 1 pM.

In some embodiments, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 pM-0.5 pM. In some embodiments, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 pM-0.5 pM, 0.1 pM-0.5 pM, 0.15 pM-0.5 pM, 0.2 pM-0.5 pM, 0.25 pM-0.5 pM, 0.3 pM-0.5 pM, 0.35 pM-0.5 pM, 0.4 pM-0.5 pM, 0.45 pM- 0.5 pM, 0.05 pM-0.4 pM, 0.1 pM-0.4 pM, 0.15 pM-0.4 pM, 0.2 pM-0.4 pM, 0.25 pM-0.4 pM, 0.3 pM-0.4 pM, 0.35 pM-0.4 pM, 0.05 pM-0.3 pM, 0.1 pM-0.3 pM, 0.15 pM-0.3 pM, 0.2 pM- 0.3 pM, 0.25 pM-0.3 pM, 0.05 pM-0.2 pM, 0.1 pM-0.2 pM, 0.15 pM-0.2 pM, or 0.05 pM-0.1 pM. In some embodiments, a BMP signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.05 pM-0.2 pM (e.g., 0.05 pM, 0.1 pM, 0.15 pM, or 0.2 pM). In some embodiments, a BMP signaling pathway inhibitor (e.g., LDN-193189) is present in the medium at a concentration of 0.1 pM.

In some embodiments, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM-10 pM. In some embodiments, a ROCK inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM-10 pM, 1 pM-9 pM, 1 pM-8 pM, 1 pM-7 pM, 1 pM-6 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-10 pM, 2 pM-9 pM, 2 pM-8 pM, 2 pM-7 pM, 2 pM-6 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-10 pM, 3 pM-9 pM, 3 pM-8 pM, 3 pM-7 pM, 3 pM-6 pM, 3 pM-5 pM, 3 pM-4 pM, 4 pM-10 pM, 4 pM-9 pM, 4 pM-8 pM, 4 pM-7 pM, 4 pM-6 pM, 4 pM-5 pM, 5 pM-10 pM, 5 pM-9 pM, 5 pM-8 pM, 5 pM-7 pM, 5 pM-6 pM, 6 pM-10 pM, 6 pM-9 pM, 6 pM-8 pM, 6 pM-7 pM, 7 pM-10 pM, 7 pM-9 pM, 7 pM-8 pM, 8 pM-10 pM, 8 pM-9 pM, or 9 pM-10 pM. In some embodiments, a ROCK inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 1 pM- 5 pM (e.g., 1 pM, 1.5 pM, 2 pM, 2.5 pM, 3 pM, 3.5 pM, 4 pM, 4.5 pM, or 5 pM). In some embodiments, a ROCK inhibitor (e.g., thiazovivin) is present in the medium at a concentration of 2.5 pM.

In some embodiments, a protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 0.5 nM- 10 nM. In some embodiments, a protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 0.5 nM-10 nM, 0.5 nM-9 nM, 0.5 nM- 8 nM, 0.5 nM-7 nM, 0.5 nM-6 nM, 0.5 nM-5 nM, 0.5 nM-4 nM, 0.5 nM-3 nM, 0.5 nM- 2 nM, 0.5 nM-1 nM, 1 nM-10 nM, 1 nM-9 nM, 1 nM- 8 nM, 1 nM-7 nM, 1 nM-6 nM, 1 nM-5 nM, 1 nM-4 nM, 1 nM-3 nM, 1 nM- 2 nM, 2 nM-10 nM, 2 nM-9 nM, 2 nM- 8 nM, 2 nM-7 nM, 2 nM-6 nM, 2 nM-5 nM, 2 nM-4 nM, 2 nM-3 nM, 3 nM-10 nM, 3 nM-9 nM, 3 nM- 8 nM, 3 nM-7 nM, 3 nM-6 nM, 3 nM-5 nM, 3 nM-4 nM, 4 nM-10 nM, 4 nM-9 nM, 4 nM- 8 nM, 4 nM-7 nM, 4 nM-6 nM, 4 nM-5 nM, 5 nM-10 nM, 5 nM-9 nM, 5 nM- 8 nM, 5 nM-7 nM, 5 nM-6 nM, 6 nM-10 nM, 6 nM-9 nM, 6 nM- 8 nM, 6 nM-7 nM, 7 nM-10 nM, 7 nM-9 nM, 7 nM- 8 nM, 8 nM-10 nM, 8 nM-9 nM, or 9 nM-10nM. In some embodiments, a protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 1 nM-5 nM (e.g., 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM). In some embodiments, a protein kinase inhibitor (e.g., staurosporine) is present in the medium at a concentration of 3 nM.

In some embodiments, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) is present in the medium at a concentration of 0.05 pM-0.5 pM. In some embodiments, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) is present in the medium at a concentration of 0.05 pM-0.5 pM, 0.1 pM-0.5 pM, 0.15 pM-0.5 pM, 0.2 pM-0.5 pM, 0.25 pM-0.5 pM, 0.3 pM-0.5 pM, 0.35 pM-0.5 pM, 0.4 pM-0.5 pM, 0.45 pM-0.5 pM, 0.05 pM-0.4 pM, 0.1 pM-0.4 pM, 0.15 pM-0.4 pM, 0.2 pM-0.4 pM, 0.25 pM-0.4 pM, 0.3 pM-0.4 pM, 0.35 pM-0.4 pM, 0.05 pM-0.3 pM, 0.1 pM-0.3 pM, 0.15 pM-0.3 pM, 0.2 pM-0.3 pM, 0.25 pM-0.3 pM, 0.05 pM-0.2 pM, 0.1 pM-0.2 pM, 0.15 pM-0.2 pM, or 0.05 pM-0.1 pM. In some embodiments, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) is present in the medium at a concentration of 0.05 pM-0.2 pM (e.g., 0.05 pM, 0.1 pM, 0.15 pM, or 0.2 pM). In some embodiments, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) is present in the medium at a concentration of 0.1 pM.

In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 pM-0.5 pM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 pM-0.5 pM, 0.05 pM-0.5 pM, 0.1 pM-0.5 pM, 0.15 pM-0.5 pM, 0.2 pM-0.5 pM, 0.25 pM-0.5 pM, 0.3 pM-0.5 pM, 0.35 pM-0.5 pM, 0.4 pM-0.5 pM, 0.45 pM-0.5 pM, 0.02 pM-0.4 pM, 0.05 pM-0.4 pM, 0.1 pM-0.4 pM, 0.15 pM-0.4 pM, 0.2 pM-0.4 pM, 0.25 pM-0.4 pM, 0.3 pM-0.4 pM, 0.35 pM-0.4 pM, 0.02 pM-0.3 pM, 0.05 pM-0.3 pM, 0.1 pM-0.3 pM, 0.15 pM-0.3 pM, 0.2 pM-0.3 pM, 0.25 pM-0.3 pM, 0.02 pM-0.2 pM, 0.05 pM-0.2 pM, 0.1 pM-0.2 pM, 0.15 pM-0.2 pM, 0.02 pM-0.1 pM, 0.05 pM-0.1 pM, or 0.02 pM-0.05 pM. In some embodiments, retinoic acid is present in the medium at a concentration of 0.02 pM-0.2 p M (e.g., 0.02 pM, 0.05 pM, 0.1 pM, 0.15 pM, or 0.2 pM). In some embodiments, retinoic acid is present in the medium at a concentration of 0.05 pM.

In some embodiments, an acetyl CoA related metabolite (e.g., acetate) is present in the medium at a concentration of 0.1 mM-10 mM. In some embodiments, an acetyl CoA related metabolite (e.g., acetate) is present in the medium at a concentration of 0.1 mM-10 mM, 0.1 mM-9 mM, 0.1 mM-8 mM, 0.1 mM-7 mM, 0.1 mM-6 mM, 0.1 mM-5 mM, 0.1 mM-4 mM, 0.1 mM-3 mM, 0.1 mM-2 mM, 0.1 mM-1 mM, 0.1 mM-0.5 mM, 0.5 mM-10 mM, 0.5 mM-9 mM, 0.5 mM-8 mM, 0.5 mM-7 mM, 0.5 mM-6 mM, 0.5 mM-5 mM, 0.5 mM-4 mM, 0.5 mM-3 mM, 0.5 mM-2 mM, 0.5 mM-1 mM, 1 mM-10 mM, 1 mM-9 mM, 1 mM-8 mM, 1 mM-7 mM, 1 mM- 6 mM, 1 mM-5 mM, 1 mM-4 mM, 1 mM-3 mM, 1 mM-2 mM, 2 mM-10 mM, 2 mM-9 mM, 2 mM-8 mM, 2 mM-7 mM, 2 mM-6 mM, 2 mM-5 mM, 2 mM-4 mM, 2 mM-3 mM, 3 mM-10 mM, 3 mM-9 mM, 3 mM-8 mM, 3 mM-7 mM, 3 mM-6 mM, 3 mM-5 mM, 3 mM-4 mM, 4 mM- 10 mM, 4 mM-9 mM, 4 mM-8 mM, 4 mM-7 mM, 4 mM-6 mM, 4 mM-5 mM, 5 mM-10 mM, 5 mM-9 mM, 5 mM-8 mM, 5 mM-7 mM, 5 mM-6 mM, 6 mM-10 mM, 6 mM-9 mM, 6 mM-8 mM, 6 mM-7 mM, 7 mM-10 mM, 7 mM-9 mM, 7 mM-8 mM, 8 mM-10 mM, 8 mM-9 mM, or 9 mM-10 mM. In some embodiments, an acetyl CoA related metabolite (e.g., acetate) is present in the medium at a concentration of 0.5 mM-5 mM (e.g., 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM). In some embodiments, an acetyl CoA related metabolite (e.g., acetate) is present in the medium at a concentration of 1 mM.

In some embodiments, an HD AC inhibitor (e.g., P-hydroxybutyrate) is present in the medium at a concentration of 0.05 pM-0.5 pM. In some embodiments, an HDAC inhibitor (e.g., P-hydroxybutyrate) is present in the medium at a concentration of 0.05 pM-0.5 pM, 0.1 pM-0.5 pM, 0.15 pM-0.5 pM, 0.2 pM-0.5 pM, 0.25 pM-0.5 pM, 0.3 pM-0.5 pM, 0.35 pM-0.5 pM, 0.4 pM-0.5 pM, 0.45 pM-0.5 pM, 0.05 pM-0.4 pM, 0.1 pM-0.4 pM, 0.15 pM-0.4 pM, 0.2 pM-0.4 pM, 0.25 pM-0.4 pM, 0.3 pM-0.4 pM, 0.35 pM-0.4 pM, 0.05 pM-0.3 pM, 0.1 pM-0.3 pM, 0.15 pM-0.3 pM, 0.2 pM-0.3 pM, 0.25 pM-0.3 pM, 0.05 pM-0.2 pM, 0.1 pM-0.2 pM, 0.15 pM-0.2 pM, or 0.05 pM-0.1 pM. In some embodiments, an HDAC inhibitor (e.g., P- hydroxybutyrate) is present in the medium at a concentration of 0.1 pM-0.5 pM (e.g., 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM , 0.5 pM). In some embodiments, an HDAC inhibitor is present in the medium at a concentration of 0.2 pM.

In some embodiments, a redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 20 pM-100 pM. In some embodiments, a redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 20 pM-100 pM, 20 pM-90 pM, 20 pM-80 pM, 20 pM-70 pM, 20 pM-60 pM, 20 pM-50 pM, 20 pM-40 pM, 20 pM-30 pM, 30 pM-100 pM, 30 pM-90 pM, 30 pM-80 pM, 30 pM-70 pM, 30 pM-60 pM, 30 pM-50 pM, 30 pM-40 pM, 40 pM-100 pM, 40 pM-90 pM, 40 pM-80 pM, 40 pM-70 pM, 40 pM-60 pM, 40 pM-50 pM, 50 pM-100 pM, 50 pM-90 pM, 50 pM-80 pM, 50 pM-70 pM, 50 pM-60 pM, 60 pM-100 pM, 60 pM-90 pM, 60 pM-80 pM, 60 pM-70 pM, 70 pM-100 pM, 70 pM-90 pM, 70 pM-80 pM, 80 pM-100 pM, 80 pM-90 pM, or 90 pM-100 pM. In some embodiments, a redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 50 pM-100 pM (e.g., 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM). In some embodiments, a redox homeostasis regulator (e.g., taurine) is present in the medium at a concentration of 90 pM.

In some embodiments, a one carbon metabolism pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 pM-100 pM. In some embodiments, a one carbon metabolism pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 pM-100 pM, 20 pM-90 pM, 20 pM-80 pM, 20 pM-70 pM, 20 pM-60 pM, 20 pM-50 pM, 20 pM-40 pM, 20 pM-30 pM, 30 pM-100 pM, 30 pM-90 pM, 30 pM-80 pM, 30 pM-70 pM, 30 pM-60 pM, 30 pM-50 pM, 30 pM-40 pM, 40 pM-100 pM, 40 pM-90 pM, 40 pM-80 pM, 40 pM-70 pM, 40 pM-60 pM, 40 pM-50 pM, 50 pM-100 pM, 50 pM-90 pM, 50 pM-80 pM, 50 pM-70 pM, 50 pM-60 pM, 60 pM-100 pM, 60 pM-90 pM, 60 pM-80 pM, 60 pM-70 pM, 70 pM-100 pM, 70 pM-90 pM, 70 pM-80 pM, 80 pM-100 pM, 80 pM-90 pM, or 90 pM-100 pM. In some embodiments, a one carbon metabolism pathway intermediate (e.g., formate) is present in the medium at a concentration of 20 pM-80 pM (e.g., 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, or 80 pM). In some embodiments, a one carbon metabolism pathway intermediate (e.g., formate) is present in the medium at a concentration of 50 pM.

In some embodiments, a vitamin (e.g., biotin) is present in the medium at a concentration of 0.1 pM-5 pM. In some embodiments, a vitamin (e.g., biotin) is present in the medium at a concentration of 0.1 pM-5 pM, 0.1 pM-4 pM, 0.1 pM-3 pM, 0.1 pM-2 pM, 0.1 pM-1 pM, 0.1 pM-0.5 pM, 0.5 pM-5 pM, 0.5 pM-4 pM, 0.5 pM-3 pM, 0.5 pM-2 pM, 0.5 pM-1 pM, 1 pM-5 pM, 1 pM-4 pM, 1 pM-3 pM, 1 pM-2 pM, 2 pM-5 pM, 2 pM-4 pM, 2 pM-3 pM, 3 pM-5 pM, 3 pM-4 pM, or 4 pM-5 pM. In some embodiments, a vitamin (e.g., biotin) is present in the medium at a concentration of 0.5 pM-2 pM (e.g., 0.5 pM, 0.8 pM, 1 pM, 1.5 pM, 2 pM). In some embodiments, a vitamin (e.g., biotin) is present in the medium at a concentration of 0.8 pM.

In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 1 mM-10 mM. In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 1 mM-10 mM, 1 mM-9 mM, 1 mM-8 mM, 1 mM-7 mM, 1 mM-6 mM, 1 mM-5 mM, 1 mM-4 mM, 1 mM-3 mM, 1 mM-2 mM, 2 mM-10 mM, 2 mM-9 niM. 2 mM-8 mM, 2 mM-7 niM. 2 mM-6 mM, 2 mM-5 mM, 2 mM-4 niM. 2 mM-3 mM, 3 mM- 10 mM, 3 mM-9 mM, 3 mM-8 mM, 3 mM-7 mM, 3 mM-6 mM, 3 mM-5 mM, 3 mM-4 mM, 4 mM-10 mM, 4 mM-9 mM, 4 mM-8 mM, 4 mM-7 mM, 4 mM-6 mM, 4 mM-5 mM, 5 mM-10 mM, 5 mM-9 mM, 5 mM-8 mM, 5 mM-7 mM, 5 mM-6 mM, 6 mM-10 mM, 6 mM-9 mM, 6 mM-8 mM, 6 mM-7 mM, 7 mM-10 mM, 7 mM-9 mM, 7 mM-8 mM, 8 mM-10 mM, 8 mM-9 mM, or 9 mM-10 mM. In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 2 mM-6 mM (e.g., 2 mM, 3 mM, 4 mM, 5 mM, or 6 mM). In some embodiments, glutamine (e.g., L-glutamine) is present in the medium at a concentration of 4 mM.

In some embodiments, the medium comprises a PKC activator (e.g., PdBu) at a concentration of 0.2 pM-1 pM, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of 0.5 pM-5 pM, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.1 pM-0.5 pM, an epidermal growth factor (e.g., betacellulin) at a concentration of 10 ng/ml-30 ng/ml, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) at a concentration of 0.5 pM-5 pM, a TGFP-R1 kinase inhibitor (e.g., ALK5i) at a concentration of 5 pM-20 pM, a thyroid hormone (e.g., GC-1) at a concentration of 0.5 pM-5 pM, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) at a concentration of 0.05 pM-0.2 pM, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 1 pM- 5 pM, a protein kinase inhibitor (e.g., staurosporine) at a concentration of 1 nM-5 nM, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) at a concentration of 0.05 pM-0.2 pM, retinoic acid at a concentration of 0.02 pM-0.2 pM, an acetyl CoA related metabolite (e.g., acetate) at a concentration of 0.5 mM-5 mM, an HD AC inhibitor (e.g., P-hydroxybutyrate) at a concentration of 0.1 pM-0.5 pM, a redox homeostasis regulator (e.g., taurine) at a concentration of 50 pM-100 pM, an one carbon metabolism pathway intermediate (e.g., formate) at a concentration of 20 pM-80 pM, a vitamin (e.g., biotin) at a concentration of 0.5 pM-2 pM, glutamine (e.g., L-glutamine) at a concentration of 2 mM-6 mM.

In some embodiments, the medium comprises a PKC activator (e.g., PdBu) at a concentration of 0.5 pM, a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656) at a concentration of 2 pM, a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1) at a concentration of 0.25 pM, an epidermal growth factor (e.g., betacellulin) at a concentration of 20 ng/ml, a notch signaling pathway inhibitor (e.g., y-secretase inhibitor such as XXI) at a concentration of 2 pM, a TGFP-R1 kinase inhibitor (e.g., ALK5i) at a concentration of 10 pM, a thyroid hormone (e.g., GC-1) at a concentration of 1 pM, a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN-193189) at a concentration of 0.1 pM, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin) at a concentration of 2.5 pM, a protein kinase inhibitor (e.g., staurosporine) at a concentration of 3 nM, a histone methyltransferase EZH2 inhibitor (e.g., DZNEP or UNC0321) at a concentration of 0.1 pM, retinoic acid at a concentration of 0.05 pM, an acetyl CoA related metabolite (e.g., acetate) at a concentration of 4 mM, an HD AC inhibitor (e.g., P-hydroxybutyrate) at a concentration of 50 pM, a redox homeostasis regulator (e.g., taurine) at a concentration of 90 pM, an one carbon metabolism pathway intermediate (e.g., formate) at a concentration of 50 pM, a vitamin (e.g., biotin) at a concentration of 0.8 pM, glutamine (e.g., L-glutamine) at a concentration of 4 mM.

In some embodiments, an in vitro composition described herein further comprises a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol (PVA), poloxamer, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(N-isopropylacrylamide), or polyacrylamide, optionally wherein the water- soluble synthetic polymer is polyvinyl alcohol. In some embodiments, the water water-soluble synthetic polymer is PVA. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% to 0.5% (w/v), 0.01% to 0.2% (w/v), 0.02% to 0.1% (w/v), or 0.03% to 0.08% (w/v) of the culture medium. In some embodiments, the water-soluble synthetic polymer has a concentration of 0.005% (w/v), 0.01% (w/v), 0.05% (w/v), 0.1% (w/v), 0.15% (w/v), 0.2% (w/v), 0.25% (w/v), 0.3% (w/v), 0.35% (w/v), to 0.4% (w/v), 0.45% (w/v), or 0.5% (w/v) of the medium. Polyvinyl alcohol described herein can refer to a water-soluble synthetic polymer that has an idealized formula [CH2CH(OH)]n, which can be either partially or completed hydrolyzed. In some cases, the polyvinyl alcohol is manufactured by either partial or complete hydrolysis of polyvinyl acetate to remove acetate groups. In some cases, the polyvinyl alcohol is at most 85% hydrolyzed, e.g., 80% hydrolyzed. The percentage of hydrolyzation measures the approximate percentage (e.g., average percentage) of acetate residue that is hydrolyzed in the polyvinyl acetate precursor polymer. In some cases, the polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the water-soluble synthetic polymer is PVA, and the PVA is at most 90% (e.g., 87%-89%) hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed (e.g., in stages 1-4). In some embodiments, the PVA is 89% hydrolyzed (e.g., in stage 5).

In some embodiments, the disclosure provides for compositions, bioreactors, and/or TFF systems comprising, or methods of culturing, using and/or generating, any of the cells or cell clusters disclosed herein. Any of the cells disclosed herein may be in a cell cluster or may be dissociated, unless the context clearly indicates otherwise.

In some embodiments, an in vitro composition or bioreactor and/or TFF system described herein comprises a population of pancreatic progenitor cells comprising cells that are PDX1- positive and NKX6.1 -positive, and insulin-negative. In some embodiments, an in vitro composition or bioreactor and/or TFF system described herein comprises a population of pancreatic progenitor cells comprising cells that are PDXl-positive and NKX6.1-positive, and insulin-positive. In some embodiments, an in vitro composition comprising described herein comprises a population of pancreatic progenitor cells comprising cells that are PDXl-positive and NKX6.1-positive, and insulin-negative and cells that are PDXl-positive and NKX6.1- positive, and insulin-positive. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

In some embodiments, the population of pancreatic progenitor cells comprises more cells that are PDXl-positive, NKX6.1-positive, and insulin-negative than cells that are PDXl- positive, NKX6.1-positive and insulin-positive. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the population of pancreatic progenitor cells are PDXl-positive, NKX6.1-positive, and insulin-negative. In some embodiments, no more than 50% (e.g., no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive, NKX6.1- positive, and insulin-positive. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive, NKX6.1- positive, and insulin-negative. In some embodiments, 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%- 40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive, NKX6.1- positive, and insulinpositive. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDXl-positive, NKX6.1- positive, and insulinnegative and 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%-40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive, NKX6.1- positive, and insulin-positive. In some embodiments, at least 50% of the population of pancreatic progenitor cells are PDXl- positive, NKX6.1- positive, and insulin-negative, and no more than 50% of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDXl-positive, NKX6.1- positive, and insulin-positive. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

In some embodiments, the population of pancreatic progenitor cells comprises more cells that are PDX1 -positive, NKX6.1- positive, and insulin-positive than cells that are PDX1- positive, NKX6.1- positive, and insulin-negative. In some embodiments, at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) of the population of pancreatic progenitor cells are PDX1 -positive, NKX6.1- positive, and insulin-positive. In some embodiments, no more than 50% (e.g., no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10%) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDX1 -positive, NKX6.1- positive, and insulin-negative. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDX1 -positive, NKX6.1- positive, and insulin-positive. In some embodiments, 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%- 40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDX1 -positive, NKX6.1- positive, and insulinnegative. In some embodiments, 50%-90% (e.g., 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-90%, 60%-80%, 60%-70%, 30%-80%, 70%-90%, 70%-80%, or 80%-90%) of the population of pancreatic progenitor cells are PDX1 -positive, NKX6.1- positive, and insulinpositive and 10%-50% (e.g., 10%-50%, 20%-50%, 30%-50%, 40%-50%, 10%-40%, 20%-40%, 30%-40%, 10%-30%, 20%-30%, or 10%-20%,) of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDX1 -positive, NKX6.1- positive, and insulin-negative. In some embodiments, at least 50% of the population of pancreatic progenitor cells are PDX1- positive, NKX6.1- positive, and insulin-positive, and no more than 50% of the population of pancreatic progenitor cells are pancreatic progenitor cells that are PDX1 -positive, NKX6.1- positive, and insulin-negative. In some embodiments, the cells are in clusters. In more embodiments, the population comprises clusters.

Methods of producing pancreatic islet cells

In embodiments, the present disclosure relates to compositions, bioreactors, TFF systems, and methods of generating endocrine cells from pancreatic progenitor cells or precursors. Certain exemplary detailed protocols of generating endocrine cells to provide at least one SC-P cell are described in U.S. Patent Application Publication No. US20150240212, US20150218522, US 20200332262, US 20210198632, US 20220090020, US 2021-0238553, US Patent 10,030,229; US Patent 10,443,042; and published application WO2022147056, each of which is herein incorporated by reference in its entirety.

In some embodiments, a method of generating a population of endocrine cells leads to increased percentage of pancreatic a and/or 5 cells and decreased percentage of pancreatic EC cells when generating pancreatic P cells. In some embodiments, a method described herein may be used to obtain an enriched population of a cells. In some embodiments, a method described herein may be used to obtain an enriched population of P cells. In some embodiments, a method described herein may be used to obtain an enriched population of a cells and P cells. In some embodiments, a method described herein may be used to obtain an increased yield of pancreatic endocrine cells. The disclosed bioreactors and/or TFF systems can be used in these methods.

The differentiation of hPSC cells to hormone-expressing pancreatic endocrine cells may be conducted by transitioning hPSC cells through major stages of embryonic development; differentiation to mesendoderm and definitive endoderm, establishment of the primitive gut endoderm, patterning of the posterior foregut, and specification and maturation of pancreatic endoderm and endocrine precursors. Through these stages, hPSC cells can obtain pancreatic endocrine phenotype and ability of glucose responsive insulin secretion in vitro.

Generally, the at least one pancreatic SC-a, SC-P and/or SC-5 cell or precursor thereof, e.g., pancreatic progenitors produced according to the methods disclosed herein can comprise a mixture or combination of different cells, e.g., for example a mixture of cells such as a PDX1- positive pancreatic progenitors, pancreatic progenitors co-expressing PDX1 and NKX6-1, a Ngn3-positive endocrine progenitor cell, an insulin-positive endocrine cell e.g., NKX6.1- positive, ISEl-positive cells, or P-like cells), and/or other pluripotent or stem cells. In some embodiments, the cells are in clusters.

The at least one pancreatic a, P and/or 5 cell or precursor thereof can be produced according to any suitable culturing protocol to differentiate a stem cell or pluripotent cell to a desired stage of differentiation. In some embodiments, the at least one pancreatic a, P and/or 5 cell or the precursor thereof are produced by culturing at least one pluripotent cell, or clusters of pluripotent cells, for a period of time and under conditions suitable for the at least one pluripotent cell to differentiate into the at least one pancreatic a, P and/or 5 cell or the precursor thereof. As disclosed herein, this culturing protocol can include TFF, such as ATF.

In some embodiments, the at least one pancreatic a, P and/or 5 cell or precursor thereof is a substantially pure population of pancreatic a, P and/or 5 cells or precursors thereof. In some embodiments, a population of pancreatic a, P and/or 5 cells or precursors thereof comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, a population pancreatic a, P and/or 5 cells or precursors thereof are substantially free or devoid of embryonic stem cells or pluripotent cells or iPS cells. In some embodiments, a method described herein produces a population of cells comprising pancreatic a, P and/or 5 cells at a ratio that resembles that of a natural pancreatic islet. These cells can be in clusters.

In some embodiments, a method described herein comprises (i) culturing a first population of cells comprising pancreatic progenitor cells (e.g., cells that are PDX1 -positive, NKX6.1-negative; or a mixture of cells that are PDX1 -positive, NKX6.1 -negative and cells that are PDX1 -positive, NKX6.1-positive) in a first medium comprising a Forkhead Box 01 (FoxOl) inhibitor and a notch signaling pathway inhibitor for a period of time to obtain a second population of cells (e.g., a population of cells that comprises more PDX1 -positive, NKX6.1- positive cells than the first population); and (ii) culturing the second population of cells in a second medium comprising a PKC activator and a Wnt signaling pathway inhibitor. In some embodiments, the method generates a population of cells comprising cells that are PDX1- positive, NKX6.1-positive, and insulin-positive. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, a method described herein comprises culturing a first population of cells in a first medium, wherein the first population of cells comprises pancreatic progenitor cells that are PDXl-positive and NKX6.1 negative, and pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive; and the first medium comprises a Forkhead Box 01 (FoxOl) inhibitor (e.g., AS1842856 or a derivative thereof). In some embodiments, the first medium further comprises a notch signaling pathway inhibitor. In some embodiments, the notch signaling pathway inhibitor is a y-secretase inhibitor (e.g., XXI, DAPT or derivatives thereof). In some embodiments, the y-secretase inhibitor is XXI. In some embodiments, the first medium does not comprise a Wnt signaling pathway inhibitor. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, the first population of cells comprises pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive. In some embodiments, the first population of cells comprises more pancreatic progenitor cells that are PDXl-positive and NKX6.1 negative than pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive. In some embodiments, the first population of cells comprises more pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive than pancreatic progenitor cells that are PDXl-positive and NKX6.1 negative. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, first medium further comprises a PKC activator (e.g., PdBU, TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or derivatives thereof). In some embodiments, the PKC activator is PdBU. In some embodiments, the first medium further comprises one or more (e.g., 1, 2, 3, 4, 5) agents selected from a fibroblast growth factor (e.g., KGF), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), and a TGF-P ligand (e.g., activin A). In some embodiments, the first medium further comprises a water-soluble synthetic polymer (e.g., PVA such as PVA80%). In some embodiments, the first medium comprises a FoxOl inhibitor (e.g., AS1842856 or a derivative thereof), a notch signaling pathway inhibitor (e.g., y- secretase inhibitor such as XXI), a PKC activator (e.g., PdBU), a fibroblast growth factor (e.g., KGF), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), and a TGF- P ligand (e.g., activin A), and a water-soluble synthetic polymer (e.g., PVA such as PVA80%).

In some embodiments, the first population of cells are cultured in the first medium for a period of about 12-72 hours (e.g., about 12-72 hours, 12-66 hours, 12-60 hours, 12-54 hours, 12- 48 hours, 12-42 hours, 12-36 hours, 12-30 hours, 12-24 hours, 12-18 hours, 18-72 hours, 18-66 hours, 18-60 hours, 18-54 hours, 18-48 hours, 18-42 hours, 18-36 hours, 18-30 hours, 18-24 hours, 24-72 hours, 24-66 hours, 24-60 hours, 24-54 hours, 24-48 hours, 24-42 hours, 24-36 hours, 24-30 hours, 30-72 hours, 30-66 hours, 30-60 hours, 30-54 hours, 30-48 hours, 30-42 hours, 30-36 hours, 36-72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours). In some embodiments, the first population of cells are cultured in the first medium for a period of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the first population of cells are cultured in the first medium for a period of about 24 hours. In some embodiments, the first population of cells are cultured in the first medium for a period of about 48 hours.

In some embodiments, culturing the first population of cells in the first media for a contacting period described herein (e.g., 24 or 48 hours) results in a second population of cells. In some embodiments, the second population of cells comprises pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive and pancreatic progenitor cells that are PDXl-positive and NKX6.1 negative. In some embodiments, the second population of cells comprises more pancreatic progenitor cells that are PDXl-positive and NKX6.1 -positive than the first population of cells. In some embodiments, the second population of cells comprises more pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive than pancreatic progenitor cells that are PDXl-positive and NKX6.1 negative. In some embodiments, the second population of cells comprises trace amounts (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of the second population of cells) of pancreatic progenitor cells that are PDX1 -positive and NKX6.1 negative. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, a method described herein further comprises culturing the second population of cells with a second medium comprising a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656). In some embodiments, the second medium comprises a PKC activator (e.g., PdBu). In some embodiments, the second medium further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) agents selected from an epidermal growth factor (e.g., betacellulin), a thyroid hormone (e.g., GC-1), a TGFP-R1 kinase inhibitor (e.g., ALK5i), a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321). In some embodiments, the second medium further comprises one or more (e.g., 1, 2, 3, 4) agents selected from an acetyl CoA related metabolite (e.g., acetate), an HDAC inhibitor (e.g., P- hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and a one carbon metabolism pathway intermediate (e.g., formate). In some embodiments, the second medium further comprises a vitamin (e.g., biotin). In some embodiments, the second medium further comprises glutamine. In some embodiments, the second medium further comprises a water soluble synthetic polymer (e.g., PVA such as PVA 87-89%). In some embodiments, the second medium does not comprise a FOXO1 inhibitor. In some embodiments, the second medium comprises a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), a PKC activator (e.g., PdBu), an epidermal growth factor (e.g., betacellulin), a thyroid hormone (e.g., GC-1), a TGFP-R1 kinase inhibitor (e.g., ALK5i), a notch signaling pathway inhibitor (e.g., a y- secretase inhibitor such as XXI), a sonic hedgehog (SHH) signaling pathway inhibitor (e.g., SANT-1), retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HDAC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine and a water soluble synthetic polymer (e.g., PVA such as PVA 87-89%), and does not comprise a FOXO1 inhibitor. In some embodiments, the second population of cells are cultured in the second medium for a period of about 12-72 hours (e.g., about 12-72 hours, 12-66 hours, 12-60 hours, 12-54 hours, 12-48 hours, 12-42 hours, 12-36 hours, 12-30 hours, 12-24 hours, 12-18 hours, 18-72 hours, 18-66 hours, 18-60 hours, 18-54 hours, 18-48 hours, 18-42 hours, 18-36 hours, 18-30 hours, 18-24 hours, 24-72 hours, 24-66 hours, 24-60 hours, 24-54 hours, 24-48 hours, 24-42 hours, 24-36 hours, 24-30 hours, 30-72 hours, 30-66 hours, 30-60 hours, 30-54 hours, 30-48 hours, 30-42 hours, 30-36 hours, 36-72 hours, 36-66 hours, 36-60 hours, 36-54 hours, 36-48 hours, 36-42 hours, 42-72 hours, 42-66 hours, 42-60 hours, 42-54 hours, 42-48 hours, 48-72 hours, 48-66 hours, 48-60 hours, 48-54 hours, 54-72 hours, 54-66 hours, 54-60 hours, 60-72 hours, 60-66 hours, or 66-72 hours). In some embodiments, the second population of cells are cultured in the second medium for a period of about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some embodiments, the second population of cells are cultured in the second medium for a period of about 48 hours.

In some embodiments, culturing the second population of cells in the second media for a contacting period described herein (e.g., 48 hours) results in a third population of cells. In some embodiments, the third population of cells comprises pancreatic progenitor cells that are PDX1- positive and NKX6.1 -positive. In some embodiments, the third population of cells comprises cells that are ISL1 -positive. In some embodiments, the third population of cells comprises cells that are ISLl-negative. In some embodiments, the third population of cells comprises cells that are ISL1 -positive. In some embodiments, the third population of cells comprises more cells that are ISLl-positive than the first and second population of cells. In some embodiments, the third population of cells comprises more cells that are ISLl-negative than cells that are ISLl-positive. In some embodiments, the third population of cells comprises cells that are insulin-negative. In some embodiments, the third population of cells comprises cells that are insulin-positive. In some embodiments, the third population of cells comprises more cells that are insulin-negative than cells that are insulin-positive. In some embodiments, the third population of cells comprises more cells that are insulin-positive than the first and second population of cells. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, the method further comprises culturing the third population of cells in a third medium comprising one or more agents selected from: a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI), a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321). In some embodiments, the third medium further comprises one or more agents selected from an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and one carbon metabolism pathway intermediate (e.g., formate). In some embodiments, the third medium further comprises a vitamin (e.g., biotin). In some embodiments, the third medium further comprises glutamine. In some embodiments, the third medium further comprises a water soluble synthetic polymer (e.g., PVA such as PVA 87-89%).

In some embodiments, the third medium does not comprise a Wnt signaling pathway inhibitor or a PKC activator. In some embodiments, the third medium comprises a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI), a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine and a water soluble synthetic polymer (e.g., PVA such as PVA 87-89%), and does not comprise a Wnt signaling pathway inhibitor and a PKC activator. In some embodiments, the third population of cells are cultured in the third medium (e.g., the third medium that does not comprise a Wnt signaling pathway inhibitor or a PKC activator) for a period of about 24-96 hours (e.g., about 24-96 hours, 24-84 hours, 24-72 hours, 24-60 hours, 24-48 hours, 24-36 hours, 36-96 hours, 36-84 hours, 36-72 hours, 36-60 hours, 36-48 hours, 48-96 hours, 48-84 hours, 48- 72 hours, 48-60 hours, 60-96 hours, 60-84 hours, 60-72 hours, 72-96 hours, 72-84 hours, or 84- 96 hours). In some embodiments, the third population of cells are cultured in the third medium for a period of about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours. In some embodiments, the third population of cells are cultured in the third medium for a period of about 96 hours.

In some embodiments, the third medium further comprises a Wnt signaling pathway inhibitor but does not comprise a PKC activator. In some embodiments, the third medium comprises Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), a notch signaling pathway inhibitor (e.g., a y-secretase inhibitor such as XXI), a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine and a water soluble synthetic polymer (e.g., PVA such as PVA 87-89%), and does not comprise a PKC activator. In some embodiments, the third population of cells are cultured in the third medium (e.g., the third medium that comprises a Wnt signaling pathway inhibitor but not a PKC activator) for a period of about 24-48 hours (e.g., about 24-48 hours, 24-36 hours, or 36-48 hours), after which the Wnt signaling pathway inhibitor is removed from the third medium and the cells are further cultured for about 24-48 hours ((e.g., about 24-48 hours, 24-36 hours, or 36- 48 hours). In some embodiments, the third population of cells are cultured in the third medium (e.g., the third medium that comprises a Wnt signaling pathway inhibitor but not a PKC activator) for a period of about 48 hours, after which the Wnt signaling pathway inhibitor is removed from the third medium and the cells are further cultured for about 48 hours.

In some embodiments, culturing the third population of cells in the third media for a contacting period described herein (e.g., 96 hours) results in a fourth population of cells. In some embodiments, the fourth population of cells comprises cells that are PDXl-positive and NKX6.1 positive. In some embodiments, the fourth population of cells comprises cells that are insulinpositive. In some embodiments, the fourth population of cells comprises cells that are PDXl- positive, NKX6.1 positive, and insulin-positive. In some embodiments, the fourth population of cells comprise cells that are ISLl-positive. In some embodiments, the fourth population of cells comprises cells that are ISL-1 negative. In some embodiments, at least 30% (e.g., at least 30%, at least 40%, at least 50%, or at least 60%)) of the fourth population of cells are insulin-positive. In some embodiments, 30%-50%, 30%-40%, or 40%-50% of the fourth population of cells are insulin-positive. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, the method further comprises culturing the fourth population of cells in a fourth medium comprising one or more agents selected from: a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321). In some embodiments, the fourth medium further comprises one or more agents selected from an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), and an one carbon metabolism pathway intermediate (e.g., formate). In some embodiments, the fourth medium further comprises a vitamin (e.g., biotin). In some embodiments, the fourth medium further comprises one or more of glutamine (e.g., L- glutamine), glutamate (e.g., L-glutamate), and carnitine (e.g., L-carnitine). In some embodiments, the fourth medium further comprises albumin (e.g., human serum albumin or HSA). In some embodiments, the fourth medium further comprises ZnSCL. In some embodiments, the fourth media does not comprise a Wnt signaling pathway inhibitor or a PKC activator. In some embodiments, the fourth medium comprises a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine, glutamate, carnitine, albumin (e.g., human serum albumin or HSA), and ZnSO4, and does not comprise a Wnt signaling pathway inhibitor or a PKC activator.

In some embodiments, the fourth population of cells are cultured in the fourth medium for a period of about 24-96 hours (e.g., about 24-96 hours, 24-84 hours, 24-72 hours, 24-60 hours, 24-48 hours, 24-36 hours, 36-96 hours, 36-84 hours, 36-72 hours, 36-60 hours, 36-48 hours, 48-96 hours, 48-84 hours, 48-72 hours, 48-60 hours, 60-96 hours, 60-84 hours, 60-72 hours, 72-96 hours, 72-84 hours, or 84-96 hours). In some embodiments, the fourth population of cells are cultured in the fourth medium for a period of about 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,

85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours. In some embodiments, the fourth population of cells are cultured in the fourth medium for a period of about 72 hours.

In some embodiments, culturing the fourth population of cells in the fourth media for a contacting period described herein (e.g., 96 hours) results in a fifth population of cells. In some embodiments, a method described herein further comprises culturing the fifth population of cells in a fifth medium comprising glutamine, albumin (e.g., human serum albumin or HSA), and ZnSO4. In some embodiments, the fifth medium comprises glutamine, albumin (e.g., human serum albumin or HSA) and ZnSO4, and does not comprise any one of the agents selected from: a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled- coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321). In some embodiments, the fifth media further comprises a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), a one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamine, glutamate, and carnitine. In some embodiments, the fifth medium comprises a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HD AC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), an one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), glutamate, glutamine, carnitine, albumin (e.g., human serum albumin or HSA), and ZnSCU, and does not comprise any one of the agents selected from: a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin), a protein kinase inhibitor (e.g., staurosporine), and a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321). In some embodiments, the fifth medium comprises albumin (e.g., human serum albumin or HSA), and ZnSO4, and does not comprise any one of the agents selected from: a TGFP-R1 kinase inhibitor (e.g., ALK5i), a thyroid hormone (e.g., GC-1), a bone morphogenetic (BMP) signaling pathway inhibitor (e.g., LDN193189), a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (e.g., triazovivin), a protein kinase inhibitor (e.g., staurosporine), a histone methyltransferase EZH2 inhibitor (e.g., DZNep or UNC0321), an acetyl CoA related metabolite (e.g., acetate), an HDAC inhibitor (e.g., P-hydroxybutyrate), a redox homeostasis regulator (e.g., taurine), an one carbon metabolism pathway intermediate (e.g., formate), a vitamin (e.g., biotin), carnitine, glutamate, and glutamine. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, the fifth population of cells are cultured in the fifth medium for a period of about 96-240 hours (e.g., about 96-240 hours, 96-216 hours, 96-192 hours, 96-168 hours, 96-144 hours, 96-120 hours; 120-240 hours, 120-216 hours, 120-192 hours, 120-168 hours, 120-144 hours, 144-240 hours, 144-216 hours, 144-192 hours, 144-168 hours, 168-240 hours, 168-216 hours, 168-192 hours, 192-240 hours, 192-216 hours, or 192-240 hours). In some embodiments, the fifth population of cells are cultured in the fifth medium for a period of about 24, 48, 72, 96, 120, 144, 168, 192, 216, or 240 hours. In some embodiments, the fifth population of cells are cultured in the fifth medium for a period of about 192 hours. In some embodiments, culturing the fifth population of cells in the fifth media for a contacting period described herein (e.g., 192 hours) results in a sixth population of cells. In some embodiments, at least 15% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or more) of the sixth population of cells are NKX6.1 -negative, ISL1- positive; and wherein less than 12% (e.g., less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2% or less) of the sixth population of cells are NKX6.1 -negative, ISLl-negative. In some embodiments, the cells are in clusters. In more embodiments, these population(s) comprise clusters.

In some embodiments, a method described herein comprises:

(i) culturing a first population of cells in a first medium to obtain a second population of cells, wherein the first population of cells comprises pancreatic progenitor cells that are PDX1- positive and NKX6.1 negative, and pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive; and the first medium comprises: a FoxOl inhibitor, a notch signaling pathway inhibitor, a PKC activator, a fibroblast growth factor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a TGF-P ligand, and a water-soluble synthetic polymer;

(ii) culturing the second population of cells obtained in (i) with a second medium to obtain a third population of cells, wherein the second medium comprises: a Wnt signaling pathway inhibitor, a PKC activator, an epidermal growth factor, a thyroid hormone, a TGFP-R1 kinase inhibitor, a notch signaling pathway inhibitor, a sonic hedgehog (SHH) signaling pathway inhibitor, retinoic acid, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a bone morphogenetic (BMP) signaling pathway inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, a one carbon metabolism pathway intermediate, a vitamin, glutamine and a water soluble synthetic polymer (e.g., PVA), and wherein the second medium does not comprise a FOXO1 inhibitor;

(iii) culturing the third population of cells obtained in (ii) with a third medium to obtain a fourth population of cells, wherein the third medium comprises: a notch signaling pathway inhibitor, a TGFP-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, and a histone methyltransferase EZH2 inhibitor, an acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, an one carbon metabolism pathway intermediate, a vitamin, glutamine and a water soluble synthetic polymer, and wherein the third medium does not comprise a Wnt signaling pathway inhibitor and a PKC activator; (iv) culturing the fourth population of cells obtained in (iii) with a fourth medium to obtain a fifth population of cells, wherein the fourth medium comprises a notch signaling pathway inhibitor, a TGFP-R1 kinase inhibitor, a thyroid hormone, a bone morphogenetic (BMP) signaling pathway inhibitor, a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, a protein kinase inhibitor, a histone methyltransferase EZH2 inhibitor, an acetyl CoA related metabolite, an HD AC inhibitor, a redox homeostasis regulator, a one carbon metabolism pathway intermediate, a vitamin, glutamine, glutamate, carnitine, albumin, and ZnSO4, and wherein the fourth medium does not comprise a Wnt signaling pathway inhibitor and a PKC activator; and

(v) culturing the fifth population of cells obtained in (iv) with a fifth medium to obtain a sixth population of cells, wherein the fifth medium comprises albumin (e.g., human serum albumin or HSA) and ZnSO4. In some embodiments, the culturing steps occur in any of the bioreactors and/or TFF systems disclosed herein. In some embodiments, the culturing steps occur using any of the culturing methods disclosed herein.

In some embodiments, cells in the first population can be in clusters. In some embodiments, the cells in the second population can be in clusters. In some embodiments, cells in the third population can be in clusters. In some embodiments, the cells in the fourth population can be in clusters. In some embodiments, cells in the fifth population can be in clusters. In some embodiments, cells in the sixth population can be in clusters. In more embodiments, the cells in the first, second, third, fourth, fifth, and/or sixth population can be in clusters.

In some embodiments, a method described herein further comprises generating the first population of cells comprising pancreatic progenitor cells that are PDX1 -positive and NKX6.1 negative and pancreatic progenitor cells that are PDXl-positive and NKX6.1 positive. In some embodiments, the first population of cells are differentiated from stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, or other pluripotent stem cells). In some embodiments, the stem cells (e.g., embryotic stem cells) are generated from the inner cell mass of blastocyst-stage embryos represent. In other embodiments, the stem cells are obtained without the destruction of a human embryo. Stem cells can be maintained in culture, renew for themselves, proliferate unlimitedly as undifferentiated ES cells, and are capable of differentiating into all cell types of the body as the ectoderm, mesoderm, and endoderm lineage cells or tissues. In some embodiments, the stem cells are dissociated. In some embodiments, the stem cells can be in clusters. Cell types during pancreatic differentiation

Embodiments of the present disclosure provide cell types of the pancreatic lineage obtained during differentiation of stem cells to generate pancreatic islet cells. Such cells include any cell that is capable of differentiating into a pancreatic islet cell, including for example, a pluripotent stem cell, a definitive endoderm cell, a primitive gut tube cell, a pancreatic progenitor cell, or endocrine progenitor cell, when cultured under conditions suitable for differentiating the precursor cell into the pancreatic islet cell. These cells can be propagated and differentiated, using any of the compositions, TFF systems, and/or methods disclosed herein.

A “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, pluripotent stem cells can differentiate into lineage-restricted progenitor cells (e.g., mesodermal stem cells or endoderm cells), which in turn can differentiate into cells that are further restricted (e.g., neuron progenitors or pancreatic progenitors), which can differentiate into end-stage cells (i.e., terminally differentiated cells, e.g., pancreatic beta cells, neurons, cardiomyocytes, etc.), which play a characteristic role in a certain tissue type, and can or cannot retain the capacity to proliferate further.

Disclosed are methods and systems for differentiating stem cells, and clusters thereof into somatic cells, specifically pancreatic endocrine cells. By “somatic cell,” it is meant any cell in an organism that, in the absence of experimental manipulation, does not ordinarily give rise to all types of cells in an organism. In other words, somatic cells are cells that have differentiated sufficiently that they do not naturally generate cells of all three germ layers of the body, i.e., ectoderm, mesoderm and endoderm. For example, somatic cells can include both neurons and neural progenitors, the latter of which is able to naturally give rise to all or some cell types of the central nervous system but cannot give rise to cells of the mesoderm or endoderm lineages.

In some embodiments, the cell is a stem cell, and the stem cell is a multipotent cell and is not a pluripotent stem cell. In some embodiments, the stem cell is a stem cell reprogrammed from a primary pancreatic islet cell. In some embodiments, the multipotent stem cell is the SR1423 cell line described in Ratiu et al., 2023, bioRxiv, https://doi.org/10.1101/ 2023.10.20.563345.

Definitive Endoderm Cells

The definitive endoderm can be generated in vivo from the inner cell mass by the process of gastrulation of embryogenesis, in which epiblast cells are instructed to form the three germ layers. Definitive endoderm can give rise to diverse cells and tissues that contribute to vital organs as the pancreatic P cells, liver hepatocytes, lung alveolar cells, thyroid, thymus, and the epithelial lining of the alimentary and respiratory tract. It is different from the primitive endoderm of extraembryonic tissues, which can give rise to the visceral and parietal endoderm. The definitive endoderm derived from ES cells is theoretically capable of becoming any endoderm derivatives.

Precise patterning of anterior-posterior axis of the definitive endoderm can eventually form the primitive gut tube. The definitive endoderm-derived primitive gut tube induces the pharynx, esophagus, stomach, duodenum, small and large intestine along the anterior-posterior axis as well as associated organs, including pancreas, lung, thyroid, thymus, parathyroid, and liver. The anterior portion of the foregut of the primitive gut tube becomes lung, thyroid, esophagus, and stomach. The pancreas, liver, and duodenum originate from the posterior portion of the foregut. The midgut and hindgut of primitive gut tube gives rise to the small and large intestine. The anterior foregut expresses developmental markers, NK2 homeobox 1 (NKX2-1) and SRY (sex determining region Y)-box 2 (SOX2); the posterior foregut expresses hematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (0NECUT1, known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and the midgut/hindgut expresses caudal type homeobox 1 (CDX1), caudal type homeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3, 19, 20).

Definitive endoderm cells of use in the method and bioreactors and/or TFF systems disclosed herein can be derived from any source or generated in accordance with any suitable protocol. In some embodiments, pluripotent stem cells, e.g., iPSCs or hESCs, are differentiated to definitive endoderm cells. In some embodiments, the definitive endoderm cells (stage 1) are further differentiated, e.g., to primitive gut tube cells (stage 2), PDXl-positive pancreatic progenitor cells (stage 3), NKX6.1-positive pancreatic progenitor cells (stage 4), or Ngn3- positive endocrine progenitor cells or insulin-positive endocrine cells (stage 5), followed by induction or maturation to SC-P cells (stage 6).

In some embodiments, definitive endoderm cells can be obtained by differentiating at least some pluripotent cells into a population into definitive endoderm cells in the bioreactors and/or TFF system disclosed herein. The pluripotent cells can be in clusters. In some embodiments, the method includes contacting a population of pluripotent cells with i) at least one growth factor from the TGF-P superfamily, and ii) a Wnt signaling pathway activator, to induce the differentiation of at least some of the pluripotent cells into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm.

Any growth factor from the TGF-P superfamily capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a Wnt signaling pathway activator) can be used in the method provided herein. In some embodiments, the growth factor from the TGF-P superfamily comprises Activin A. In some embodiments, the growth factor from the TGF-P superfamily comprises growth differentiating factor 8 (GDF8). Any Wnt signaling pathway activator capable of inducing the pluripotent stem cells to differentiate into definitive endoderm cells (e.g., alone, or in combination with a growth factor from the TGF-P superfamily) can be used in the method provided herein. In some embodiments, the Wnt signaling pathway activator comprises CHIR99021. In some embodiments, the Wnt signaling pathway activator comprises Wnt3a recombinant protein.

In some embodiments, differentiating at least some pluripotent cells in a population into definitive endoderm cells is achieved by a process of contacting a population of pluripotent cells with i) Activin A, and ii) CHIR99021 for a suitable period of time, e.g., about 2 days, about 3 days, about 4 days, or about 5 days to induce the differentiation of at least some of the pluripotent cells in the population into definitive endoderm cells, wherein the definitive endoderm cells express at least one marker characteristic of definitive endoderm. In some embodiments, the process comprises contacting a population of pluripotent cells with activin A and CHIR99021 for 1 day, and then with activin A (in the absence of CHIR99021) for a further 1 or 2 days.

In some examples, the method comprises differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a suitable concentration of the growth factor from the TGF-P superfamily (e.g., Activin A), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some embodiments, the method comprises use of about 70-130 ng. ml, 80- 120 ng/ml, or 90-110 ng/ml Activin A for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 100 ng/mL Activin A for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 200 ng/mL Activin A for differentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cells into definitive endoderm cells by contacting a population of pluripotent cells with a suitable concentration of the Wnt signaling pathway activator (e.g., CHIR99021), such as, about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.5 pM, about 0.8 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 5 pM, about 8 pM, about 10 pM, about 12 pM, about 15 pM, about 20 pM, about 30 pM, about 50 pM, about 100 pM, or about 200 pM. In some embodiments, the method comprises use of about 1-5 pM or 2-4 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 2 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 3 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells. In some embodiments, the method comprises use of about 5 pM CHIR99021 for differentiation of pluripotent cells into definitive endoderm cells.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is PVA. In some cases, the PVA is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the PVA is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein expresses at least one marker selected from the group consisting of: Nodal, Tmprss2, Tmem30b, Stl4, Spink3, Sh3gl2, Ripk4, RablS, Npnt, Clic6, Cldn5, Cacnalb, Bnipl, Anxa4, Emb, FoxAl, Soxl7, and Rbm35a, wherein the expression of at least one marker is upregulated to by a statistically significant amount in the definitive endoderm cell relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Gata4, SPARC, AFP and Dab2 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein does not express by a statistically significant amount at least one marker selected the group consisting of: Zicl, Pax6, Flkl and CD31 relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein has a higher level of phosphorylation of Smad2 by a statistically significant amount relative to the pluripotent stem cell from which it was derived. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein has the capacity to form gut tube in vivo. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein can differentiate into a cell with morphology characteristic of a gut cell, and wherein a cell with morphology characteristic of a gut cell expresses FoxA2 and/or Claudin6. In some embodiments, a definitive endoderm cell produced by the methods and bioreactors and/or TFF systems as disclosed herein can be further differentiated into a cell of endoderm origin. In some embodiments, a population of stem cells or stem cell clusters are cultured in the presence of at least one P cell differentiation factor prior to any differentiation or during the first stage of differentiation. One can use any pluripotent stem cell, such as a human pluripotent stem cell, or a human iPS cell or any of pluripotent stem cell as discussed herein or other suitable pluripotent stem cells. In some embodiments, a P cell differentiation factor as described herein can be present in the culture medium of a population of pluripotent stem cells or may be added in bolus or periodically during growth (e.g. replication or propagation) of the population of pluripotent stem cells in the bioreactor and/or TFF system. In certain examples, a population of pluripotent stem cells can be exposed to at least one P cell differentiation factor prior to any differentiation in the bioreactor and/or TFF system. In other examples, a population of pluripotent stem cells may be exposed to at least one P cell differentiation factor during the first stage of differentiation in the bioreactor and/or TFF system.

Primitive Gut Tube Cells

Embodiments of the disclosure involve primitive gut tube cells. Primitive gut tube cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some embodiments, definitive endoderm cells, or clusters including these cells are differentiated to primitive gut tube cells in the bioreactor and/or TFF system. In some embodiments, the primitive gut tube cells are further differentiated, e.g., to PDXl-positive pancreatic progenitor cells, NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation to SC-P cells. One or more of these processes can be performed in the disclosed bioreactors and/or TFF systems, optionally using cell clusters.

In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells in the disclosed bioreactors and/or TFF systems. The primitive gut tube cells can be in clusters. In some embodiments, the method includes contacting definitive endoderm cells with at least one growth factor from the fibroblast growth factor (FGF) family in the bioreactor and/or TFF system, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells, wherein the primitive gut tube cells express at least one marker characteristic of primitive gut tube cells.

Any growth factor from the FGF family capable of inducing definitive endoderm cells to differentiate into primitive gut tube cells (e.g., alone, or in combination with other factors) can be used in the method provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family comprises FGF2. In some embodiments, the at least one growth factor from the FGF family comprises FGF8B. In some embodiments, the at least one growth factor from the FGF family comprises FGF10. In some embodiments, the at least one growth factor from the FGF family comprises FGF21.

In some embodiments, primitive gut tube cells can be obtained by differentiating at least some definitive endoderm cells in a population into primitive gut tube cells in the disclosed bioreactors and/or TFF systems. In some embodiments, the method includes contacting definitive endoderm cells with KGF for a certain period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days, to induce the differentiation of at least some of the definitive endoderm cells into primitive gut tube cells.

In some embodiments, the method comprises differentiating definitive endoderm cells into primitive gut tube cells by contacting definitive endoderm cells with a suitable concentration of the growth factor from the FGF family (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some embodiments, the method comprises use of about 20-80 ng/ml, 30-70 ng/ml, or 40-60 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 50 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells. In some embodiments, the method comprises use of about 100 ng/mL KGF for differentiation of definitive endoderm cells into primitive gut tube cells.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is polyvinyl alcohol. In some cases, the polyvinyl alcohol is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol (PVA) is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 80% hydrolyzed.

PDX1 -positive Pancreatic Progenitor Cells

Embodiments of the disclosure involve PDX1 -positive pancreatic progenitor cells. The PDX-1 positive pancreatic progenitor cells can be in clusters. PDX1 -positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some embodiments, primitive gut tube cells are differentiated to PDX1 - positive pancreatic progenitor cells in the disclosed bioreactors and/or TFF systems. In some embodiments, the PDXl-positive pancreatic progenitor cells are NKX6.1 negative, and can be further differentiated to, e.g., NKX6.1-positive pancreatic progenitor cells, Ngn3-positive endocrine progenitor cells, insulin-positive endocrine cells, followed by induction or maturation to SC-P cells. These cells can be in clusters.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells in the disclosed bioreactors and/or TFF systems. In some embodiments, the method includes contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from TGF-P superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator; vi) at least one protein kinase C activator, and vii) a ROCK inhibitor to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) a growth factor from TGF-P superfamily, iii) at least one growth factor from the FGF family, iv) at least one SHH pathway inhibitor, v) at least one retinoic acid (RA) signaling pathway activator; and vi) at least one protein kinase C activator, to induce the differentiation of at least some of the primitive gut tube cells into PDXl- positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with one or more of i) at least one BMP signaling pathway inhibitor, ii) at least one growth factor from the FGF family, iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid (RA) signaling pathway activator; and v) at least one protein kinase C activator, to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDXl-positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with i) at least one SHH pathway inhibitor, ii) at least one retinoic acid (RA) signaling pathway activator; and iii) at least one protein kinase C activator, wherein the PDX1 -positive pancreatic progenitor cells express PDX1.

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with i) at least one growth factor from the FGF family, and ii) at least one retinoic acid (RA) signaling pathway activator, to induce the differentiation of at least some of the primitive gut tube cells into PDXl-positive pancreatic progenitor cells, wherein the PDX1 -positive pancreatic progenitor cells express PDX1.

Any BMP signaling pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of a growth factor from TGF-P superfamily, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used in the method provided herein. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 pM, about 0.02pM, about 0.05pM, about O.lpM, about 0.2pM, about 0.5 pM, about 0.8 pM, about 1 pM, about 1.2 pM, about 1.5pM, about 1.75pM, about 2 pM, about 2.2 pM, about 2.5pM, about 2.75pM, about 3 pM, about 3.25 pM, about 3.5 pM, about 3.75 pM, about 4 pM, about 4.5 pM, about 5 pM, about 8 pM, about 10 pM, about 15 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of BMP signaling pathway inhibitor (e.g., DMH-1) about 250 nM.

Any growth factor from the TGF-P superfamily capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, a growth factor from the FGF family, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the growth factor from TGF-P family comprises Activin A. In some embodiments, the growth factor from TGF-P family comprises GDF8. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 5 ng/mL, about 7.5 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15 ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL, about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24 ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL, about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, or about 100 ng/mL. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF- P superfamily (e.g., Activin A), such as, about 17-23 ng/ml, about 18-22 ng/ml, or about 19-21 ng/ml. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A) of about 20 ng/ml.

Any growth factor from the FGF family capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, a growth factor from TGF-P superfamily, at least one SHH pathway inhibitor, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 20-80 ng/ml, about 30-70 ng/ml, about 40-60 ng/ml, or about 45-55 ng/ml. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng/ml.

Any SHH pathway inhibitor capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, a growth factor from TGF-P superfamily, at least one retinoic acid signaling pathway activator, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240- 260 nM, or about 245-255 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a SHH pathway inhibitor (e.g., Santl) of about 250 nM.

Any RA signaling pathway activator capable of inducing primitive gut tube cells to differentiate into PDXl-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one protein kinase C activator, and ROCK inhibitor) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about O.lpM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 2 pM.

Any PKC activator capable of inducing primitive gut tube cells to differentiate into PDX1 -positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one BMP signaling pathway inhibitor, at least one growth factor from the FGF family, at least one SHH pathway inhibitor, at least one RA signaling pathway activator, and ROCK inhibitor) can be used. In some embodiments, the PKC activator comprises PdBU. In some embodiments, the PKC activator comprises TPPB. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 pM, 10 pM, about 20 pM, about 50 pM, about 75 pM, about 80 pM, about 100 pM, about 120 pM, about 140 pM, about 150 pM, about 175 pM, about 180 pM, about 200 pM, about 210 pM, about 220 pM, about 240 pM, about 250 pM, about 260 pM, about 280 pM, about 300 pM, about 320 pM, about 340 pM, about 360 pM, about 380 pM, about 400 pM, about 420 pM, about 440 pM, about 460 pM, about 480 pM, about 500 pM, about 520 pM, about 540 pM, about 560 pM, about 580 pM, about 600 pM, about 620 pM, about 640 pM, about 660 pM, about 680 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of 10 nM-1 mM, 10 nM-500 pM, 10 nM-1 pM, 10-800 nM, 100-900 nM, 300-800 nM, 300-600 nM, 400-600 nM, 450-550 nM, or about 500 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting primitive gut tube cells with a concentration of a PKC activator (e.g., PdBU or TPPB) of about 500 nM. In some embodiments, primitive gut tube cells are not treated with a PKC activator (e.g., PDBU).

In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells in the disclosed bioreactors and/or TFF systems. In some embodiments, the method includes contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A, for a suitable period of time, e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A, for about 2 days. In some embodiments, PDXl-positive pancreatic progenitor cells can be obtained by differentiating at least some primitive gut tube cells in a population into PDXl-positive pancreatic progenitor cells, e.g., by contacting primitive gut tube cells with retinoic acid, KGF, Santl, DMH-1, PdBU, thiazovivin, and Activin A for 1 day, followed by contacting the cells with retinoic acid, KGF, Santl, PdBU, thiazovivin, and Activin A for 1 day (in the absence of DMH-1). NKX6.1 -positive Pancreatic Progenitor Cells

Embodiments of the disclosure involve NKX6.1-positive pancreatic progenitor cells. NKX6.1 -positive pancreatic progenitor cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some embodiments, PDX1 -positive, NKX6.1 -negative pancreatic progenitor cells are differentiated to PDX1 -positive, NKX6.1- positive pancreatic progenitor cells. These cells can be in clusters. In some embodiments, the NKX6.1 -positive pancreatic progenitor cells are further differentiated, e.g., to Ngn3-positive endocrine progenitor cells, or insulin-positive endocrine cells, followed by induction or maturation to SC-P cells in the disclosed bioreactors and/or TFF systems.

In some embodiments, a method of producing a NKX6.1-positive pancreatic progenitor cell from a PDX1 -positive pancreatic progenitor cell comprises contacting a population of cells or cell clusters (e.g., under conditions that promote cell clustering and/or promoting cell survival) comprising PDX1 -positive pancreatic progenitor cells with at least two P celldifferentiation factors comprising a) at least one growth factor from the fibroblast growth factor (FGF) family, b) a sonic hedgehog pathway inhibitor, and optionally c) a low concentration of a retinoic acid (RA) signaling pathway activator, to induce the differentiation of at least one PDXl-positive pancreatic progenitor cell in the population into NKX6.1-positive pancreatic progenitor cells, wherein the NKX6.1-positive pancreatic progenitor cells expresses NKX6.1.

In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, to induce the differentiation of at least some of the PDXl-positive pancreatic progenitor cells into PDXl-positive, NKX6.1-positive pancreatic progenitor cells, wherein the PDXl-positive, NKX6.1- positive pancreatic progenitor cells express PDX1 and NKX6.1.

In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF- P superfamily, to induce the differentiation of at least some of the PDXl-positive pancreatic progenitor cells into PDXl-positive, NKX6.1-positive pancreatic progenitor cells. In some embodiments, following 3, 4, or 5 days of contacting the PDXl-positive, NKX6.1-positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-P superfamily; the cells are then contacted with i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, and optionally iii) a RA signaling pathway activator, iv) ROCK inhibitor, and v) at least one growth factor from the TGF-P superfamily, and vi) a PKC activator and optionally vii) a gamma-secretase inhibitor. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells under conditions that promote cell clustering with at least one growth factor from the FGF family. In some embodiments, the growth factor from the FGF family is KGF.

In some embodiments, the disclosure provides for a method in which a first population of cells comprising PDX1 -positive, NKX6.1 -negative cells is cultured in a media comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a RA signaling pathway activator, iv) a ROCK inhibitor, and v) a growth factor from the TGF-P superfamily for a period of about 1, 2, 3, 4 or 5 days (e.g., 2-4, 3-4, or 4-5 days); thereby generating a second population of cells. In some embodiments, the second population of cells is then incubated in a composition comprising any one or combination of: i) at least one growth factor from the FGF family, ii) at least one SHH pathway inhibitor, iii) a RA signaling pathway activator, iv) a ROCK inhibitor, v) a growth factor from the TGF-P superfamily, vi) a PKC activator, vii) a FoxOl inhibitor, and optionally viii) a notch signaling inhibitor for about 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days).

In some embodiments, in the media for culturing the first population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng/ml, about 46- 54 ng/ml, about 47-53 ng/ml, about 48-52 ng/ml, or about 49-51 ng/ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, the RA signaling pathway activator is present at a concentration of about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM, the ROCK inhibitor is present at a concentration of about 2-3 pM, about 2.2-2.8 pM, or about 2.4-2.6 pM, and/or the growth factor from the TGF-P superfamily is present at a concentration of about 2-8 ng/ml, about 3-7 ng/ml or about 4-6 ng/ml.

In some embodiments, in the media for culturing the second population of cells, the growth factor from the FGF family is present at a concentration of about 45-55 ng/ml, about 46- 54 ng/ml, about 47-53 ng/ml, about 48-52 ng/ml, or about 49-51 ng/ml, the SHH pathway inhibitor is present at a concentration of about 200-300 nM, about 220-280 nM, or about 240-260 nM, the RA signaling pathway activator is present at a concentration of about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM, the ROCK inhibitor is present at a concentration of about 2-3 pM, about 2.2-2.8 pM, or about 2.4-2.6 pM, the growth factor from the TGF-P superfamily is present at a concentration of 2 about -8 ng/ml, about 3-7 ng/ml or about 4-6 ng/ml, the PKC activator is present at a concentration of about 0.2-0.8 pM, about 0.3-0.7 pM, or about 0.4-0.6 pM, and the FoxOl inhibitor is present at a concentration of about 0.7- 1.3 pM, about 0.8- 1.2 pM, or about 0.9- 1.1 pM, and optionally the notch signaling inhibitor is present at a concentration of about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM.

In some embodiments, the PDXl-positive pancreatic progenitor cells are produced from a population of pluripotent cells. In some embodiments, the PDXl-positive pancreatic progenitor cells are produced from a population of iPS cells. In some embodiments, the PDXl- positive pancreatic progenitor cells are produced from a population of ESC cells. In some embodiments, the PDXl-positive pancreatic progenitor cells are produced from a population of definitive endoderm cells. In some embodiments, the PDXl-positive pancreatic progenitor cells are produced from a population of primitive gut tube cells.

Any growth factor from the FGF family capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one SHH pathway inhibitor, a ROCK inhibitor, a growth factor from the TGF-P superfamily, and at least one retinoic acid signaling pathway activator) can be used in the method provided herein. In some embodiments, the at least one growth factor from the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the at least one growth factor from the FGF family is selected from the group consisting of FGF8B, FGF 10, and FGF21. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from PGP family (e.g., KGF), such as, about 20-80 ng/ml, about 30-70 ng/ml, about 40-60 ng/ml, or about 45-55 ng/ml. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a growth factor from FGF family (e.g., KGF) of about 50 ng/ml.

Any SHH pathway inhibitor capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, a retinoic acid signaling pathway activator, ROCK inhibitor, and at least one growth factor from the TGF-P superfamily) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting PDX1- positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl) of about 250 nM.

Any RA signaling pathway activator capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, ROCK inhibitor, and at least one growth factor from the TGF-P superfamily) can be used. In some embodiments, the RA signaling pathway activator comprises retinoic acid. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about O.lpM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 70-130 nM, about 80- 120 nM, about 90-110 nM, or about 95-105 nM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid) of about 100 nM. Any ROCK inhibitor capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and at least one growth factor from the TGF-P superfamily) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y- 27632, Fasudil/HA1077, or 14-1152. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y- 27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y- 27632 or Thiazovivin) of about 2.5 pM.

Any activator from the TGF-P superfamily capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one SHH pathway inhibitor, a RA signaling pathway activator, and ROCK inhibitor) can be used. In some embodiments, the activator from the TGF-P superfamily comprises Activin A or GDF8. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about 0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8 ng/mL, about 1 ng/mL, about 1.2 ng/mL, about 1.4 ng/mL, about 1.6 ng/mL, about 1.8 ng/mL, about 2 ng/mL, about 2.2 ng/mL, about 2.4 ng/mL, about 2.6 ng/mL, about 2.8 ng/mL, about 3 ng/mL, about 3.2 ng/mL, about 3.4 ng/mL, about 3.6 ng/mL, about 3.8 ng/mL, about 4 ng/mL, about 4.2 ng/mL, about 4.4 ng/mL, about 4.6 ng/mL, about 4.8 ng/mL, about 5 ng/mL, about 5.2 ng/mL, about 5.4 ng/mL, about 5.6 ng/mL, about 5.8 ng/mL, about 6 ng/mL, about 6.2 ng/mL, about 6.4 ng/mL, about 6.6 ng/mL, about 6.8 ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL, about 20 ng/mL, about 30 ng/mL, or about 50 ng/mL. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 2-8 ng/ml, about 3-7 ng/ml, about 4-6 ng/ml, or about 4.5-5.5 ng/ml. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a growth factor from TGF-P superfamily (e.g., Activin A), such as, about 5 ng/mL.

Any FoxOl inhibitor capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, PKC activator, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the FoxOl inhibitor is AS 1842856. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, about or 0.9- 1.1 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a FoxOl inhibitor (e.g., AS1842856), such as, about 1 pM.

Any PKC activator capable of inducing PDXl-positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, FoxOl inhibitor, and Notch signaling inhibitor) can be used in the method provided herein. In some embodiments, the PKC activator is PDBU. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.2-0.8 pM, about 0.3-0.7 pM, about 0.4-0.6 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a PKC activator (e.g., PDBU), such as, about 0.5 pM.

Any Notch signaling inhibitor capable of inducing PDX1 -positive pancreatic progenitor cells to differentiate into NKX6.1-positive pancreatic progenitor cells (e.g., alone, or with any combination of at least one growth factor from the FGF family, at least one retinoic acid signaling pathway activator, ROCK inhibitor, at least one growth factor from the TGF-P superfamily, FoxOl inhibitor, and PKC activator) can be used in the method provided herein. In some embodiments, the Notch signaling inhibitor is XXI. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21 pM, about 0.22 pM, about 0.23 pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9- 2.1 pM. In some examples, the method comprises contacting PDXl-positive pancreatic progenitor cells with a concentration of a Notch signaling inhibitor (e.g., XXI), such as, about 2 pM.

In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Santl, and RA, for a period of 5 days or 6 days. In some embodiments, the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDXl-positive pancreatic progenitor cells under conditions that promote cell clustering with KGF, Santl, RA, thiazovivin, and Activin A, for a period of 5 or 6 days. In some embodiments, the PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 5 days. In some embodiments, the PDX1 -positive, NKX6.1- positive pancreatic progenitor cells are obtained by contacting PDX1 -positive pancreatic progenitor cells under conditions that promote cell clustering with KGF for a period of 6 days. In some embodiments, the PDX1 -positive, NKX6.1-positive pancreatic progenitor cells are obtained by: a) contacting PDXl-positive pancreatic progenitor cells with KGF, Santl, RA, thiazovivin, and Activin A, for a period of 3, 4 or 5 days (e.g., 4 days), followed by; b) contacting the cells of a) with PDBU, XXI, KGF, Santl, RA, thiazovivin, and Activin A and optionally AS1842856 for a period of 1, 2 or 3 days (e.g., 2 days).

Insulin-positive Endocrine Cells

Embodiments of the disclosure involve insulin-positive endocrine cells (e.g., NKX6.1- positive, ISL1 -positive cells, or P-like cells) and additional methods of generating insulinpositive endocrine cells in the disclosed bioreactors and/or TFF systems. Insulin-positive endocrine cells of use herein can be derived from any source or generated in accordance with any suitable protocol. In some embodiments, NKX6.1 -positive pancreatic progenitor cells, or clusters containing these cells, are differentiated to insulin-positive endocrine cells (e.g., NKX6.1-positive, ISLl-positive cells, or P-like cells) in the disclosed bioreactors and/or TFF systems. In some embodiments, the insulin-positive endocrine cells are further differentiated, e.g., by induction or maturation to SC-P cells in the disclosed bioreactors and/or TFF systems.

In some embodiments, a method of producing an insulin-positive endocrine cell from an NKX6.1-positive pancreatic progenitor cell comprises contacting a population of cells (e.g., under conditions that promote cell clustering) comprising NKX6-l-positive pancreatic progenitor cells with a) a TGF-P signaling pathway inhibitor, b) a thyroid hormone signaling pathway activator, , c) a BMP pathway inhibitor, and/or d) a protein kinase inhibitor to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine ceil expresses insulin. In some embodiments, insulin-positive endocrine cells express PDX1, NKX6.1, ISL1, NKX2.2, Mafb, glis3, Suri, Kir6.2, Znt8, SLC2A1, SLC2A3 and/or insulin.

Any TGF-P signaling pathway inhibitor capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used. In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about

3.5 pM, about 4 pM, about 4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 10.5 pM, about 11 pM, about 11.5 pM, about 12 pM, about 12.5 pM, about 13 pM, about

13.5 pM, about 14 pM, about 14.5 pM, about 15 pM, about 15.5 pM, about 16 pM, about 16.5 pM, about 17 pM, about 17.5 pM, about 18 pM, about 18.5pM, about 19 pM, about 19.5 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 pM, about 8-12 pM, about 9-11 pM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 pM.

Any thyroid hormone signaling pathway activator capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulinpositive endocrine cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a TGF-P signaling pathway inhibitor) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC- 1), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, or about 0.9-1.1 pM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 pM. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with at least one additional factor in a disclosed bioreactor and/or TFF system. In some embodiments, the method comprises contacting the PDXl-positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a y-secretase inhibitor, iii) at least one growth factor from the epidermal growth factor (EGF) family, iv) a TGF-P signaling pathway inhibitor, or vii) a thyroid hormone signaling pathway activator. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with at least one additional factor. In some embodiments, the method comprises contacting the PDXl-positive NKX6.1 -positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a Wnt signaling pathway inhibitor, or ix) a PKC activator.

In some embodiments, the method comprises contacting the PDXl-positive NKX6.1- positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) a protein kinase inhibitor, or ix) a ROCK inhibitor.

In some embodiments, the method comprises contacting the PDXl-positive NKX6.1- positive pancreatic progenitor cells with at least one of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, or x) a ROCK inhibitor. In some embodiments, the method comprises contacting the PDXl-positive, NKX6.1 -positive pancreatic progenitor cells in a culture with a i) a SHH pathway inhibitor, ii) a RA signaling pathway activator, iii) a y-secretase inhibitor, iv) at least one growth factor from the epidermal growth factor (EGF) family, v) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, vi) a TGF-P signaling pathway inhibitor, vii) a thyroid hormone signaling pathway activator, viii) an epigenetic modifying compound, ix) a protein kinase inhibitor, x) a ROCK inhibitor, xi) a PKC activator and xii) a Wnt signaling pathway inhibitor for 1, 2, or 3 days (e.g., 1-2, 1-3, or 2-3 days), and then contacting the cells in the culture with i) a y-secretase inhibitor, ii) at least one growth factor from the epidermal growth factor (EGF) family, iii) at least one bone morphogenetic protein (BMP) signaling pathway inhibitor, iv) a TGF-P signaling pathway inhibitor, v) a thyroid hormone signaling pathway activator, vi) an epigenetic modifying compound, vii) a protein kinase inhibitor, and viii) a ROCK inhibitor for a period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 1-7, 1- 5, 1-3, 3-7, 3-5, 5-7, or 4-6 days) in the absence of a SHH pathway inhibitor, a RA signaling pathway activator, a Wnt signaling pathway inhibitor, PKC activator, and/or growth factor from the epidermal growth factor (EGF) family.

In some embodiments, in the method of generating the insulin-positive endocrine cells from the PDXl-positive NKX6.1-po stive pancreatic progenitor cells, some of the differentiation factors are present only for the first 1, 2, 3, 4, or 5 days during the differentiation step. In some embodiments, some of the differentiation factors, such as the SHH pathway inhibitor, the RA signaling pathway activator, the PKC activator, and the at least one growth factor from the EGF family are removed from the culture medium after the first 1, 2, or 3 days of incubation.

Any y-secretase inhibitor that is capable of inducing the differentiation of NKX6.1- positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the y-secretase inhibitor comprises XXI. In some embodiments, the y-secretase inhibitor comprises DAPT. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a y-secretase inhibitor (e.g., XXI), such as, about 0.01 pM, about 0.02 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 2.9 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.2 pM, about 5.4 pM, about 5.6 pM, about 5.8 pM, about 6 pM, about 6.2 pM, about 6.4 pM, about 6.6 pM, about 6.8 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 20 pM, about 30 pM, or about 50 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a y-secretase inhibitor (e.g., XXI), such as, about 1.7-2.3 pM, about 1.8- 2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting NKX6.1- positive pancreatic progenitor cells with a concentration of a y-secretase inhibitor (e.g., XXI), such as about 2 pM. Any growth factor from the EGF family capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGE-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the at least one growth factor from the EGF family comprises betacellulin. In some embodiments, at least one growth factor from the EGF family comprises EGF. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGF family (e.g., betacellulin), such as, about 1 ng/mL, about 2 ng/mL, about 4 ng/mL, about 6 ng/mL, about 8 ng/mL, about 10 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18 ng/mL, about 20 ng/mL, about 22 ng/mL, about 24 ng/mL, about 26 ng/mL, about 28 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGE family (e.g., betacellulin), such as, about 17-23 ng/ml, about 18-22 ng/ml, or about 19-21 ng/ml. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a growth factor from EGE family (e.g., betacellulin), such as, about 20 ng/ml.

Any RA signaling pathway activator capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGE-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the RA signaling pathway activator comprises RA. In some examples, the method comprises contacting NKX6.1- positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 0.02 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1.4 pM, about 1.5 pM, about 1.6 pM, about 1.7 pM, about 1.8 pM, about 1.9 pM, about 2 pM, about 2.1 pM, about 2.2 pM, about 2.3 pM, about 2.4 pM, about 2.5 pM, about 2.6 pM, about 2.7 pM, about 2.8 pM, about 3 pM, about 3.2 pM, about 3.4 pM, about 3.6 pM, about 3.8 pM, about 4 pM, about 4.2 pM, about 4.4 pM, about 4.6 pM, about 4.8 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 12 pM, about 14 pM, about 15 pM, about 16 pM, about 18 pM, about 20 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 20-80 nM, about 30-70 nM, or about 40-60 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of an RA signaling pathway activator (e.g., retinoic acid), such as, about 50 nM.

Any SHH pathway inhibitor capable of inducing the differentiation of NKX6.1-positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the method provided herein. In some embodiments, the SHH pathway inhibitor comprises Santl. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 0.001 pM, about 0.002 pM, about 0.005 pM, about 0.01 pM, about 0.02 pM, about 0.03pM, about 0.05pM, about 0.08 pM, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.2 IpM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 220-280 nM, about 230-270 nM, about 240-260 nM, or about 245-255 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a SHH pathway inhibitor (e.g., Santl), such as, about 250 nM.

Any BMP signaling pathway inhibitor capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells to differentiate into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

Any ROCK inhibitor that is capable of inducing the differentiation of NKX6.1-positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-l 152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 pM.

Any epigenetic modifying compound that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HD AC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HD AC inhibitor, e.g., KD5170. In some examples, the method comprises contacting PDX1- positive, NKX6.1-positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 0.01 pM, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 |jM, about 3 |aM, about 4 |jM, about 5 |aM, about 6 |aM, about 7 |jM, about 7.5 |aM, about 8 |aM, about 9 |aM, about 10 |aM, about 15 |aM, about 20 |aM, about 25 |jM, about 30 |aM, about 35 |aM, about 40 |aM, about 50 |aM, or about 100 |aM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 70-130 nM, about 80-120 nM, or about 90-110 nM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 100 nM.

Any Wnt signaling pathway inhibitor that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the Wnt signaling pathway inhibitor comprises a tankyrase inhibitor. In some embodiments, the tankyrase inhibitor is NVP-TNKS656. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about O.lpM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 pM, about 0.6 pM, about 0.65 pM, about 0.7 pM, about 0.75 pM, about 0.8 pM, about 0.85 pM, about 0.9 pM, about 0.95 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 4.5 pM, or about 5 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 1.7-2.3 pM, about 1.8-2.2 pM, or about 1.9-2.1 pM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1-positive pancreatic progenitor cells with a concentration of a Wnt signaling pathway inhibitor (e.g., a tankyrase inhibitor such as NVP-TNKS656), such as, about 2 pM.

Any PKC activator that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used. In some embodiments, the PKC activator is TPB or PDBU. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 0.01 pM, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.55 |aM, about 0.6 |aM, about 0.65 |aM, about 0.7 |jM, about 0.75 |aM, about 0.8 |jM, about 0.85 |aM, about 0.9 |aM, about 0.95 |aM, about 1 |jM, about 2 |jM, about 3 |jM, about 4 |jM, about 5 |aM, about 6 |aM, about 7 |jM, about 7.5 |aM, about 8 |aM, about 9 |aM, about 10 |jM, about 15 |aM, or about 20 |aM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1-positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 450-550 mM, about 475-525 nM, about 490-510 nM, or about 495-505 nM. In some examples, the method comprises contacting PDX1 -positive, NKX6.1 -positive pancreatic progenitor cells with a concentration of a PKC activator (TPB or PDBU), such as, about 500 nM.

In some embodiments, the population of cells is optionally contacted with a protein kinase inhibitor. In some embodiments, the population of cells is not contacted with the protein kinase inhibitor. In some embodiments, the population of cells is contacted with the protein kinase inhibitor. Any protein kinase inhibitor that is capable of inducing the differentiation of NKX6.1 -positive pancreatic progenitor cells in a population into insulin-positive endocrine cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator). In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 pM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 pM, about 4.9 nM, or about 5 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 1-5 nM, about 2-4 nM, or about 2.5- 3.5 nM. In some examples, the method comprises contacting NKX6.1 -positive pancreatic progenitor cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 3 nM.

In some embodiments, the cells are further contacted with a water-soluble synthetic polymer. In some embodiments, the water-soluble synthetic polymer is PVA. In some cases, the PVA is at least 78% hydrolyzed, e.g., 79-81% hydrolyzed, 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the PVA is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. In some embodiments, the PVA is 89% hydrolyzed.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Santl, and betacellulin, PDBU, and NVP-TNKS656 for a period of 7 days, to induce the differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell in the disclosed bioreactors and/or TFF systems, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3 or GC-1, RA, Santl, betacellulin, and LDN193189 for a period of 7 days, to induce the differentiation of at least one NKX6.1 -positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. In some embodiments, one or more differentiation factors are added in a portion of the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6 days of the period of time for Stage 5. In one example, the cells are contacted with SHH signaling pathway inhibitor the PKC activator, the retinoic acid, and/or the Wnt signaling pathway inhibitor for only the first 2, 3, 4, or 5 days during Stage 5, after which the SHH signaling pathway inhibitor, the PKC activator, the retinoic acid, and/or the Wnt signaling pathway inhibitor are not included in or removed from the culture medium. In another example, the cells are contacted with BMP signaling pathway inhibitor for only the first 1, 2, or 3 days during Stage 5, after which the BMP signaling pathway inhibitor is removed from the culture medium.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) with one or more metabolites in the disclosed bioreactor and/or TFF systems. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive pancreatic progenitor cells) with one or more of an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, and/or glutamine. Examples of metabolites include glutamine, taurine, acetate, beta-hydroxybutyrate, biotin, and formate.

In some embodiments, a composition (e.g., medium), bioreactor and/or TFF system of the disclosure comprises an acetyl CoA-related metabolite. Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl- CoA, and P-hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 pM, about 10 pM, about 100 pM, about 500 pM, about 800 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8- 2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 50- 1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 160 nM.

In some embodiments, a composition (e.g., medium), bioreactors and/or TFF system of the disclosure comprises one or more vitamins. Exemplary vitamins include, but are not limited to biotin, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the vitamin is biotin present at a concentration of about 800 nM. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM. In some embodiments, a composition (e.g., medium), bioreactor and/or TFF system of the disclosure comprises a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P-Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC- 1, HD AC-2, HD AC-3, HD AC-4, HD AC-5, HD AC-6, HD AC-7, HD AC-8, HD AC-9, HD AC- 10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI- 24781, CRA-024781), RGFP966, AR-42 (HDAC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD- 123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC-907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VPA), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Sulforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, Splitomicin, HPOB, LMK-235, Biphenyl-4- sulfonyl chloride (p-Phenylbenzenesulfonyl, 4- Phenylbenzenesulfonyl, p-Biphenylsulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, Raddeanin A, Tinostamustine(EDO-SlOl), Domatinostat (4SC-202), and BG45. In some embodiments, the HDACi is P-Hydroxybutyrate. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the HDACi is P-Hydroxybutyrate present at a concentration of about 200 nM. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

In some embodiments, a composition (e.g., medium), bioreactor and/or TFF system of the disclosure comprises a redox homeostasis regulator. Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx- I, catalase, and co-enzyme Q10. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 110 pM, about 150 pM, or about 200 pM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90 pM. In some embodiments, the redox homeostasis regulator intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 30 pM to 1 mM, 40 pM to 1 mM, 50 pM to 1 mM, 60 pM to 1 mM, 70 pM to 1 mM, 80 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 30 pM to 250 pM, 40 pM to 250 pM, 50 pM to 250 pM, 60 pM to 250 pM, 70 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 40 pM to 100 pM, 50 pM to 100 pM, 60 pM to 100 pM, 70 pM to 100 pM, or 80 pM to 100 pM.

In some embodiments, a composition (e.g., medium), bioreactor and/or TFF system of the disclosure comprises a one carbon metabolism pathway intermediate. Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10-formylTHF; 5,10-meTHF; 5,10-meTHF; and 10-formylTHF. In some embodiments, the one carbon metabolism pathway intermediate is formate present at a concentration of about 50 pM. In some embodiments, the one carbon metabolism pathway intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 100 nM to 60 pM, 500 nM to 60 pM, 1 pM to 60 pM, 10 pM to 60 pM, 20 pM to 60 pM, 30 pM to 60 pM, 40 pM to 60 pM, or 45 pM to 55 pM.

In some embodiments, a composition (e.g., medium), bioreactors and/or TFF system of the disclosure comprises glutamine. Thus in some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability, such as a free glutamine form, such as a non-dipeptide form, a non-alanine-glutamine dipeptide form (e.g., a non-alanyl-1- glutamine form), a non-glycine-glutamine dipeptide form (e.g., a non-glycyl-l-glutamine form), a form that in which glutamine is not conjugated to another amino acid or stabilizing moiety, a monomeric form, a free form, or a combination thereof. In some embodiments, glutamine is provided as a protein hydrolysate. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of from 0.5-20 mM, 0.5-10 mM, 0.5-5 mM, 1-5 mM, 2-5 mM, or 1 mM to 10 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 3.8-4.2 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of 1-10, 1- 7, 1-8, 1-6, 1-5, 1-4, 2-10, 2-7, 2-8, 2-6, 2-5, 2-4, 3-10, 3-7, 3-8, 3-6, 3-5, 3-4, 3.5-4.5, 3.8-4.2, or 3.9-4.1 mM. In some embodiments, glutamine is present or is added to a composition of the disclosure at a concentration of about 4 mM. In some embodiments, at least 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM of the glutamine is not in a dipeptide form. In some embodiments, at least 500 pM, at least 750 pM, at least 1 mM, at least 1.5 mM, at least 2 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 5 mM, at least 5.5 mM, at least 6 mM, at least 6.5 mM, at least 7 mM, at least 7.5 mM, at least 8 mM, at least 8.5 mM, at least 9 mM, at least 9.5 mM, or at least 10 mM of the glutamine is in a free form.

In some embodiments, the method comprises culturing the population of cells (e.g., NKX6.1 -positive pancreatic progenitor cells) in a medium in the bioreactor and/or TFF system, to induce the differentiation of at least one NKX6.1-positive pancreatic progenitor cell in the population into an insulin-positive endocrine cell, wherein the insulin-positive endocrine cell expresses insulin. The cells can be in clusters.

Embodiments of the disclosure involve treatment of cell population comprising PDX1- positive, NKX6.1-positive pancreatic progenitor cells with PKC activator and/or Wnt signaling pathway inhibitor, which can lead to increase in percentage of pancreatic a cells, increase in percentage of pancreatic 5 cells, increase in percentage of pancreatic P cells, reduction in percentage of EC cells, or any combination thereof, in the cell population of pancreatic endocrine cells generated according to the methods disclosed herein.

In some embodiments, the method comprises contacting a population of cells comprising PDX1 -positive, NKX6.1-positive pancreatic progenitor cells with a first composition comprising a F0X01 inhibitor, notch signaling inhibitor, a PKC activator, a ROCK inhibitor, a growth factor from TGFP superfamily, a growth factor from FGF family, a RA signaling pathway activator, and a SHH pathway inhibitor, for one to two days, thereby obtaining a first transformation cell population comprising PDX1 -positive, NKX6.1-positive pancreatic progenitor cells; and contacting the first transformation cell population comprising PDX1- positive, NKX6.1-positive pancreatic progenitor cells with a second composition comprising the PKC activator, notch signaling inhibitor, a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, BMP pathway inhibitor, ROCK inhibitor, retinoic acid, and EGF-family growth factor, Wnt signaling pathway inhibitor, and/or an epigenetic modifying compound, for one to two days, thereby obtaining a second transformation cell population comprising NKX6.1- positive, ISL1 -positive endocrine cells.

Pancreatic f> Cells

Embodiments of the disclosure involve generating pancreatic P cells (e.g., non-native pancreatic P cells/SC-P cells) and additional methods of generating them in the disclosed bioreactor and/or TFF systems. In some embodiments, resemble endogenous mature P cells in form and function, but nevertheless are distinct from native P cells.

In some embodiments, the insulin-positive pancreatic endocrine cells generated using the method provided herein can form a cell cluster, alone or together with other types of cells, e.g., precursors thereof, e.g., stem cell, definitive endoderm cells, primitive gut tube cell, PDX1- positive pancreatic progenitor cells, or NKX6.1-positive pancreatic progenitor cells.

In some embodiments, any of the cells or populations of cells disclosed herein are in a cell cluster. In some embodiments, the disclosure provides for a composition comprising one or more cell clusters. In some embodiments, the composition comprises 500-20000, 500-15000, 500-10000, 500-5000, 500-2000, 500-1000, 1000-20000, 1000-15000, 1000-10000, 1000-5000, 1000-2000, 2000-20000, 2000-15000, 2000-10000, 2000-5000, 5000-20000, 5000-15000, 5000- 10000, 10000-20000, 10000-15000, 15000-20000, or 3000-9000 cell clusters. In some embodiments, provided herein are cell clusters that resemble the functions and characteristics of endogenous pancreatic islets. Such cell clusters can mimic the function of endogenous pancreatic islets in regulating metabolism, e.g., glucose metabolism in a subject. In some embodiments, a composition or cell population of the present disclosure comprises NKX6.1-positive, ISLl-positive cells that express lower levels of MAFA than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISLl- positive cells that express higher levels of MAFB than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISLl-positive cells that express higher levels of SIX2, HOPX, IAPP and/or UCN3 than NKX6.1 -positive, ISLl-positive cells from the pancreas of a healthy control adult subject.

In some embodiments, a composition or cell population of the present disclosure comprises NKX6.1-positive, ISLl-positive cells that do not express MAFA. In some embodiments, the composition or cell population comprises NKX6.1-positive, ISLl-positive cells that express MAFB.

In some embodiments, the cell population comprising the insulin-positive endocrine cells can be directly induced to mature into SC-P cells without addition of any exogenous differentiation factors (such as inhibitor of TGF-P signaling pathway, thyroid hormone signaling pathway activator, PKC activator, growth factors from TGF-P superfamily, FGF family, or EGF family, SHH signaling pathway inhibitor, y-secretase inhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor). In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISLl-positive endocrine cells with a serum albumin protein, a TGF-P signaling pathway inhibitor, a SHH pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and/or an epigenetic modifying compound. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISLl-positive endocrine cells with human serum albumin protein. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISLl-positive endocrine cells with a PKC activator.

In some embodiments, the cell population comprising the insulin-positive endocrine cells can be induced to mature into SC-P cells by contacting the insulin-positive endocrine cells with differentiation factors. The differentiation factors can comprise at least one inhibitor of TGF-P signaling pathway and thyroid hormone signaling pathway activator as described herein. In some embodiments, SC-P cells can be obtained by contacting a population of cells comprising insulin-positive endocrine cells with Alk5i and T3 or GC-1 in the disclosed bioreactors and/or TFF systems. In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISLl-positive endocrine cells with (i) a TGF-P signaling pathway inhibitor, (ii) a thyroid hormone signaling pathway activator, (iii) an epigenetic modifying compound, (iv) a BMP signaling pathway inhibitor, (v) a ROCK inhibitor, and/or (vi) a protein kinase inhibitor (e.g., staurosporine) in the disclosed bioreactors and/or TFF systems.

In some embodiments, the method provided herein comprises contacting a cell population comprising NKX6.1-positive, ISLl-positive endocrine cells with (i) a growth factor from the FGF family, (ii) a TGF-P signaling pathway inhibitor, (iii) a thyroid hormone signaling pathway activator, (iv) an epigenetic modifying compound, (v) a protein kinase inhibitor, (vi) a ROCK inhibitor, (vii) a BMP signaling pathway inhibitor, and (viii) a lipase inhibitor for about one two five days in the disclosed bioreactors and/or TFF systems. In some embodiments, the contacting is for about three days.

Any TGF-P signaling pathway inhibitor capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a thyroid hormone signaling pathway activator) can be used in the disclosed methods and bioreactors and/or TFF systems. In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises Alk5 inhibitor II. In some examples, the method comprises contacting insulin-positive endocrine cells in the disclosed bioreactors and/or TFF systems with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 0.1 pM, about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about

4.5 pM, about 5 pM, about 5.5 pM, about 6 pM, about 6.5 pM, about 7 pM, about 7.5 pM, about 8 pM, about 8.5 pM, about 9 pM, about 9.5 pM, about 10 pM, about 10.5 pM, about 11 pM, about 11.5 pM, about 12 pM, about 12.5 pM, about 13 pM, about 13.5 pM, about 14 pM, about 14.5 pM, about 15 pM, about 15.5 pM, about 16 pM, about 16.5 pM, about 17 pM, about

17.5 pM, about 18 pM, about 18.5pM, about 19 pM, about 19.5 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, or about 50 pM. In some examples, the method comprises contacting insulin-positive endocrine cells in the disclosed bioreactors and/or TFF systems with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 7-13 pM, about 8-12 pM , or about 9-11 pM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a TGF-P signaling pathway inhibitor (e.g., Alk5 inhibitor such as Alk5 inhibitor II), such as, about 10 pM. Any thyroid hormone signaling pathway activator capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with other P cell-differentiation factors, e.g., a TGF-P signaling pathway inhibitor) can be used. In some embodiments, the thyroid hormone signaling pathway activator comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator comprises GC-1. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about O.lpM, about 0.12 pM, about 0.13 pM, about 0.14 pM, about 0.15 pM, about 0.16 pM, about 0.17 pM, about 0.18 pM, about 0.19 pM, about 0.2 pM, about 0.21pM, about 0.22pM, about 0.23pM, about 0.24 pM, about 0.25 pM, about 0.26 pM, about 0.27 pM, about 0.28 pM, about 0.29 pM, about 0.3 pM, about 0.31 pM, about 0.32 pM, about 0.33 pM, about 0.34 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, about 0.5 pM, about 0.6 pM, about 0.8 pM, about 1 pM, about 2 pM, or about 5 pM. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 0.7-1.3 pM, about 0.8-1.2 pM, or about 0.9-1.1 pM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of thyroid hormone signaling pathway activator (e.g., GC-1), such as, about 1 pM.

Any BMP signaling pathway inhibitor capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the disclosed bioreactors and/or TFF systems. In some embodiments, the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In some examples, the method comprises contacting insulin-positive endocrine cells in the disclosed bioreactors and/or TFF systems with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about IpM. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 70-130 nM, about 80-120 nM, about 90-110 nM. In some examples, the method comprises contacting NKX6.1-positive pancreatic progenitor cells with a concentration of BMP signaling pathway inhibitor (e.g., LDN1931189), such as, about 100 nM.

Any ROCK inhibitor that is capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the disclosed bioreactors and/or TFF systems. In some embodiments, the ROCK inhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-1152. In some embodiments, the ROCK inhibitor comprises Y-27632. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 11 pM, about 12 pM, about 13 pM, about 14 pM, about 15 pM, about 16 pM, about 17 pM, about 18 pM, about 19 pM, about 20 pM, about 21 pM, about 22 pM, about 23 pM, about 24 pM, about 25 pM, about 26 pM, about 27 pM, about 28 pM, about 29 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.2-2.8 pM, about 2.3-2.7 pM, or about 2.4-2.6 pM. In some embodiments, the ROCK inhibitor comprises Thiazovivin. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of a ROCK inhibitor (e.g., Y-27632 or Thiazovivin), such as, about 2.5 pM.

Any epigenetic modifying compound that is capable of inducing the differentiation of insulin-positive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the disclosed bioreactors and/or TFF systems. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor or a HD AC inhibitor. In some embodiments, the epigenetic modifying compound comprises a histone methyltransferase inhibitor, e.g., DZNep. In some embodiments, the epigenetic modifying compound comprises a HD AC inhibitor, e.g., KD5170. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 0.01 M, about 0.025 pM, about 0.05 pM, about 0.075 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.5 pM, about 0.75 pM, about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 7.5 pM, about 8 pM, about 9 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 50 pM, or about 100 pM. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 70-130 nM, about 80-120 nM, or about 90-110 nM. In some examples, the method comprises contacting insulin-positive endocrine cells to mature into SC-P cells with a concentration of an epigenetic modifying compound (e.g., DZNep or KD5170), such as, about 100 nM.

Any protein kinase inhibitor that is capable of inducing the differentiation insulinpositive endocrine cells to mature into SC-P cells (e.g., alone, or in combination with any of a TGF-P signaling pathway inhibitor and/or a thyroid hormone signaling pathway activator) can be used in the disclosed bioreactors and/or TFF systems. In some embodiments, the protein kinase inhibitor comprises staurosporine. In some examples, the method comprises contacting insulinpositive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2.0 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 pM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, about 3.5 nM, about 3.6 nM, about 3.7 nM, about 3.8 nM, about 3.9 nM, about 4.0 nM, about 4.1 nM, about 4.2 nM, about 4.3 nM, about 4.4 nM, about 4.5 nM, about 4.6 nM, about 4.7 nM, about 4.8 pM, about 4.9 nM, or about 5 nM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 1-5 nM, about 2-4 nM, or about 2.5-3.5 nM. In some examples, the method comprises contacting insulin-positive endocrine cells with a concentration of a protein kinase inhibitor (e.g., staurosporine), such as, about 3 nM.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells) with one or more metabolites in the disclosed bioreactors and/or TFF systems. In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells) with one or more of an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine. Examples of metabolites include taurine, acetate, betahydroxybutyrate, biotin, carnitine, glutamate, and formate.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF systems of the disclosure comprises an acetyl CoA-related metabolite. Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl- CoA, and P-hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 10 nM, about 50 nM, about 80 nM, about 100 nM, about 120 nM, about 140 nM, about 150 nM, about 200 nM, about 300 nM, about 500 nM, about 800 nM, about 1 pM, about 10 pM, about 100 pM, about 500 pM, about 800 pM, about 900 pM, about 1 mM, about 2 mM, about 3 mM, about 5 mM, or about 10 mM. In some embodiments, the acetyl CoA-related metabolite is present in or is added to a composition of the disclosure at a concentration of about 0.01-50 mM, 0.1-50 mM, 0.5-50 mM, 0.01-20 mM, 0.1-20 mM, 0.5-20 mM, 0.01-10 mM, 0.1-10 mM, 0.5-10 mM, 0.8-25 mM, 0.8-10 mM, 0.8-5 mM, 0.8- 2 mM, 0.8-1.5 mM, 0.8-1.2 mM, 0.9-1.1 mM, or 0.95-1.05 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 1 mM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 50- 1000 nM, 50-800 nM, 50-500 nM, 50-300 nM, 50-250 nM, 100-200 nM, or 125-175 nM. In some embodiments, the acetyl CoA-related metabolite is acetate present at a concentration of about 160 nM.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises one or more vitamins. Exemplary vitamins include, but are not limited to biotin, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the vitamin is biotin present at a concentration of about 800 nM. In some embodiments, the vitamin is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM. In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises a histone deacetylase inhibitor (HDACi). Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P-Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC- 1, HD AC-2, HD AC-3, HD AC-4, HD AC-5, HD AC-6, HD AC-7, HD AC-8, HD AC-9, HD AC- 10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS-275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI-34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI- 24781, CRA-024781), RGFP966, AR-42 (HDAC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD- 123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC-907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VPA), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Sulforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, Splitomicin, HPOB, LMK-235, Biphenyl-4- sulfonyl chloride (p-Phenylbenzenesulfonyl, 4- Phenylbenzenesulfonyl, p-Biphenylsulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, Raddeanin A, Tinostamustine(EDO-SlOl), Domatinostat (4SC-202), and BG45. In some embodiments, the HDACi is P-Hydroxybutyrate. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 300 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 pM, about 1.5 pM, about 3 pM, about 5 pM, about 10 pM, or about 100 pM. In some embodiments, the HDACi is P-Hydroxybutyrate present at a concentration of about 200 nM. In some embodiments, the HDACi is present in or is added to a composition of the disclosure at a concentration of about 1 nM to 500 pM, 1 nM to 100 pM, 1 nM to 10 pM, 1 nM to 1 pM, 1 nM to 800 nM, 1 nM to 600 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 25 nM to 500 pM, 25 nM to 100 pM, 25 nM to 10 pM, 25 nM to 1 pM, 25 nM to 800 nM, 25 nM to 600 nM, 25 nM to 400 nM, 25 nM to 300 nM, 25 nM to 200 nM, 50 nM to 500 pM, 50 nM to 100 pM, 50 nM to 10 pM, 50 nM to 1 pM, 50 nM to 800 nM, 50 nM to 600 nM, 50 nM to 400 nM, 50 nM to 300 nM, 50 nM to 200 nM, 100 nM to 500 pM, 100 nM to 100 pM, 100 nM to 10 pM, 100 nM to 1 pM, 100 nM to 800 nM, 100 nM to 600 nM, 100 nM to 400 nM, 100 nM to 300 nM, or 100 nM to 200 nM.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises a redox homeostasis regulator. Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx- I, catalase, and co-enzyme Q10. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 110 pM, about 150 pM, or about 200 pM. In some embodiments, the redox homeostasis regulator is taurine. In some embodiments, the redox homeostasis regulator is taurine present at a concentration of about 90 pM. In some embodiments, the redox homeostasis regulator intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 30 pM to 1 mM, 40 pM to 1 mM, 50 pM to 1 mM, 60 pM to 1 mM, 70 pM to 1 mM, 80 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 30 pM to 250 pM, 40 pM to 250 pM, 50 pM to 250 pM, 60 pM to 250 pM, 70 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 40 pM to 100 pM, 50 pM to 100 pM, 60 pM to 100 pM, 70 pM to 100 pM, or 80 pM to 100 pM.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises a one carbon metabolism pathway intermediate. Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10-formylTHF; 5,10-meTHF; 5,10-meTHF; and 10-formylTHF. In some embodiments, the one carbon metabolism pathway intermediate is formate present at a concentration of about 50 pM. In some embodiments, the one carbon metabolism pathway intermediate is present or is added at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM, 1 pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 100 nM to 60 pM, 500 nM to 60 pM, 1 pM to 60 pM, 10 pM to 60 pM, 20 pM to 60 pM, 30 pM to 60 pM, 40 pM to 60 pM, or 45 pM to 55 pM.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises glutamate (e.g., L-glutamate). In some embodiments, glutamate can be present in a composition of the disclosure at a concentration of about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 450 pM, about 500 pM, about 550 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 4 mM, or about 5 mM. In some embodiments, glutamate is present or is added to a composition of the disclosure at a concentration of about 500 pM. In some embodiments, glutamate is present or is added to a composition of the disclosure at a concentration of from about 100 pM to 5mM, 200 pM to 5mM, 300 pM to 5mM, 400 pM to 5mM, 100 pM to 3mM, 200 pM to 3mM, 300 pM to 3mM, 400 pM to 3mM, 100 pM to 2mM, 200 pM to 2mM, 300 pM to 2mM, 400 pM to 2mM, 100 pM to ImM, 200 pM to ImM, 300 pM to ImM, 400 pM to ImM, 100 pM to 700 pM, 200 pM to 700 pM, 300 pM to 700 pM, 400 pM to 700 pM, 100 pM to 600 pM, 200 pM to 600 pM, 300 pM to 600 pM, or 400 pM to 600 pM.

In some embodiments, a composition (e.g., medium) or bioreactor and/or TFF system of the disclosure comprises carnitine. In some embodiments, carnitine is present in or is added to a composition of the disclosure at a concentration of about 100 nM, about 500 nM, about 1 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 75 pM, or about 100 pM. In some embodiments, carnitine is present or is added at a concentration of about 40 pM. In some embodiments, carnitine is present in or is added to a composition of the disclosure at a concentration of about 100 nM to 1 mM, 500 nM to 1 mM,l pM to 1 mM, 10 pM to 1 mM, 20 pM to 1 mM, 30 pM to 1 mM, 100 nM to 250 pM, 500 nM to 250 pM, 1 pM to 250 pM, 10 pM to 250 pM, 20 pM to 250 pM, 30 pM to 250 pM, 100 nM to 100 pM, 500 nM to 100 pM, 1 pM to 100 pM, 10 pM to 100 pM, 20 pM to 100 pM, 30 pM to 100 pM, 100 nM to 60 pM, 500 nM to 60 pM, 1 pM to 60 pM, 10 pM to 60 pM, 20 pM to 60 pM, 30 pM to 60 pM, 35 pM to 60 pM, or 30 pM to 50 pM.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells or cell clusters comprising these cells) with a serum albumin protein (e.g., HSA) in the disclosed bioreactors and/or TFF systems. In some embodiments, the serum albumin is present at a concentration of 0.01-2% HSA. In some embodiments, the serum albumin is present at a concentration of 0.03-0.1%, 0.03-0.07%, or 0.04-0.05%. In some embodiments, the serum albumin is present at a concentration of 0.05%. In some embodiments, the serum albumin is present at a concentration of 0.7- 1.3%, 0.8- 1.2%, 0.9- 1.1% or at 1%. In some embodiments, the serum albumin is present at a concentration of 1%.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells or cell clusters comprising these cells) with ZnSO4. In some embodiments, the method comprises contacting the cells with 1-100 pM, 1-50 pM, 1-20 pM, 1-12 pM, 5-15 pM, 8-12 pM or 9-11 pM of ZnSC . In some embodiments, the method comprising contacting the cells with about 10 pM of ZnSC .

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells or cell clusters comprising these cells) with one or more of an a serum albumin protein, a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, an epigenetic modifying compound, acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the method further comprises contacting the population of cells or clusters comprising these cells following the first period with one or more of a serum albumin protein, an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, and/or an epigenetic modifying compound. In some embodiments, the cells are contacted with a higher concentration of the serum albumin in the second period as compared to the first period. In some embodiments, the compositions further comprise ZnSO4. In some embodiments, the method further comprises contacting the population of cells or clusters comprising these cells following the first period with human serum albumin, but in the absence of a TGF-P signaling pathway inhibitor, a TH signaling pathway activator, a protein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathway inhibitor, an epigenetic modifying compound, an acetyl CoA-related metabolite, a vitamin, histone deacetylase inhibitor (HDACi), a redox homeostasis regulator, a one carbon metabolism pathway intermediate, glutamate, and/or carnitine.

In some embodiments, the method comprises contacting the population of cells (e.g., NKX6.1-positive, ISLl-positive, insulin-positive cells or clusters comprising these cells) with one or more of HSA, Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a first period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 4 days). In some embodiments, the method further comprises contacting the population of cells following the first period with one or more of HSA, taurine, acetate, beta-hydroxybutyrate, biotin, carnitine, glutamate, and formate for a second period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days) or more in the absence of an Alk5 inhibitor II, GC-1, staurosporine, thiazovivin, LDN193189, DZNEP. In some embodiments, the compositions further comprise ZnSCU. In some embodiments, the cells are contacted with a higher concentration of the HSA (e.g., about 1.0%) in the second period as compared to the first period (e.g., about 0.05%).

In some examples, insulin-positive endocrine cells can be matured in a NS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium in the disclosed bioreactors and/or TFF systems. In some embodiments, the insulin-positive endocrine cells can be matured in a CMRE medium supplemented with 10% FBS. In some embodiments, the insulin-positive endocrine cells can be matured in a DMEM/F12 medium supplemented with 1% HSA. In other cases, SC-P cells can be obtained by culturing the population of cells containing the insulinpositive endocrine cells in a MCDB131 medium that can be supplemented by 2% BSA. In some embodiments, the MCDB131 medium with 2% BSA for maturation of insulin-positive endocrine cells into SC-P cells can be comprise no small molecule factors as described herein. In some case, the MCDB131 medium with 2% BSA for maturation of insulin-positive endocrine cells into SC-P cells can comprise no serum (e.g., no FBS). In other cases, SC-P cells can be obtained by culturing the population of cells containing the insulin-positive endocrine cells in a MCDB131 medium that can be supplemented by 0.05% HSA and vitamin C. In some embodiments, SC-P cells can be obtained by culturing the population of cells containing the insulin-positive endocrine cells in a MCDB131 medium that can be supplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gin, e.g., 4mM). In some embodiments, the type of culture medium may be changed during S6. For instance, the S6 cells are cultured in a MCDB131 medium that can be supplemented by 0.05% HSA and vitamin C for the first two to four days, and then followed by a DMEM/F12 medium supplemented with 1% HSA. In some embodiments, additional factors are introduced into the culture medium. For instance, S6 cells can be cultured in a MCDB131 medium that can be supplemented by 0.05% HSA, ITS-X, vitamin C, and glutamine (Gin, e.g., 4mM) throughout the 10-12 days, during which ZnSO4 is introduced from day 4 of S6.

In some embodiments, the medium used to culture the cells in the bioreactor and/or TFF system can be xeno-free. A xeno-free medium for culturing cells and/or cell clusters of originated from an animal can have no product from non-human animals. In some embodiments, a xeno-free medium for culturing human cells and/or cell clusters can have no products from any non-human animals. For example, a xeno-free medium for culturing human cells and/or cell clusters can comprise human platelet lysate (PLT) instead of fetal bovine serum (FBS). For example, a medium can comprise from about 1% to about 20%, from about 5% to about 15%, from about 8% to about 12%, from about 9 to about 11% serum. In some embodiments, medium can comprise about 10% of serum. In some embodiments, the medium can be free of small molecules and/or FBS. For example, a medium can comprise MCDB131 basal medium supplemented with 2% BSA. In some embodiments, the medium is serum-free. In some examples, a medium can comprise no exogenous small molecules or signaling pathway agonists or antagonists, such as, growth factor from fibroblast growth factor family (FGF, such as FGF2, FGF8B, FGF 10, or FGF21), Sonic Hedgehog Antagonist (such as Santl, Sant2, Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol, cyclopamine, or derivatives thereof), Retinoic Acid Signaling agonist (e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961, AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314), inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK) (e.g., Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152), activator of protein kinase C (PKC) (e.g., phorbol 12,13- dibutyrate (PDBU) , TPB, phorbol 12-myristate 13-acetate, bryostatin 1, or derivatives thereof), antagonist of TGF P super family (e.g., Alk5 inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB-525334, SD- 208, SB-505124, or derivatives thereof), inhibitor of BMP type 1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormone signaling pathway activator (e.g., T3, GC-1 or derivatives thereof), gamma-secretase inhibitor (e.g., XXI, DAPT, or derivatives thereof), activator of TGF- P signaling pathway (e.g., WNT3a or Activin A) growth factor from epidermal growth factor (EGF) family (e.g., betacellulin or EGF), broad kinase (e.g., staurosporine or derivatives thereof), non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine, vitamin D, vitamin C, vitamin A, or derivatives thereof), or other additions like N- acetyl cysteine, zinc sulfate, or heparin. In some embodiments, the reaggregation medium can comprise no exogenous extracellular matrix molecule. In some embodiments, the reaggregation medium does not comprise MATRIGEL™. In some embodiments, the reaggregation medium does not comprise other extracellular matrix molecules or materials, such as, collagen, gelatin, poly-L-lysine, poly- D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, for example, or lysed cell membrane preparations.

A person of ordinary skill in the art will appreciate that the concentration of serum albumin supplemented into the medium may vary. For example, a medium (e.g., MCDB131) can comprise about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% BSA. In some embodiments, the media comprises 0.05-0.5%, 0.1-0.5%, 0.1-0.3%, 0.15-0.25%, 0.18-0.22%, 0.5-5%, 0.5-3%, 1-3%, 1.5-2.5%, or 1.8-2.2% BSA. In other cases, a medium can comprise about 0.01%, 0.05%, 0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA. In some embodiments, the media comprises 0.05-0.5%, 0.1-0.5%, 0.1-0.3%, 0.15-0.25%, 0.18-0.22%, 0.5-5%, 0.5-3%, 1-3%, 1.5-2.5%, or 1.8-2.2% HSA. In some embodiments, the media comprises glucose. In some embodiments, the media comprises 0.1-20 mM, 0.1-10 mM, 0.1-5 mM, 0.1-1 mM, 1-20 mM, 1-10 mM, 1-5 mM, 3-20 mM, 3-10 mM, 3-5 mM, or 4-5 mM glucose. In some embodiments, the media comprises 10-20 mM, 10-15 mM, or 11-13 mM glucose. In some embodiments, the media comprises 20-30 mM, or 22-27 mM glucose. In some embodiments, the media comprises Glutamax. In some embodiments, the media comprises 0.1-10%, 0.1-5%, 0.1-2%, 1-10%, 1-5%, Glutamax. In particular embodiments, the media comprises 0.8- 1.2% Glutamax. In some embodiments, the media comprises B27. In some embodiments, the media comprises Glutamax. In some embodiments, the media comprises 0.1-10%, 0.1-5%, 0.1-2%, 1-10%, 1-5% B27. In particular embodiments, the media comprises 0.8-1.2% B27. In some embodiments, the media comprises a ROCK inhibitor (e.g., Y-27632 or thiazovivin). In some embodiments, the media comprises 1- 100 pM, 1-50, 1-20, 1-10, 5-20, 5-15, or 8-12 pM of the ROCK inhibitor. In some embodiments, the media comprises bFGF. In some embodiments, the media comprises 1-1000 ng/ml, 1-500 ng/mL, 1-200 ng/mL, 50-1000 ng/mL, 50-500 ng/mL, 50-200 ng/mL, or 80-120 ng/mL bFGF. In particular embodiments, the media comprises 80-120 ng/mL bFGF. The medium used (e.g., MCDB131 medium) can contain components not found in traditional basal media, such as trace elements, putrescine, adenine, thymidine, and higher levels of some amino acids and vitamins. These additions can allow the medium to be supplemented with very low levels of serum or defined components. The medium can be free of proteins and/or growth factors, and may be supplemented with EGF, hydrocortisone, and/or glutamine. The medium can comprise one or more extracellular matrix molecules (e.g., extracellular proteins). Non-limiting exemplary extracellular matrix molecules used in the medium can include collagen, placental matrix, fibronectin, laminin, merosin, tenascin, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan, biglycan, thrombospondin, vitronectin, and decorin. In some embodiments, the medium comprises laminin, such as LN-332. In some embodiments, the medium comprises heparin. Any of these media can be used in the disclosed bioreactor and/or TFF system.

The medium can be changed periodically via methods such as centrifugation or settling in addition to media exchange via TFF, e.g., to provide optimal environment for the cells in the medium. When culturing the cells dissociated from the first cell cluster for re-aggregation, the medium can be changed at least or about every 4 hours, 12 hours, 24 hours, 48 hours, 3 days or 4 days. For example, the medium can be changed about every 48 hours. In some embodiments, cells can be cultured in a bioreactor and/or TFF system under dynamic conditions (e.g., under conditions in which the cells are subject to constant movement or stirring while in the suspension culture). For dynamic culturing of cells, the cells can be cultured in a container (e.g., an non-adhesive container such as a spinner flask (e.g., of 200 ml to 3000 ml, for example 250 ml; of 100 ml; or in 125 ml Erlenmeyer), which can be connected to a control unit and thus present a controlled culturing system. Alternatively, the cells can be cultured in a bioreactor. In some embodiments, cells can be cultured under non-dynamic conditions (e.g., a static culture) while preserving their proliferative capacity. For non-dynamic culturing of cells, the cells can be cultured in an adherent culture vessel. An adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells. The substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used). The substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixtures thereof, for example, Matrigel™, and lysed cell membrane preparations.

Medium in a dynamic cell culture vessel (e.g., a spinner flask or bioreactor) can be stirred (e.g., by a stirrer). The spinning speed can correlate with the size of the re-aggregated second cell cluster. The spinning speed can be controlled so that the size of the second cell cluster can be similar to an endogenous pancreatic islet. In some embodiments, the spinning speed is controlled so that the size of the second cell cluster can be from about 75 pm to about 250 pm. The spinning speed of a dynamic cell culture vessel (e.g., a spinner flask or bioreactor) can be about 20 rounds per minute (rpm) to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40 rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some embodiments, the spinning speed can be about 50 rpm.

Stage 6 cells as provided herein may or may not be subject to the dissociation and reaggregation process as described herein. In some embodiments, the cell cluster comprising the insulin-positive endocrine cells can be reaggregated. The reaggregation of the cell cluster can enrich the insulin-positive endocrine cells. In some embodiments, the insulin-positive endocrine cells in the cell cluster can be further matured into pancreatic P cells. For example, after reaggregation, the second cell cluster can exhibit in vitro GSIS, resembling native pancreatic islet. For example, after reaggregation, the second cell cluster can comprise non-native pancreatic P cell that exhibits in vitro GSIS. In some embodiments, the reaggregation process can be performed according to the disclosure of PCT application PCT/US2018/043179, which is incorporated herein by reference in its entirety. Stage 6 cells obtained according to methods provided herein can have high recovery yield after cryopreservation and reaggregation procedures. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery yield after cryopreservation post stage 5, as compared to a corresponding cell population without such treatment. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a higher recovery yield after cryopreservation post stage 5, as compared to a corresponding cell population without treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3. In some embodiments, stage 6 cells that are obtained in a differentiation process that involves treatment of a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-P superfamily (e.g., Activin A) at stage 3 and treatment of an epigenetic modifying compound (e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have a recovery yield after cryopreservation post stage 5 that is at least about 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50%. The recovery yield can be calculated as a percentage of cells that survive and form reaggregated cell clusters after cryopreservation, thawing and recovery, and reaggregation procedures, as compared to the cells before the cryopreservation.

In some embodiments, the present disclosure relates to cryopreservation of the nonnative pancreatic P cells or precursors thereof obtained using the methods provided herein. In particular embodiments, the cells are cryopreserved following stage 5 and before stage 6. In some embodiments, the cell population comprising non-native pancreatic P cells can be stored via cryopreservation. For instances, the cell population comprising non-native P cells, e.g., Stage 6 cells are thawed. In some embodiments, the cells can be dissociated into cell suspension, e.g., single cell suspension, and the cell suspension can be cryopreserved, e.g., frozen in a cryopreservation solution. The dissociation of the cells can be conducted by any of the technique provided herein, for example, by enzymatic treatment. The cells can be frozen at a temperature of at highest -20 °C, at highest -30 °C, at highest -40 °C, at highest -50 °C, at highest -60 °C, at highest -70 °C, at highest -80 °C, at highest -90 °C, at highest -100 °C, at highest -110 °C, at highest -120 °C, at highest -130 °C, at highest -140 °C, at highest -150 °C, at highest -160 °C, at highest -170 °C, at highest -180 °C, at highest -190 °C, or at highest -200 °C. In some embodiments, the cells are frozen at a temperature of about -80 °C. In some embodiments, the cells are frozen at a temperature of about -195 °C. Any cooling methods can be used for providing the low temperature needed for cryopreservation, such as, but not limited to, electric freezer, solid carbon dioxide, and liquid nitrogen. In some embodiments, any cryopreservation solution available to one skilled in the art can be used for incubating the cells for storage at low temperature, including both custom made and commercial solutions. For example, a solution containing a cryoprotectant can be used. The cryoprotectant can be an agent that is configured to protect the cell from freezing damage. For instance, a cryoprotectant can be a substance that can lower the glass transition temperature of the cryopreservation solution. Exemplary cryoprotectants that can be used include DMSO (dimethyl sulfoxide), glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran (e.g., dextran-40), and trehalose. Additional agents can be added in to the cryopreservation solution for other effects. In some embodiments, commercially available cryopreservation solutions can be used in the method provided herein, for instance, FrostaLife™, pZerve™, Prime-XV®, Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLB ANKER®, CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, and CryoDefend® Stem Cells Media.

During the differentiation process, the cells can be subject to irradiation treatment. In some embodiments, the cell population at Stage 6, e.g., the cell population or cell cluster that has cells being differentiated from insulin-positive endocrine cells into pancreatic P cells, is irradiated for a period of time. In some embodiments, the cell population at Stage 6 after reaggregation following the recovery from cryopreservation is irradiated for a period of time. In some embodiments, the cryopreserved cells (e.g., the cells that are cryopreserved at the end of Stage 5) are irradiated for a certain period of time prior to thawing and recovery for subsequent differentiation process.

In some embodiments, the stage 6 cells comprise NKX6.1-positive, insulin-positive cells. In some embodiments, the stage 6 cells comprise NKX6.1-positive, insulin-negative cells. In some embodiments, the stage 6 cells comprise C-peptide positive cells. In some embodiments, Stage 6 cells or cells that have characteristics of stage 6 cells are incubated in NS- GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In some embodiments, the stage 6 cells or cells that have characteristics of stage 6 cells are contacted with any one or more of a vitamin or anti-oxidant (e.g., vitamin C), an albumin protein (e.g., a human serum albumin protein), a TGF-beta pathway inhibitor (e.g., an ALK5 inhibitor II), a bone morphogenic protein (BMP) type 1 receptor inhibitor (e.g., LDN193189), a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (e.g., thiazovivin), a histone methyltransferase inhibitor (e.g., DZNEP), and a protein kinase inhibitor (e.g., staurosporine). See, e.g.., W02020264072. In some embodiments, the stage 6 cells are contacted with a PKC activator (see, e.g., WO2019217487, which is incorporated by reference herein in its entirety).

Differentiation factors

Embodiments of the disclosure relate to contacting progenitor cells (e.g., stem cells, e.g., iPS cells, definitive endoderm cells, primitive gut tube cells, PDXl-positive pancreatic progenitor cells, NKX6.1 -positive pancreatic progenitor cells, insulin-positive endocrine cells) with P cell differentiation factors, for example, to induce the maturation of the insulin-positive endocrine cells or differentiation of other progenitor cells into SC-P cells (e.g., mature pancreatic P cells). In some embodiments, the differentiation factor can induce the differentiation of pluripotent cells (e.g., iPSCs or hESCs) into definitive endoderm cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor can induce the differentiation of definitive endoderm cells into primitive gut tube cells, e.g., in accordance with methods and bioreactors and/or TFF systems described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of primitive gut tube cells into PDXl- positive pancreatic progenitor cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of PDXl-positive pancreatic progenitor cells into NKX6-1 -positive pancreatic progenitor cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the differentiation of NKX6-1 -positive pancreatic progenitor cells into insulin-positive endocrine cells, e.g., in accordance with a method described herein. In some embodiments, the differentiation factor(s) can induce the maturation of insulin-positive endocrine cells into pancreatic islet cells, e.g., in accordance with a method described herein. The cells can be in clusters.

At least one differentiation factor described herein can be used alone, or in combination with other differentiation actors, to generate pancreatic islet cells (e.g., SC-beta cells) according to the methods as disclosed herein. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten differentiation factors described herein are used in the methods of generating pancreatic islet cells.

In some embodiments, a composition described herein does not comprise one or more of the differentiation factors provided herein. Forkhead Box 01 ( FoxOl ) inhibitor

Embodiments of the disclosure relate to the use of Forkhead Box 01 (FoxOl) inhibitors as differentiation factors. In some embodiments, the FoxOl inhibitor used in the compositions and methods described herein is a compound of Formula (I): or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof, wherein:

R¹ is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group;

R² is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; each instance of R³ is independently optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R³ are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; each instance of R⁴ is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -OR^cl, -NO2, -N(R^c2)2, -SR^cl, - CN, or -SCN;

R⁵ is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; each instance of R⁶ is independently halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -OR^cl, -NO2, -N(R^c2)2, -SR^cl, - CN, or -SCN; wherein R^cl is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; wherein each instance of R^c2 is independently hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally two instances of R^c2 are taken together with their intervening atoms to form a substituted or unsubstituted heterocyclic or substituted or unsubstituted heteroaryl ring; x is 0, 1, or 2; y is 0 or 1 ; and z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, as valency permits.

In some embodiments, the compound is of Formula (I-A): or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof, wherein:

R¹ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R² is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; each instance of R³ is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R⁴ is halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl; and R⁵ is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In some embodiments, R¹ is hydrogen. In some embodiments, R² is optionally substituted alkyl. In some embodiments, R² is ethyl. In some embodiments, at least one instance of R³ is hydrogen. In some embodiments, both instances of R³ are hydrogen. In some embodiments, at least one instance of R⁴ is halogen. In some embodiments, at least one instance of R⁴ is fluorine. In some embodiments, x is 1. In some embodiments, R⁵ is hydrogen. In some embodiments, y is 1. In some embodiments, z is 0.

In some embodiments, the compound is of formula: or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, prodrug, composition, or mixture thereof.

In some embodiments, the compound is AS 1842856.

In some embodiments, a medium described herein does not comprise a FoxOl inhibitor.

Transforming Growth F actor- f (TGF-/>) Superfamily

Embodiments of the disclosure relate to the use of growth factors from the transforming growth factor-P (TGF-P) superfamily as differentiation factors. The “TGF-P superfamily” means proteins having structural and functional characteristics of known TGFP family members. The TGFP family of proteins can include the TGFP series of proteins, the Inhibins (including Inhibin A and Inhibin B), the Activins (including Activin A, Activin B, and Activin AB), MIS (Mullerian inhibiting substance), BMP (bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF (monoclonal nonspecific suppressor factor), and others. Activity of this family of proteins can be based on specific binding to certain receptors on various cell types. Members of this family can share regions of sequence identity, particularly at the C-terminus, that correlate to their function. The TGFP family can include more than one hundred distinct proteins, all sharing at least one region of amino acid sequence identity. Members of the family that can be used in the method disclosed herein can include, but are not limited to, the following proteins, as identified by their GenBank accession numbers: P07995, P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529, P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, 088959, 008717, P58166, 061643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643, P49001, P21274, 046564, 019006, P22004, P20722, Q04906, Q07104, P30886, P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885, P34820, Q29607, P12644, Q90752, 046576, P27539, P48969, Q26974, P07713, P91706, P91699, P27091, 042222, Q24735, P20863, 018828, P55106, Q9PTQ2, 014793, 008689, 042221, 018830, 018831, 018836, 035312, 042220, P43026, P43027, P43029, 095390, Q9R229, 093449, Q9Z1W4, Q9BDW8, P43028, Q7Z4P5, P50414, P17246, P54831, P04202, P01137, P09533, P18341, 019011, Q9Z1Y6, P07200, Q9Z217, 095393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737, AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1 S4Y, NP-001009856.1, NP- 1-032406.1, NP-999193.1, XP-519063.1, AAG17260.1, CAA40806.1, NP- 1-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1, AAC59738.1, CAI46003.1, B40905, AAQ55811.1, AAK40342.1, XP-540364.1, P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504, BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG05339.1, BAB17601.1, CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1, AAA59169.1, XP-541000.1, NP-990537.1, NP- 1-002184.1, AAC14187.1, AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C, AAH30029.1, CAA49326.1, XP-344131.1, AA-148845.1, XP-1-148966.3, 148235, B41398, AAH77857.1, AAB26863.1, 1706327A, BAA83804.1, NP-571143.1, CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506, CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1, BAC81672.1, BAA12694.1, BAA08494.1, B36192, C36192, BAB 16971.1, NP-034695.1, AAA49160.1, CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1, XP-522443.1, AAM27448.1, XP-

538247.1, AAD30133. 1, AAC36741.1, AAH10404.1, NP-032408.1, AAN03682.1, XP-

509161.1, AAC32311.1, NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-

571023.1, CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1, AAH73508.1, AADO2201.1, NP-999793.1, NP-990542.1, AAF19841.1, AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1, EAL41229.1, AAT07302.1, CAI19472.1, NP- 031582.1, AAA40548.1, XP-535880.1, NP-1-037239.1, AAT72007.1, XP-418956.1, CAA41634.1, BAC30864.1, CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1, NP-990479.1, NP-999820.1, AAB27335.1, S45355, CAB82007.1, XP-534351.1, NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1, BAA89012.1, IES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1, AAQ89234.1, AAM27000.1, AAH30959.1, CAGO1491.1, NP-571435.1, 1REU, AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1, CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.1, AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-

342592.1, XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1, CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1, AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AATI2416.1, NP-

571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1, BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1, BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1, AAB94786.1, NP- 001483.2, XP-528195.1, NP-571417.1, NP-001001557. 1, AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2, P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1, NP-1-038554.1, CAG04089.1, CAD60936.2, NP- 031584.1, B55452, AAC60285.1, BAA06410.1, AAH52846.1, NP-031580.1, NP-1-036959.1, CAA45836.1, CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1, AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1, AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1, BAC02895.1, AAWS

1451.1, AAF82188.1, XP-544189.1, NP-990568.1, BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1, AAT99303.1, AAD28038.1, AAH52168.1, NP- 001004122.1, CAA72733.1, NP-032133.2, XP-394252.1, XP-224733.2, JH0801, AAP97721.1, NP-989669.1, S43296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032135.1, AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1, AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1, CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1, CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1, AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1, AAW47740.1, AAW29442.1, NP-722813.1, AARO8901.1, AAO 15420.2, CAC59700.1, AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1, AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1, NP-005802.1, XP-

343149.1, AW34055.1, XP-538221.1, AAR27580.1, XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1 W4, NP-1-031585.2, NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1, CAE46407.1, XP-1-417667.1, BAC53989.1, BAB19659.1, AAM46922.1, AAA81169.1, AAK28707.1, AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1, BAD16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1, AAK08152.2, 2009388A, AAR12161.1, CAGO1961.1, CAB63656.1, CAD67714.1, CAF94162.1, NP-477340.1, EAL24792.1, NP- 1-001009428.1, AAB86686.1, AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1, Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1, XP-525772.1, NP- 060525.2, AAH33585.1, AAH69080.1, CAG12751.1, AAH74757.2, NP-034964.1, NP- 038639.1, 042221, AAF02773.1, NP-062024.1, AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1, AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1, BAB16046.1, AAN63522.1, NP-571041.1, AAB04986.2, AAC26791.1, AAB95254.1, BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1, XP- 1-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1, AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1, AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-

999600.2, NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1 AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997. 1, AAN03842.1, NP-

571951.2, CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1, NP-

624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2, NP-001192.1, XP-

544948.1, AAQ18013.1, AAV38739.1, NP-851298.1, CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1, XP-1-421648.1, CAB63704.1, NP- 037306.1, A55706, AAF02780.1, CAG09623.1, NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1, AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP-1- 001009400.1, AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1, AAA3 1526.1.

The growth factor from the TGF-P superfamily in the methods and compositions provided herein can be naturally obtained or recombinant. In some embodiments, the growth factor from the TGF-P superfamily comprises Activin A. The term “Activin A” can include fragments and derivatives of Activin A. The sequence of an exemplary Activin A is provided as SEQ ID NO: 1. Other non-limiting examples of Activin A are provided in SEQ ID NO: 3-16, and non-limiting examples of nucleic acids encoding Activin A are provided in SEQ ID NO: 2, SEQ ID NO: 17, and SEQ ID NO: 18 . In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide comprising an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 and 3-16, or functional fragments thereof. In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide comprising the amino acid any one of SEQ ID NOs: 1 and 3-16.

SEQ ID NO: 1 - Homo sapiens Inhibin beta A subunit (Activin A) amino acid sequence: GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHY RMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 2 - Homo sapiens Inhibin beta A chain (Activin A) nucleic acid sequence: GGCTTGGAGTGTGATGGCAAGGTCAACATCTGCTGTAAGAAACAGTTCTTTGTCAGTTTCAA GGACATCGGCTGGAATGACTGGATCATTGCTCCCTCTGGCTATCATGCCAACTACTGCGAGG GTGAGTGCCCGAGCCATATAGCAGGCACGTCCGGGTCCTCACTGTCCTTCCACTCAACAGTC ATCAACCACTACCGCATGCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTGCC CACCAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGG

ACATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCATAG

SEQ ID NO: 3 - Homo sapiens Inhibin beta A chain preproprotein sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEV

GLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKK

KKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCK

KQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS

CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 4 - Homo sapiens Inhibin B subunit amino acid sequence:

ARQSEDHPHRRRRRGLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGT

SGSSLSFHSTVINHYRMRGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 5 - Homo sapiens Inhibin B subunit in testis Homo sapiens amino acid sequence:

GLECDGKVNICCKKQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHY

ACGHSPFANLKSCCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 6 - Homo sapiens Inhibin beta A chain preproprotein sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGEEAEEV

GLKGERSELLLSEKVVDARKSTWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGKK

KKKEEEGEGKKKGGGEGGAGADEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCK

KQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 7 - Mus musculus (Mouse) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHGSAPDCPSCALATLPKDGPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDTGDEAEE

MGLKGERSELLLSEKVVDARKSTWHIFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGK

KKKKEVDGDGKKKDGSDGGLEEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKK

QFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSC CVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 8 - Rattus norvegicus (Rat) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHGAAPDCPSCALATLPKDGPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIRLFQQQKHPQGSLDMGDEAEE

MGLKGERSELLLSEKVVDARKSTWHIFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLGK

KKKKEVDGDGKKKDGSDGGLEEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKK

QFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSC

CVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS SEQ ID NO: 9 - Gallus (Chicken) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWKRGFLLVICWIIVRSSPTPGSEGHSSVADCPSCALTTLSKDVPSSQPEMVEAVKKHILNM

LHLRDRPNITQPVPKAALLNATKKLHVGKVGDDGYVEIEDDVGRRAEMNEVVEQTSEIITFAES

GTPKKTLHFEISKEGSELSVVEHAEVWLFLKVSKANRSRTKVTIRLFQQQRQPKGNSEAAEDME

DMGLKGERSETLISEKAVDARKSTWHIFPISSSVQRLLDQGQSSLDVRIACDLCQETGASLVLLG

KKKKKEDDGEGKEKDGGELTGEEEKEQSHRPFLMMLARHSEDRQHRRRERGLECDGKVNICCK

KQFFVSFKDIGWSDWIIAPTGYHANYCEEECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 10 - Bos taurus (Bovine) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALATLPKDVPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISKEGSDLSVVERAEIWLFLKVPKANRTRSKVTIRLFQQQKHLQGSLDAGEEAEEV

GLKGEKSEMLISEKVVDARKSTWHIFPVSSCIQRLLDQGKSSLDIRIACEQCQETGASLVLLGKKK

KKEEEGEGKKRDGEGGAGGDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQ

FFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCC VPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 11 - Equus caballus (Horse) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVKSSPTPGSEGHSAAPNCPSCALATLPKDVPNAQPEMVEAVKKHILN

MLHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAES

GTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRSKVTIRLLQQQKHPQGSSDTREEAEE

ADLMEERSEQLISEKVVDARKSTWHIFPVSSSIQRLLDQGKSSLDIRIACDQCHETGASLVLLGKK

KKKEEEGEGKKKDGGEAGAGVDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCK

KQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINQYRLRGHNPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 12 - Sus scrofa (Pig) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSGGHSAAPDCPSCALATLPKDVPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVELEDDIGRRAEMNELMEQTSEIITFAEA

GTARKTLRFEISKEGSDLSVVERAEIWLFLKVPKANRTRTKVSIRLFQQQRRPQGSADAGEEAED

VGFPEEKSEVLISEKVVDARKSTWHIFPVSSSIQRLLDQGKSALDIRTACEQCHETGASLVLLGKK

KKKEEEAEGRKRDGEGAGVDEEKEQSHRPFLMLQARQSEEHPHRRRRRGLECDGKVNICCKKQ

SEQ ID NO: 13 - Ovis aries (Sheep) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGHSAAPDCPSCALATLPKDVPNSQPEMVEAVKKHILNM

LHLKKRPDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG

TARKTLHFEISQEGSDLSVVERAEIWLFLKVPKANRTRSKVTIRLFQQQKHLQGSLDAGEEAEEV

GLKGEKSEMLISEKVVDARKSTWHIFPVSSCIQRLLDQGKSSLDIRIACEQCQETGASLVLLGKKK

RKEEEGEGKKRDGEGGAGGDEEKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCKKQ

FYVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKSCC VPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 14 - Felis catus (cat) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MPLLWLRGFLLASCWIIVRSSPTPGSEGPGAAPDCPSCALATLPKDVPNSQPEMVEAVKKHILNM LHLKKRPEVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIGRRAEMNELMEQTSEIITFAESG TARKTLHFEISKEGSDLSVVERAEVWLFLKVPKANRTRTKVTIQLLQKQPQGGVDAGEEAEEMG LMEERNEVLISEKVVDARKSTWHIFPVSSSIQRLLDQGKSSLDVRIACEQCHETGASLVLLGKKK KKEEEGEGKKKDGGDGGAGADEDKEQSHRPFLMLQARQSEDHPHRRRRRGLECDGKVNICCK KQFFVSFKDIGWNDWIIAPSGYHANYCEGECPSHIAGTSGSSLSFHSTVINHYRMRGHSPFANLKS CCVPTKLRPMSMLYYDDGQNIIKKDIQNMIVEECGCS

SEQ ID NO: 15 - Danio rerio (zebrafish) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MSPLPLLSGILLLLIRSCSLSAMVTKGSLPMSEQQAGATVCPSCALARFRKGVSESEDEGAQQDV VEAVKRHILNMLHLQERPNITHPVPRAALLNAIRKVHVGRVAKDGSVLIEDEASNRAETEQAEQ TEIITFAETGEAPGIVNFLISKEGGEMSVVDQANVWIFLRLPKGNRTRANVNIRLLLQQGAGEKIL AEKSVDTRRSGWHTFPASESVQSLLQRGGSTLSLRVSCPLCADARATPVLVSPGGSEREQSHRPF LMAVVRQMDELSLRRRRKRGLECDGKARVCCKRQFYVNFKDIGWNDWIIAPSGYHANYCEGD CASNVASITGNSLSFHSTVISHYRIRGYSPFTNIKSCCVPTRLRAMSMLYYNEEQKIVKKDIQNMI VEECGCS

SEQ ID NO: 16 - Carassius auratus (goldfish) Inhibin beta A chain (Activin beta-A chain) amino acid sequence:

MSSLTLVNRGTAALRLFVRGLLTHSSREWLSGDGEPDDPVTPCPSCALAQRQKDSEEQTDMVEA VKRHILNMLHLNTRPNVTHPVPRAALLNAIRRLHVGRVGEDGTVEMEEDGGGLGEHREQSEEQ PFEIITFAEPGDAPDIMKFDISMEGNTLSVVEQANVWLLLKVAKGSRGKGKVSVQLLQHGKADP GSADGPQEAVVSEKTVDTRRSGWHTLPVSRTVQTLLDGDSSMLSLRVSCPMCAEAGAVPILVPT ESNKGKEREQSHRPFLMVVLKPAEEHPHRRSKRGLECDGKIRVCCKRQFYVNFKDIGWSDWIIA PSGYHANYCEGDCPSHVASITGSALSFHSTVINHYRMRGYSPFNNIKSCCVPTRLRAMSMLYYNE

EQKIIKKDIQNMIVEECGCS

SEQ ID NO: 17 - Recombinant Inhibin B subunit nucleic acid sequence GCCCGGCAGTCTGAAGACCACCCTCATCGCCGGCGTCGGCGGGGCTTGGAGTGTGATGGCA AGGTCAACATCTGCTGTAAGAAACAGTTCTTTGTCAGTTTCAAGGACATCGGCTGGAATGAC TGGATCATTGCTCCCTCTGGCTATCATGCCAACTACTGCGAGGGTGAGTGCCCGAGCCATAT AGCAGGCACGTCCGGGTCCTCACTGTCCTTCCACTCAACAGTCATCAACCACTACCGCATGC

GGGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTGCCCACCAAGCTGAGACCCATG TCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACATTCAGAACATGATCGT GGAGGAGTGTGGGTGCTCATAGAGTTGCCCAGCCCAGGGGGAAAGGGAGCAAGA

SEQ ID NO: 18 - Homo sapiens mature subunit beta(A) inhibin in testis nucleic acid sequence GGCCTGGAGTGCGACGGCAAGGTCAACATCTGCTGTAAGAAACAGTTCTTTGTCAGTTTCAA GGACATCGGCTGGAATGACTGGATCATTGCTCCCTCTGGCTATCATGCCAACTACTGCGAGG GTGAGTGCCCGAGCCATATAGCAGGCACGTCCGGGTCCTCACTGTCCTTCCACTCAACAGTC ATCAACCACTACGCATGCGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGTGCCCAC

CAAGCTGAGACCCATGTCCATGTTGTACTATGATGATGGTCAAAACATCATCAAAAAGGACA TTCAGAACATGATCGTGGAGGAGTGCGGGTGCTCCTAA

In some embodiments, the growth factor from the TGF-P superfamily comprises growth differentiation factor 8 (GDF8). The term “GDF8” can include fragments and derivatives of GDF8. The sequences of GDF8 polypeptides are available to the skilled artisan. In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human GDF8 polypeptide sequence (GenBank Accession EAX1O88O).

In some embodiments, the growth factor from the TGF-P superfamily comprises a growth factor that is closely related to GDF8, e.g., growth differentiation factor 11 (GDF11). In some embodiments, the growth factor from the TGF-P superfamily comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human GDF11 polypeptide sequence (GenBank Accession AAF21630).

In some embodiments, the growth factor from the TGF-P superfamily can be replaced with an agent mimics the at least one growth factor from the TGF-P superfamily. Exemplary agents that mimic the at least one growth factor from the TGF-P superfamily, include, without limitation, IDE1 and IDE2.

In some embodiments, a medium described herein does not comprise a TGF-P superfamily protein.

BMP Signaling Pathway Inhibitors

Embodiments of the disclosure relate to the use of BMP signaling pathway inhibitors (which also may be referred to as “BMP inhibitors” herein) as cell differentiation factors. The BMP signaling family is a diverse subset of the TGF-P superfamily (Sebald et al. Biol. Chem. 385:697-710, 2004). Over twenty known BMP ligands are recognized by three distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least three type I (AEK2, AEK3, and AEK6) receptors. Dimeric ligands facilitate assembly of receptor heteromers, allowing the constitutively-active type II receptor serine/threonine kinases to phosphorylate type I receptor serine/threonine kinases. Activated type I receptors phosphorylate BMP-responsive (BR-) SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4, a co-SMAD that also facilitates TGF signaling. In addition, BMP signals can activate intracellular effectors such as MAPK p38 in a SMAD-independent manner (Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists such as noggin, chordin, gremlin, and follistatin limit BMP signaling by ligand sequestration.

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises DMH-1, or a derivative, analogue, or variant thereof. In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises LDN193189 (also known as LDN193189, 1062368-24- 4, LDN-193189, DM 3189, DM-3189, IUPAC Name: 4-[6-(4-piperazin-l- y Ipheny l)pyrazolo [1,5- a] pyrimidin- 3 -yl] quinolone). In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises the following compound or a derivative, analogue, or variant of the following compound:

In some embodiments, DMH-1 can be more selective as compared to LDN193189. In some embodiments of the present disclosure, DMH-1 can be particularly useful for the methods provided herein. In some embodiments, the methods and compositions provided herein, or specific stages of the methods disclosed herein (e.g., stage 3), exclude use of LDN193189. In some embodiments, the methods and compositions provided herein exclude use of LDN193189, or a derivative, analogue, or variant thereof for generating PDX1 -positive pancreatic progenitor cells from primitive gut tube cells. In some embodiments, the methods and compositions provided herein relate to use of DMH-1, or a derivative, analogue, or variant thereof for generating PDXl-positive pancreatic progenitor cells from primitive gut tube cells.

In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprise an analog or derivative of LDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug of LDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189 comprises LDN193189 hydrochloride. In some embodiments, the BMP signaling pathway inhibitor in the methods and composition provided herein comprises a compound of Formula I from U.S. Patent Publication No. 2011/0053930.

In some embodiments, a medium described herein does not comprise a BMP signaling pathway inhibitor.

TGF-[> Signaling Pathway Inhibitors

Embodiments of the disclosure relate to the use of TGF-P signaling pathway inhibitors as cell differentiation factors.

In some embodiments, the TGF-P signaling pathway comprises TGF-P receptor type I kinase (TGF-P RI) signaling. In some embodiments, the TGF-P signaling pathway inhibitor comprises ALK5 inhibitor II (CAS 446859-33-2, an ATP-competitive inhibitor of TGF-B RI kinase, also known as RepSox, IUPAC Name: 2-[5-(6-methylpyridin-2-yl)-lH-pyrazol-4-yl]- 1,5-naphthyridine. In some embodiments, the TGF-P signaling pathway inhibitor is an analog or derivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II (also named “ALK5i”) is a compound of Formula I as described in U.S. Patent Publication No. 2012/0021519, incorporated by reference herein in its entirety.

In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is a TGF-P receptor inhibitor described in U.S. Patent Publication No. 2010/0267731. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein comprises an ALK5 inhibitor described in U.S. Patent Publication Nos. 2009/0186076 and 2007/0142376. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is A 83-01. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is not A 83-01. In some embodiments, the compositions and methods described herein exclude A 83-01. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 431542. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 431542. In some embodiments, the compositions and methods described herein exclude SB 431542. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is D 4476. In some embodiments, the TGF-P signaling pathway inhibitor is not D 4476. In some embodiments, the compositions and methods described herein exclude D 4476. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor is not GW 788388. In some embodiments, the compositions and methods described herein exclude GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is LY 364947. In some embodiments, the TGF-P signaling pathway inhibitor is not LY 364947. In some embodiments, the compositions and methods described herein exclude LY 364947. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is LY 580276. In some embodiments, the TGF-P signaling pathway inhibitor is not LY 580276. In some embodiments, the compositions and methods described herein exclude LY 580276. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 525334. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 525334. In some embodiments, the compositions and methods described herein exclude SB 525334. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SB 505124. In some embodiments, the TGF-P signaling pathway inhibitor is not SB 505124. In some embodiments, the compositions and methods described herein exclude SB 505124. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is SD 208. In some embodiments, the TGF-P signaling pathway inhibitor is not SD 208. In some embodiments, the compositions and methods described herein exclude SD 208. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 6604. In some embodiments, the TGF-P signaling pathway inhibitor is not GW 6604. In some embodiments, the compositions and methods described herein exclude GW 6604. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is GW 788388. In some embodiments, the TGF-P signaling pathway inhibitor in the methods and compositions provided herein is not GW 788388. In some embodiments, the compositions and methods described herein exclude GW 788388.

From the collection of compounds described above, the following can be obtained from various sources: LY-364947, SB-525334, SD-208, and SB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo., 63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals, Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604 available from GlaxoSmithKline, 980 Great West Road, Brentford, Middlesex, TW8 9GS, United Kingdom; LY580276 available from Lilly Research, Indianapolis, Ind. 46285; and SMI 6 available from Biogen Idee, P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C., 27709-4627.

In some embodiments, a medium described herein does not comprise a TGF-P signaling pathway inhibitor. Wnt Signaling Pathway

Embodiments of the disclosure relate to the use of activators of the Wnt signaling pathway as cell differentiation factors.

In some embodiments, the Wnt signaling pathway activator in the methods and compositions provided herein comprises CHIR99021. In some embodiments, the Wnt signaling pathway activator in the methods and compositions provided herein comprises a derivative of CHIR99021, e.g., a salt of CHIR99021, e.g., trihydrochloride, a hydrochloride salt of CHIR99021. In some embodiments, the Wnt signaling pathway activator in the methods and compositions provided herein comprises Wnt3a recombinant protein. In some embodiments, the Wnt signaling pathway activator in the methods and compositions provided herein comprises a glycogen synthase kinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, without limitation, 3F8, A 1070722, AR-A 014418, BIO, BlO-acetoxime, FRATide, 10Z- Hymenialdisine, Indirubin-3 'oxime, kenpaullone, L803, L803-mts, lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TWS 119, and analogs or derivatives of any of these. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a Wnt signaling pathway activator.

In some embodiments, a medium described herein does not comprise a Wnt signaling pathway activator.

Embodiments of the disclosure relate to the use of inhibitors of the Wnt signaling pathway as P cell differentiation factors.

In some embodiments, the Wnt signaling inhibitor is a tankyrase inhibitor that inhibits expression or activity of at least one tankyrase (TNKS) protein. In some embodiments, the at least one tankyrase protein is tankyrase 1 or tankyrase 2. In some embodiments, the Wnt signaling inhibitor inhibits binding of a substrate to a nicotinamide subsite or an adenosine subsite, or both, of a tankyrase protein. In some embodiments, the tankyrase inhibitor is AZ 6102, JW55, MN64, IWR-l-endo, TC-E5001, WIKI4, TNKS 22, TNKS 49, 2X-121 (E7449), XAV-939 (XAV), G007-LK, NVP-TNKS656, decernotinib, (VX-509), vismodegib (GDC- 0449), IM- 12, GSK429286A, INO-1001, Ofloxacin, TG101209, FG-4592, l-BET-762, LY2157299, MK- 0752, Wnt-C59 (C59), MC1568, Pacritinib (SB 1518), SB415286, Drocinostat, IWR-l-endo, Norfloxacin, SH-4-54, Nexturastat A, SB216763, UNCO 79, dephnetin, GF109203X, RepSox, Sotrastaurin, SB431542, tofacitinib (CP-690550, Tasocitinib), AG-14361, CI994 (tacedinaline), Ro 31-8220 mesylate, resveratrol, NVP-TNKS656, or YO- 01027. In some embodiments, said tankyrase inhibitor is AZ 6102, NVP-TNKS656, or IWR-l- endo. In some embodiments, the tankyrase inhibitor is NVP-TNKS656 (NVP). In some embodiments, the tankyrase inhibitor selectively inhibits tankyrase 1 over tankyrase 2. In some embodiments, the tankyrase inhibitor selectively inhibits tankyrase 2 over tankyrase 1.

In some embodiments, a medium described herein does not comprise a Wnt signaling pathway inhibitor.

FGF Family

Embodiments of the disclosure relate to the use of growth factors from the FGF family as cell differentiation factors.

In some embodiments, the growth factor from the FGF family in the methods and compositions provided herein comprises keratinocyte growth factor (KGF). The polypeptide sequences of KGF are available to the skilled artisan. An example of human KGF amino acid sequence is provided in GenBank Accession No. AAB21431, provided as SEQ ID NO: 19). In some embodiments, the growth factor from the FGF family comprises a polypeptide comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 19, or a functional fragment thereof. In some embodiments, the growth factor from the FGF family comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 19.

Human KGF amino acid sequence (GenBank Accession AAB21431; SEQ ID NO: 19) MHKWILTWILPTLLYRSCFHIICLVGTISLACNDMTPEQMATNVNCSSPERHTRSYDYMEGGDIR VRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAIKGVESEFYLAMNKEGKLY AKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFVALNQKGIPVRGKKTKKEQKTAHFLP MAIT

In some embodiments, the growth factor from the FGF family in the methods and composition provided herein comprises FGF2. The polypeptide sequences of FGF2 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF2 polypeptide sequence (GenBank Accession NP 001997).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF8B. The polypeptide sequences of FGF8B are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF8B polypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF10. The polypeptide sequences of FGF10 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF10 polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF family in the methods and composition provided herein comprises FGF21. The polypeptide sequences of FGF21 are available to the skilled artisan. In some embodiments, the growth factor from the FGF family comprises a polypeptide having an amino acid sequence at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or greater identical to the human FGF21 polypeptide sequence (GenBank Accession AAQ89444.1).

In some embodiments, a medium described herein does not comprise a FGF family protein.

Sonic Hedgehog ( SHH) Signaling Pathway

Embodiments of the disclosure relate to the use of SHH signaling pathway inhibitors as cell differentiation factors.

In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises Santl. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT2. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT3. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises SANT4. In some embodiments, the SHH signaling pathway inhibitor comprises Cur61414. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises forskolin. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises tomatidine. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises AY9944. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises triparanol. In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises compound A or compound B (as disclosed in U.S. Pub. No. 2004/0060568). In some embodiments, the SHH signaling pathway inhibitor in the methods and composition provided herein comprises a steroidal alkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or a derivative thereof) as disclosed in U.S. Pub. No. 2006/0276391. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a SHH signaling pathway inhibitor.

Rho Kinase (ROCK) Signaling Pathway

Embodiments of the disclosure relate to the use of ROCK signaling pathway inhibitors (ROCK inhibitors) as cell differentiation factors.

In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Y-27632 or Thiazovivin. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Thiazovivin. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises Y-27632. In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises the following compound or a derivative thereof:

In some embodiments, the ROCK inhibitor in the methods and composition provided herein comprises the following compound or a derivative thereof:

Non- limiting examples of ROCK inhibitor that can be used in the methods and compositions provided herein include Thiazovivin, Y-27632, Fasudil/HA1077, H-1152, Ripasudil, Y39983, Wf-536, SLx-2119, Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines, Indazoles, and pyridinealkene derivatives, ROKa inhibitor, XD-4000, HMN- 1152, 4-(l-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, and quinazoline.

In some embodiments, a medium described herein does not comprise a ROCK pathway inhibitor. Retinoic Acid Signaling Pathway

Embodiments of the disclosure relate to the use of modulators of retinoic acid signaling as cell differentiation factors.

In some embodiments, the modulator of retinoic acid signaling in the methods and composition provided herein comprises an activator of retinoic acid signaling. In some embodiments, the RA signaling pathway activator in the methods and composition provided herein comprises retinoic acid. In some embodiments, the RA signaling pathway activator in the methods and composition provided herein comprises a retinoic acid receptor agonist. Exemplary retinoic acid receptor agonists in the methods and composition provided herein include, without limitation, CD 1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80, BMS 753, tazarotene, adapalene, and CD 2314.

In some embodiments, the modulator of retinoic acid signaling in the methods and composition provided herein comprises an inhibitor of retinoic acid signaling. In some embodiments, the retinoic acid signaling pathway inhibitor comprises DEAB (IUPAC Name: 2- [2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In some embodiments, the retinoic acid signaling pathway inhibitor comprises an analog or derivative of DEAB.

In some embodiments, the retinoic acid signaling pathway inhibitor in the methods and composition provided herein comprises a retinoic acid receptor antagonist. In some embodiments, the retinoic acid receptor antagonist in the methods and composition provided herein comprises (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoic acid, (E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid, (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]-benzoic acid, and (E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoic acid. In some embodiments, the retinoic acid receptor antagonist comprises BMS 195614 (CAS#253310-42-8), ER 50891 (CAS#187400-85-7), BMS 493 (CAS#170355-78-9), CD 2665 (CAS#170355-78-9), LE 135 (CAS#155877-83-l), BMS 453 (CAS #166977-43-1), or MM 11253 (CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosed herein exclude a modulator of retinoic acid signaling. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a retinoic acid signaling pathway activator. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a retinoic acid signaling pathway inhibitor.

In some embodiments, a medium described herein does not comprise retinoic acid. Protein Kinase C Activator

Embodiments of the disclosure relate to the use of protein kinase C activators as cell differentiation factors. Protein kinase C is one of the largest families of protein kinase enzymes and is composed of a variety of isoforms. Conventional isoforms include a, pi, pil, y; novel isoforms include 5, a, q, 0; and atypical isoforms include PKC enzymes are primarily cytosolic but translocate to the membrane when activated. In the cytoplasm, PKC is phosphorylated by other kinases or autophosphorylated. In order to be activated, some PKC isoforms (e.g., PKC-a) require a molecule to bind to the diacylglycerol (“DAG”) binding site or the phosphatidylserine (“PS”) binding site. Others are able to be activated without any secondary binding messengers at all. PKC activators that bind to the DAG site include, but are not limited to, bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin. PKC activators that bind to the PS site include, but are not limited to, polyunsaturated fatty acids and their derivatives. It is contemplated that any protein kinase C activator that is capable, either alone or in combination with one or more other P cell differentiation factors, of inducing the differentiation of at least one insulin-producing, endocrine cell or precursor thereof into a SC-P cell can be used in the methods, compositions, and kits described herein.

In some embodiments, any of the PKC activators disclosed herein is a PKC activator capable of binding to a DAG binding site on a PKC. In some embodiments, the PKC activator is capable of binding to a Cl domain of a PKC. In some embodiments, the PKC activator is a benzolactam-derivative. In some embodiments, the benzolactam-derivative is ((2S,5S)-(E,E)-8- (5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam), which may be referred to herein as TPPB or TPB. In some embodiments, contacting a population of cells with a benzolactam-derivative PKC activator (e.g., TPPB) increases cell yield as compared to a population of cells not treated with the benzolactam-derivative PKC activator. In some embodiments, the PKC activator is a phorbol ester. In some embodiments, the phorbol ester is Phorbol 12,13-dibutyrate, which may be referred to herein as PDBU or PdbU. In some embodiments, contacting a population of cells with a benzolactam-derivative PKC activator (e.g., TPPB) increases cell yield as compared to a population of cells treated with a phorbol ester PKC activator (e.g., PdbU). In some embodiments, the PKC activator in the methods and composition provided herein comprises PdbU. In some embodiments, the PKC activator in the methods and composition provided herein comprises TPB. In some embodiments, the PKC activator in the methods and composition provided herein comprises cyclopropanated polyunsaturated fatty acids, cyclopropanated monounsaturated fatty acids, cyclopropanated polyunsaturated fatty alcohols, cyclopropanated monounsaturated fatty alcohols, cyclopropanated polyunsaturated fatty acid esters, cyclopropanated monounsaturated fatty acid esters, cyclopropanated polyunsaturated fatty acid sulfates, cyclopropanated monounsaturated fatty acid sulfates, cyclopropanated polyunsaturated fatty acid phosphates, cyclopropanated monounsaturated fatty acid phosphates, macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V, gnidimacrin, iripallidal, ingenol, napthalenesulfonamides, diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18), insulin growth factor, hormones, and growth factor activators, as described in WIPO Pub. No. WO/2013/071282. In some embodiments, the bryostain comprises bryostatin-1, bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16, bryo statin- 17, or bryostatin-18. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a protein kinase C activator.

In some embodiments, a medium described herein does not comprise a protein kinase C activator. y-Secretase Inhibitors

Embodiments of the disclosure relate to the use of y-secretase inhibitors as cell differentiation factors.

In some embodiments, the y-secretase inhibitor in the methods and composition provided herein comprises XXI. In some embodiments, the y-secretase inhibitor in the methods and composition provided herein comprises DAPT. Additional exemplary y-secretase inhibitors in the methods and composition provided herein include, without limitation, the y-secretase inhibitors described in U.S. Pat. Nos. 7,049,296, 8,481,499, 8,501,813, and WIPO Pub. No. WO/2013/052700. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a y-secretase inhibitor.

In some embodiments, a medium described herein does not comprise a y-secretase inhibitor.

Thyroid. Hormone Signaling Pathway Activators

Embodiments of the disclosure relate to the use of thyroid hormone signaling pathway activators as cell differentiation factors.

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises triiodothyronine (T3). In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises GC-1. In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises an analog or derivative of T3 or GC-1. Exemplary analogs of T3 in the methods and composition provided herein include, but are not limited to, selective and non-selective thyromimetics, TRP selective agonist-GC-1, GC-24,4- Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid (DITPA); the selective TR-P agonist GC-1; 3-Iodothyronamine (T(l)AM) and 3,3',5-triiodothyroacetic acid (Triac) (bioactive metabolites of the hormone thyroxine (T(4)); KB-2115 and KB- 141; thyronamines; SKF L-94901; DIBIT; 3'-AC-T2; tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (via oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chain), 3,3',5'-triiodothyronine (rT3) (via T4 and T3 deiodination), 3,3'- diiodothyronine (3,3'-T2) and 3,5-diiodothyronine (T2) (via T4, T3, and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine (TOAM) (via T4 and T3 deiodination and amino acid decarboxylation), as well as for TH structural analogs, such as 3,5,3'-triiodothyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-l-thyronine (L-940901), N-[3,5-dimethyl-4-(4'- hydroxy-3'-isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4-[(4'-hydroxy- 3 '-isopropylbenzyl)-phenoxy] acetic acid (GC-1), 3,5-dichloro-4-[(4-hydroxy-3- isopropylphenoxy)phenyl] acetic acid (KB-141), and 3, 5 -diiodo thyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein comprises a prodrug or prohormone of T3, such as T4 thyroid hormone (e.g., thyroxine or L-3,5,3',5'-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator in the methods and composition provided herein is an iodothyronine composition described in U.S. Pat. No. 7,163,918.

In some embodiments, a medium described herein does not comprise a thyroid hormone.

Epidermal Growth Factor (EGF) Family

Embodiments of the disclosure relate to the use of growth factors from the EGF family as cell differentiation factors.

In some embodiments, the at least one growth factor from the EGF family in the methods and composition provided herein comprises betacellulin. An example of human betacellulin amino acid sequence is provided in GenBank Accession No.: AAB25452.1 (SEQ ID NO: 20). In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, or a functional fragment thereof. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises the amino acid sequence of SEQ ID NO: 20.

Human betacellulin amino acid sequence (GenBank: AAB25452.1; SEQ ID NO: 20) MDRAARCSGASSLPLLLALALGLVILHCVVADGNSTRSPETNGLLCGDPEENCAATTTQSKRKG HFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYIGARCERVDLFYLRGDRGQILVICLIAVMV VFIILVIGVCTCCHPLRKRRKRKKKEEEMETLGKDITPINEDIEETN

In some embodiments, at least one growth factor from the EGF family in the methods and composition provided herein comprises EGF. Epidermal growth factor (EGF) is a 53 amino acid cytokine which is proteolytically cleaved from a large integral membrane protein precursor. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises a variant EGF polypeptide, for example an isolated epidermal growth factor polypeptide having at least 90% amino acid identity to the human wild-type EGF polypeptide sequence, as disclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises an engineered EGF mutant that binds to and agonizes the EGF receptor, as is disclosed in U.S. Pat. No. 8,247,531. Non-limiting examples of amino acid sequences of growth factors from the EGF family that may be used in the compositions and methods described are provided below. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises an amino acid sequence that is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-32, or a functional fragment thereof. In some embodiments, the growth factor from the EGF family used in the compositions and methods described herein comprises the amino acid sequence of any one of SEQ ID NO: 21-32.

Homo sapiens epidermal growth factor (WT) (Genbank: AAS83395.1; SEQ ID NO: 21) NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 22) NSDSECPLSHDGYCLHGGVCMYIKAVDRYACNCVVGYIGERCQYRDLTWWGPR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 23) NSDSECPLSHDGYCLHDGVCMYIKALDKYACNCVVGYTGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 24) NSNSECPLSHDGYCLHDGVCRYIEALDRYACNCVVGYIGERCQYGDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 25) NSDSGCPLSHSGYCLHDGVCMYIKALDRYACNCVVGYAGERCQYRDLRWWARR Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 26) TRGSECPLSHDGYCLHDGVCMYIGALDRYACNCVVGYTGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 27) NSDFGCPLSYDGYCLHDGVCMYIKALDKYACNCVVGYAGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 28) SRGSKCPPSHDGYCLHDGVCMYIEALDRYACNCVVGYAGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 29) SSGSECPSSHDGYCLHDGACMYIEALDRYACNCAVGYAGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 30) SSNSECPPSHDGYCLHDGVCMYIEALDRYACNCVVGYAGERCQYRDLRWWARR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 31) NSYSECPPSYDGYCLHDGVCRYIEALDSYACNCVVGYAGERCQYRDLRWWGRR

Homo sapiens epidermal growth factor (mutant) (SEQ ID NO: 32) SSGSECPLSHDGYCLNDGVCMYIEALDKYACNCVVGYVGERCQYRDLRWWARR

In some embodiments, the at least one growth factor from the EGF family in the methods and composition provided herein is replaced with an agent that activates a signaling pathway in the EGF family. In some embodiments, the growth factor from the EGF family in the methods and composition provided herein comprises a compound that mimics EGF. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a growth factor from the EGF family.

In some embodiments, a medium described herein does not comprise a EGF family growth factor.

Epigenetic Modifying Compounds

Embodiments of the disclosure relate to the use of epigenetic modifying compound as cell differentiation factors.

The term “epigenetic modifying compound” can refer to a chemical compound that can make epigenetic changes genes, z.e., change gene expression(s) without changing DNA sequences. Epigenetic changes can help determine whether genes are turned on or off and can influence the production of proteins in certain cells, e.g., beta-cells. Epigenetic modifications, such as DNA methylation and histone modification, can alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. These processes can be crucial to normal development and differentiation of distinct cell lineages in the adult organism. They can be modified by exogenous influences, and, as such, can contribute to or be the result of environmental alterations of phenotype or pathophenotype. Importantly, epigenetic modification can have a crucial role in the regulation of pluripotency genes, which become inactivated during differentiation. Non-limiting exemplary epigenetic modifying compound include a DNA methylation inhibitor, a histone acetyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor, a bromodomain inhibitor, or any combination thereof.

In an embodiment, the histone methyltransferase inhibitor is an inhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is a histone-lysine N-methyltransferase enzyme. Non-limiting examples of an EZH2 inhibitor that can be used in the methods provided herein include 3- deazaneplanocin A (DZNep), EPZ6438, EPZ005687 (an S-adenosylmethionine (SAM) competitive inhibitor), Ell, GSK126, and UNC1999. DZNep can inhibit the hydrolysis of S- adenosyl-L-homocysteine (SAH), which is a product-based inhibitor of all protein methyltransferases, leading to increased cellular concentrations of SAH which in turn inhibits EZH2. DZNep may not be specific to EZH2 and can also inhibit other DNA methyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor that has 150-fold selectivity over EZH1. UNC1999 is an analogue of GSK126, and it is less selective than its counterpart GSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. In some embodiments, the histone methyltransferase inhibitor is UNC0321. In an embodiment, the HD AC inhibitor is a class I HD AC inhibitor, a class II HD AC inhibitor, or a combination thereof. In an embodiment, the HD AC inhibitor is KD5170 (mercaptoketone-based HD AC inhibitor), MC1568 (class Ila HDAC inhibitor), TMP195 (class Ila HDAC inhibitor), or any combination thereof. In some embodiments, HDAC inhibitor is vorinostat, romidepsin (Istodax), chidamide, panobinostat (farydak), belinostat (PXD101), panobinostat (LBH589), valproic acid, mocetinostat (MGCD0103), abexinostat (PCI- 24781), entinostat (MS-275), SB939, resminostat (4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), HB 1-8000, (a benzamide HD I), kevetrin, CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME- 344, sulforaphane, or any variant thereof.

In some embodiments, a medium described herein does not comprise an epigenetic modifying compound.

Protein Kinase Inhibitors

Embodiments of the disclosure relate to the use of protein kinase inhibitors as cell differentiation factors.

In some embodiments, the protein kinase inhibitor in the methods and composition provided herein comprises staurosporine. In some embodiments, the protein kinase inhibitor in the methods and composition provided herein comprises an analog of staurosporine. Exemplary analogs of staurosporine in the methods and composition provided herein include, without limitation, Ro-31-8220, a bisindolylmaleimide (Bis) compound, 10'-{5"-

[(methoxycarbonyl)amino]-2 -methyl }-phenylaminocarbonylstaurosponne, a staralog (see, e.g., Lopez et al., “Staurosporine-derived inhibitors broaden the scope of analog- sensitive kinase technology”, J. Am. Chem. Soc. 2013; 135(48): 18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor in the methods and composition provided herein is an inhibitor of PKCp. In some embodiments, the protein kinase inhibitor in the methods and composition provided herein is an inhibitor of PKCP with the following structure or a derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKCP is a GSK-2 compound with the following structure or a derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKC in the methods and composition provided herein is a bisindolylmaleimide. Exemplary bisindolylmaleimides include, without limitation, bisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide Ill, hydrochloride, or a derivative, analogue or variant thereof.

In some embodiments, the PKC inhibitor in the methods and composition provided herein is a pseudohypericin, or a derivative, analogue, or variant thereof. In some embodiments, the PKC inhibitor in the methods and composition provided herein is indorublin-3-monoximc, 5- Iodo or a derivative, analogue or variant thereof. In certain embodiments, the methods, compositions, and kits disclosed herein exclude a protein kinase inhibitor.

In some embodiments, a medium described herein does not comprise a protein kinase inhibitor, or more specifically, does not comprise staurosporine.

Acetyl CoA-related metabolite

In some embodiments, a composition (e.g., medium) of the disclosure comprises an acetyl CoA-related metabolite. Metabolism of acetyl-coenzyme A (acetyl-CoA) can confer numerous metabolic functions, including energy production, lipid synthesis, and protein acetylation.

Exemplary acetyl CoA-related metabolites include, but are not limited to acetate, pyruvate, ketogenic amino acids, valine, leucine, isoleucine, phenylalanine, tyrosine, lysine, tryptophan, fatty acids, CoA, Isovaleryl-CoA, and P-hydroxybutyrate. In some embodiments, the acetyl CoA-related metabolite is acetate. In some embodiments, a composition of the disclosure contains two or more different acetyl CoA related metabolites, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different acetyl CoA-related metabolites. In some embodiments, the acetyl CoA- related metabolite is acetate.

In some embodiments, a medium described herein does not include an acetyl CoA-related metabolite (e.g., does not include acetate).

Histone deacetylase inhibitor (HDACi)

In some embodiments, a composition (e.g., medium) of the disclosure comprises a histone deacetylase inhibitor (HDACi). Histone deacetylase inhibitors (HDACi) are a class of compounds that increase acetylation of lysine residues on histone proteins as well as other, nonhistone, proteins by inhibiting the activity of HD AC enzymes.

Exemplary histone deacetylase inhibitors (HDACi) include, but are not limited to P- Hydroxybutyrate, butyric acid, class I HDACi, class IIA HDACi, class IIB HDACi, class III HDACi, class IV HDACi, HDAC-1, HD AC-2, HD AC-3, HD AC-4, HD AC-5, HD AC-6, HDAC- 7, HDAC-8, HDAC-9, HDAC-10, HDAC-11, sirtuins, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, Vorinostat (suberoylanilide hydroxamic acid, SAHA, MK0683), Entinostat (MS- 275, SNDX-275), Panobinostat (LBH589, NVP-LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103, MG0103), GSK3117391 (GSK3117391A, HDAC-IN-3), BRD3308, BRD3308, Tubastatin A TFA (Tubastatin A trifluoroacetate salt), Tubastatin A, SIS 17, NKL 22, BML-210 (CAY10433), TC-H 106, SR-4370, Belinostat (PXD101, NSC726630, PX-105684), Romidepsin (FK228, Depsipeptide, FR 901228, NSC 630176), MC1568, Givinostat (ITF2357), Dacinostat (LAQ824, NVP-LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), PCI- 34051, Droxinostat (NS 41080), Abexinostat (PCI- 24781), Abexinostat (PCI-24781, CRA- 024781), RGFP966, AR-42 (HDAC-42), Ricolinostat (ACY-1215, Rocilinostat), Valproic acid sodium salt (Sodium valproate), Tacedinaline (CI994, PD- 123654, GOE-5549, Acetyldinaline), Fimepinostat (CUDC-907), Sodium butyrate (NaB), Curcumin, Diferuloylmethane, M344, Tubacin, RG2833 (RGFP109), RG2833 (RGFP109), Resminostat (RAS2410), Divalproex Sodium, Scriptaid (GCK 1026), Sodium Phenylbutyrate, Sinapinic acid (Sinapic acid), TMP269, Santacruzamate A (CAY10683), TMP195 (TFMO 2), Valproic acid (VPA), UF010, Tasquinimod (ABR-215050), SKLB-23bb, Isoguanosine, Iforaphane, BRD73954, Citarinostat (ACY-241, HDAC-IN-2), Suberohydroxamic acid, plitomicin, HPOB, LMK-235, Biphenyl-4- sulfonyl chloride (p-Phenylbenzenesulfonyl, 4-henylbenzenesulfonyl, p-Biphenylsulfonyl), Nexturastat A, TH34, Tucidinostat (Chidamide, HBI-8000, CS-055), (-)-Parthenolide, WT161, CAY10603, CAY10603, ACY-738, RaddeaninA, Tinostamustine(EDO-SlOl), Domatinostat (4SC-202), and BG45.

In some embodiments, the HDACi is P-Hydroxybutyrate. P-Hydroxybutyric acid is a ketone body that, along with butyric acid, is an agonist of hydroxycarboxylic acid receptor 2 (HCA2), a Gi/o-coupled GPCR. In some embodiments, an HDACi inhibitor is an agonist of hydroxycarboxylic acid receptor 2.

In some embodiments, a medium described herein does not comprise an HDACi (e.g., does not include P-Hydroxybutyrate).

Supplemental Compositions

In some embodiments, any of the media or compositions disclosed herein comprises a supplemental composition. In some embodiments, the supplemental composition comprises insulin. In some embodiments, the supplemental composition comprises transferrin. In some embodiments, the supplemental composition comprises selenium (e.g., sodium selenite). In some embodiments, the supplemental composition comprises ethanolamine. In some embodiments, the supplemental composition comprises ITS-X. “ITS-X” refers to insulin- transferrin- selenium-ethanolamine supplement (e.g., as may be purchased from Gibco). In some embodiments, the ITS-X is equivalent to a (v:v) 1: 100-1:2000, 1:100-1:1500, 1:500-1:1500, 1:800-1:1200, 1:900-1:1100, or 1:1000 dilution of a 100X concentration composition comprising 1000 mg/L insulin, 550 mg/L transferring, 0.67 mg/L, and 200 mg/L ethanolamine. In some embodiments, the supplemental composition comprises B27.

Redox homeostasis regulator

In some embodiments, a composition (e.g., medium) of the disclosure comprises a redoxhomeostasis regulator. Exemplary redox homeostasis regulators include, but are not limited to taurine, respiratory chain regulators, free radical scavengers, regulators of mitochondrial protein synthesis, allium sulphur compounds, anthocyanins, beta-carotene, catechins, copper, cryptoxanthins, flavonoids, indoles, isoflavonoids, lignans, lutein, lycopene, alpha lipoic acid, ellagic acid, manganese, polyphenols, selenium, glutathione, vitamin A, vitamin C, vitamin E, zinc, superoxide disutases, GSHPx, Prx-I, catalase, and co-enzyme Q10.

In some embodiments, the redox homeostasis regulator is taurine.

In some embodiments, a medium described herein does not comprise a redox homeostasis regulator.

Taurine is a non-proteinogenic B -amino sulfonic acid that can be derived from methionine and cysteine metabolism. In some embodiments, taurine can inhibit ROS generation within the respiratory chain.

In some embodiments, a medium described herein does not comprise a redox homeostasis regulator (e.g., does not include taurine).

One carbon metabolism pathway intermediate

In some embodiments, a composition (e.g., medium) of the disclosure comprises a one carbon metabolism pathway intermediate. One-carbon metabolism mediated by folate cofactors, supports multiple physiological processes including amino acid homeostasis (methionine, glycine and serine), biosynthesis of nucleotides (purines, thymidine), epigenetic maintenance, and redox defense.

Exemplary one carbon metabolism pathway intermediates include, but are not limited to formate, tetrahydrofolate (THF), 10-formylTHF; 5,10-meTHF; 5,10-meTHF; and 10- formylTHF.

In some embodiments, a medium described herein does not comprise a one carbon metabolism pathway intermediate (e.g., does not include formate).

Glutamine

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamine. Glutamine (Gin or Q) is an alpha-amino acid. Glutamine can be an essential amino acid within in vitro cell cultures. Glutamine supports the growth of cells, including cells that have high energy demands and synthesize large amounts of proteins and nucleic acids. It is an alternative energy source for rapidly dividing cells and cells that use glucose inefficiently.

In some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability. Because of its chemical instability and importance for cell growth and function, it is important that delivery of L-glutamine be tailored to each unique cell culture process. Glutamine (e.g., L- glutamine) in a free form can be unstable at physiological pH in liquid media, breaking down to ammonium and pyroglutamate at rates that make it a problem in many cell culture and biomanufacturing applications. Therefore, many cell culture media contain stabilized forms of glutamine, including dipeptide forms, such as alanyl-l-glutamine and glycyl-l-glutamine. However, these more stable forms of L-glutamine can also have limited bioavailability, for example, due to a requirement for processing by enzymes, such as cell surface peptidases. Thus in some embodiments, compositions and methods of the disclosure utilize glutamine in a form with increased bioavailability, such as a free glutamine form, such as a non-dipeptide form, a non-alanine-glutamine dipeptide form (e.g., a non-alanyl-l-glutamine form), a non-glycine-glutamine dipeptide form (e.g., a non-glycyl-l-glutamine form), a form that in which glutamine is not conjugated to another amino acid or stabilizing moiety, a monomeric form, a free form, or a combination thereof. In some embodiments, glutamine is provided as a protein hydrolysate.

In some embodiments, a basal media contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a media that already contains glutamine. In some embodiments, glutamine in a form as disclosed herein is added to a basal media that contains no glutamine or only low levels of glutamine to increase the bioavailability of glutamine.

In some embodiments, a medium described herein does not comprise glutamine.

Glutamate

In some embodiments, a composition (e.g., medium) of the disclosure comprises glutamate (e.g., L- glutamate). Glutamate can be converted into, for example, g-amino butyric acid (GABA), ornithine, 2-oxoglutarate, glucose or glutathione. Glutamate and metabolites generated therefrom can contribute to, for example, redox homeostasis, cell signaling, nitrogen assimilation, amine catabolism, amino acid biosynthesis, nucleoside biosynthesis, and cofactor production.

In some embodiments, contacting cells with glutamate can improve production of SC-P cells in vitro, for example, providing higher cell yields and recoveries, increased numbers and relative percentages of SC-P cells, enhanced stability and shelf-life of SC-P cells, SC-islet clusters with advantageous characteristics such as reduced size and increased uniformity, improved function of the SC-P cells in vitro, improved cell viability, improved cell function, reduced immunogenicity after transplantation, or a combination thereof, e.g., relative to a composition that lacks glutamate, or contains a lower concentration of glutamate.

In some embodiments, a medium described herein does not comprise glutamate. Vitamins

In some embodiments, a composition (e.g., medium) of the disclosure comprises one or more vitamins.

Exemplary vitamins include, but are not limited to biotin, vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine) and vitamin B12 (cyanocobalamin). In some embodiments the vitamin modulates fatty acid synthesis. In some embodiments the vitamin modulates branched-chain amino acid metabolism. In some embodiments the vitamin modulates or participates as a co-factor in the TCA cycle, e.g., as a cofactor for pyruvate carboxylase. In some embodiments, the vitamin is biotin. In some embodiments, a composition of the disclosure contains two or more different vitamins, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different vitamins.

In some embodiments, a medium described herein does not comprise a vitamin.

Water-soluble synthetic polymer

Water-soluble polymer described herein can refer to any polymer that has hydrophilic property and is soluble in aqueous solution at room temperature. The water-soluble polymer can be either naturally occurring or synthetic. In some embodiments, a water-soluble polymer is an albumin protein (e.g., human serum albumin or bovine serum albumin). In some embodiments, the water-soluble polymer is a water-soluble synthetic polymer. Water-soluble synthetic polymers described herein can refer to any synthetic polymer that has hydrophilic property and is soluble in aqueous solution at room temperature. Water-soluble synthetic polymers applicable in the subject methods and compositions include, but not limited to, poloxamer (e.g., P188 or PF68), polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol (PEG), PEG copolymers, poly(Nisopropylacrylamide), and polyacrylamide. The water-soluble synthetic polymer can refer to a polymer compound or a mixture of polymer compounds that may have an idealized chemical formula but a variety of derivatives and/or precursors of the idealized formula, depending on the applicable manufacturing method. In some embodiments, the water-soluble synthetic polymer is used to replace at least partially serum or serum albumin, e.g., BSA or HSA, that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the water-soluble synthetic polymer replaces 100% of serum albumin, e.g., BSA or HSA, that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the water-soluble synthetic polymer reduces the amount of serum albumin, e.g., BSA or HSA, by at least 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of that is typically utilized in cell differentiation, e.g., differentiation of pancreatic P cells or precursor cells thereof. In some embodiments, the disclosure provides for a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; primitive gut cells; PDX1 -positive, NKX6.1- negative pancreatic progenitor cells; PDX1 -positive, NKX6.1-positive pancreatic progenitor cells; insulin-positive cells; and/or pancreatic beta cells) and water soluble polymers, wherein at least 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of the water soluble polymers in the composition are water-soluble synthetic polymers (e.g., any of the PVA molecules disclosed herein) and wherein the remainder of the water soluble polymers are human serum albumin polypeptides. In some embodiments, the disclosure provides for a composition comprising a population of any of the cells disclosed herein (e.g., pluripotent stem cells; endoderm cells; primitive gut cells; PDX1 -positive, NKX6.1 -negative pancreatic progenitor cells; PDX1- positive, NKX6.1-positive pancreatic progenitor cells; insulin-positive cells; and/or pancreatic beta cells) and water soluble polymers, wherein no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% of the water soluble polymers are naturally occurring water-soluble polymers (e.g., HSA or BSA). In some embodiments, more than 90%, 95%, 99%, and up to 100% of the water-soluble polymers in the composition are water-soluble synthetic polymers (e.g., PVA).

In some embodiments, the media comprises a poloxamer. In some embodiments, the poloxamer comprises 50-90%, 60-70%, 70-90%, 70-80%, 80-90%, 75-85%, or 78-82% polyoxyethylene content. In some embodiments, the poloxamer comprises greater than 75% polyoxyethylene content. In some embodiments, the poloxamer comprises 75-85% polyoxyethylene content. In some embodiments, the poloxamer comprises 1-50%, 1-30%, 1- 20%, 10-20%, 10-30%, 15-25%, or 18-22% polyoxypropylene. In some embodiments, the poloxamer comprises a polyoxypropylene core having a molecular mass of 3,000-7,000 g/mol, 3,000-6,000 g/mol, 4,000-6,000 g/mol, 5,000-6,000 g/mol, 5,300-5,500 g/mol or around 5,400 g/mol. In some embodiments, the molecular weight of the poloxamer is 6,000-10,000 kDa, 6,000-9,000 kDa, 7,000-9,000 kDa, 8,000-10,000 kDa, 8,000-9,000 kDa, 8,200-8,800 kDa, 8,300-8,700 kDa, 8,400-8,600 kDa, or around 8,500 kDa. In some embodiments, the poloxamer is poloxamer- 188. In some embodiments, the poloxamer is poloxamer 407. In some embodiments, the poloxamer is present at a concentration of 0.01-5%, 0.01-2%, 0.01-1%, 0.01- 0.1%, 0.1-1%, or 0.1-0.6%, 0.2-0.4%, or around 0.3%. In particular embodiments, the poloxamer is present at a concentration of 0.2-0.4%. In some embodiments, the poloxamer is present at a concentration of 0.5%-5%, 0.5%-3%, 0.5-1.5%, 0.8-1.2%, 0.9-1.1%, or around 1%. In some embodiments, the poloxamer is present at a concentration of 0.9- 1.1%.

In some embodiments, the water-soluble synthetic polymer applicable to the subject compositions and methods includes polyvinyl alcohol (PVA). Polyvinyl alcohol described herein can refer to a water-soluble synthetic polymer that has an idealized formula [CH2CH(0H)]n, which can be either partially or completed hydrolyzed. In some embodiments, the polyvinyl alcohol is manufactured by either partial or complete hydrolysis of polyvinyl acetate to remove acetate groups. In some embodiments, the polyvinyl alcohol is at most 85% hydrolyzed, e.g., 80% hydrolyzed. The percentage of hydrolyzation measures the approximate percentage (e.g., average percentage) of acetate residue that is hydrolyzed in the polyvinyl acetate precursor polymer. In some embodiments, the polyvinyl alcohol is at least 85% hydrolyzed, e.g., 87-89% hydrolyzed, 87-90% hydrolyzed, or 99% hydrolyzed. In some embodiments, the polyvinyl alcohol is 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% hydrolyzed. Without wishing to be bound by a certain theory, the polyvinyl alcohol can assume a function of carrier-molecule in the culture medium, which is typically carried out by serum or serum albumin, e.g., HSA. The percentage of hydrolyzation of polyvinyl alcohol can be determined by the manufacturing method utilized to produce the polyvinyl alcohol, e.g., how polyvinyl acetate precursor polymer is converted into polyvinyl alcohol, e.g., conversion by base-catalyzed transesterification with ethanol. In some embodiments, the water-soluble synthetic polymer preparation, e.g., polyvinyl alcohol, that is used in the subject method or present in the subject composition has purity of at least 90%, such as at least 92%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or nearly 100%. Purity of polyvinyl alcohol measures the percentage of synthetic polymer that has the idealized formula [CH2CH(OH)]n in the preparation, which includes polyvinyl alcohol of any percentage of hydrolyzation. Impurity of polyvinyl alcohol preparation can include other polymer materials that do not have the idealized formula [CH2CH(OH)]n, or other organic inorganic materials.

In some embodiments, a medium described herein does not comprise a water-soluble synthetic polymer.

Stem cell derived pancreatic islet cells, compositions and method of use

In some embodiments, a population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) produced using the compositions, methods, and bioreactors and/or TFF systems described herein are also provided. In some embodiments, the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) comprises NKX6.1-positive, ISLl-positive cells and NKX6.1 -negative, ISLl-positive cells. In some embodiments, the population comprises more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl- positive cells. In some embodiments, at least 15% of the cells in the population are NKX6.1- negative, ISLl-positive cells; and wherein less than 12% of the cells in the population are NKX6.1 -negative, ISLl-negative cells. In some embodiments, methods of using the population of in vitro differentiated cells (e.g., stem cell-derived pancreatic islet cells) described herein to treat diseases (e.g., diabetes) are provided. In some embodiments, the cells in any of the cell populations disclosed herein have not been previously subjected to a cell-sorting process (e.g., affinity binding purification or FACS).

In some embodiments, the disclosure provides for a composition comprising any of the cell populations described herein (e.g., any of the cell populations described in this section). In some embodiments, the disclosure provides for method of contacting/culturing any of the cell populations disclosed herein with any of the reagents disclosed herein.

In some embodiments, the disclosure provides for a composition comprising any of the cell populations described in any of WO2012175633; WO2015028614; WO2017144695; W02019048690; W02020207998; WO2022043518; W02023203208; W02024008810; WO2025029826; WO2025093467; Wang et al., 2024, Cell, 187:1-13; Wu et al., 2024, Cell Discovery, 10(45); WO2025117331; US11,274,28O; Hogrebe et al., Nat Protoc., 2021, 16(9): 4109-4143; Vegas et al., 2016, Nat Med. 2016 Mar; 22(3): 306-311; Wu et al., 2024, Cell Discovery, 10(45); and Ratiu et al., 2023, bioRxiv, https://doi.org/10.1101/2023.10.20.563345, each of which is herein incorporated by reference in its entirety. In some embodiments, the disclosure provides for any of the bioreactors, TFF systems, and or methods disclosed herein for use with the cell growth and/or differentiation methods described in any of WO2012175633; WO2015028614; WO2017144695; W02019048690; W02020207998; WO2022043518; W02023203208; W02024008810; US11,274,280; Hogrebe et al., Nat Protoc., 2021, 16(9): 4109-4143; Vegas et al., 2016, Nat Med. 2016 Mar; 22(3): 306-311; Wu et al., 2024, Cell Discovery, 10(45); and Ratiu et al., 2023, bioRxiv, https://doi.org/10.1101/2023.10.20.563345, each of which is herein incorporated by reference in its entirety.

In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1-positive, ISLl-positive cells and NKX6.1 -negative, ISLl-positive cells. In some embodiments, the population comprises more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl-positive cells.

In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein at least 30% of the cells in the composition are NKX6.1-positive, ISLl-positive cells; wherein at least 25% of the cells in the composition are NKX6.1 -negative, ISLl-positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl-positive cells in the composition; and wherein less than 12% of the cells in the composition are NKX6.1- negative, ISLl-negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-60%, 35- 55%, 35-50%, 35-45%, 35-40%, 40-60%, 40-55%, 40-50%, 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, or 50-55% of the cells in the composition are NKX6.1-positive, ISLl-positive cells; wherein 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-45%, 25-40%, 25- 35%, 25-30%, 30-50%, 30-45%, 30-40%, 30-35%, 35-50%, 35-35%, 35-40%, 40-50%, 40-45%, or 45-50% of the cells in the composition are NKX6.1 -negative, ISLl-positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl-positive cells in the composition; and wherein 0-1%, 0-0.5%, 0.5-1%, 1-12%, 1-10%, 1-8%, 1-6%, 1-4%, 3- 5%, 1-2%, 2-12%, 2-10%, 2-8%, 2-6%, 2-4%, 4-12%, 4-10%, 4-8%, 4-6%, 6-12%, 6-10%, 6- 8%, 8-12%, 8-10%, or 10-12% of the cells in the composition are NKX6.1 -negative, ISL1- negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein 35-50% of the cells in the composition are NKX6.1-positive, ISLl-positive cells; wherein 30-45% of the cells in the composition are NKX6.1 -negative, ISLl-positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1-negative, ISLl- positive cells in the composition; and wherein 2-12% of the cells in the composition are NKX6.1 -negative, ISLl-negative cells. In some embodiments, between 5-25%, 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-15%, 15-25%, 15-20% or 20-25% of the cells in the composition are NKX6.1-positive, ISLl-negative cells.

In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein at least 30% of the cells in the composition are NKX6.1-positive, ISLl-positive cells; wherein at least 25% of the cells in the composition are NKX6.1 -negative, ISLl-positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl-positive cells in the composition; and wherein between 9-25% of the cells in the composition are NKX6.1- positive, ISLl-negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-60%, 35- 55%, 35-50%, 35-45%, 35-40%, 40-60%, 40-55%, 40-50%, 40-45%, 45-60%, 45-55%, 45-50%, 50-60%, or 50-55% of the cells in the composition are NKX6.1-positive, ISLl-positive cells; wherein 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 25-50%, 25-45%, 25-40%, 25- 35%, 25-30%, 30-50%, 30-45%, 30-40%, 30-35%, 35-50%, 35-35%, 35-40%, 40-50%, 40-45%, or 45-50% of the cells in the composition are NKX6.1 -negative, ISLl-positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1 -negative, ISLl-positive cells in the composition; and wherein 9-30%, 9-25%, 9-20%, 9-15%, 9-12%, 12-30%, 12-25%, 12- 20%, 12-15%, 15-30%, 15-25%, 15-20%, 20-30%, 20-25% or 25-30% of the cells in the composition are NKX6.1 -positive ISLl-negative cells. In some embodiments, 1-12%, 1-10%, 1- 8%, 1-6%, 1-4%, 3-5%, 1-2%, 2-12%, 2-10%, 2-8%, 2-6%, 2-4%, 4-12%, 4-10%, 4-8%, 4-6%, 6-12%, 6-10%, 6-8%, 8-12%, 8-10%, or 10-12% of the cells in the composition are NKX6.1- negative, ISLl-negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of cells (e.g., a composition comprising a cluster of cells or multiple clusters of cells); wherein 35-50% of the cells in the composition are NKX6.1-positive, ISL1- positive cells; wherein 30-45% of the cells in the composition are NKX6.1 -negative, ISL1- positive cells; wherein there are more NKX6.1-positive, ISLl-positive cells than NKX6.1- negative, ISLl-positive cells in the composition; and wherein 9-25% of the cells in the composition are NKX6.1 -positive, ISLl-negative cells.

In some embodiments, less than 12% of the cells (e.g., about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) in the population are NKX6.1 -negative, ISLl-negative cells. In some embodiments, less than 10%, less than 8%, less than 6%, less than 4%, 1%- 11%, 2%-10%, 2%-12%, 4%-12%, 6%-12%, 8%- 12%, 2%-8%, 4%-8%, 3%-6% or 3%-5% of the cells in the population are NKX6.1 -negative, ISLl-negative cells. In some embodiments, 2%-12%, 4%-12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6% or 3%-5% of the cells in the population are NKX6.1 -negative, ISLl-negative cells.

In some embodiments, at least 15% of the cells (e.g., about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% or more) in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, 15%-60%, 15%-45%,15%-30%, 30%-60%, 30%-45%, 45%-60% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, 20%- 60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the cells in the population are NKX6.1 -negative, ISLl-positive cells.

In some embodiments, at least 15% (e.g., 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60%) of the cells in the population are NKX6.1 -negative, ISLl-positive cells and less than 12% (e.g., 2%-12%, 4%- 12%, 6%-12%, 8%-12%, 2%-8%, 4%-8%, 3%-6% or 3%-5%) of the cells in the population are NKX6.1 -negative, ISLl-negative cells.

In some embodiments, at least 60%, at least 65%, at least 70%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISLl-positive cells. In some embodiments, 50-90%, 50-85%, 50- 80%, 50-75%, 50-70%, 50-60%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-90%, 70-85%, 70-80%, 70-75%, 75-90%, 75-85%, 75-80%, 80- 90%, 80-85%, or 85-90% of the cells in the population are ISLl-positive cells. In some embodiments, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, about 85-95%, or about 90-95% of the cells in the population are ISLl-positive cells. In some embodiments, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% of the cells in the population are ISLl-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises more NKX6.1 -negative, ISLl-positive cells than NKX6.1-positive, ISLl-positive cells. In some embodiments, the population comprises more NKX6.1-positive, ISLl-positive cells that NKX6.1 -negative, ISLl-positive cells. In some embodiments, at least 40% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, at least 45%, at least 50%, about 40-50%, about 45-55%, or about 50-55% of the cells in the population are NKX6.1 -negative, ISLl-positive cells. In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or about 55% of the cells in the population are NKX6.1 -negative, ISLl-positive cells.

In some embodiments, at least 20% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60% or more) of the ISLl-positive cells are NKX6.1 -negative. In some embodiments, about 20%-60%, 20%-50%, 20%-40%, 20%-30%, 30%-60%, 30%-50%, 30%-40%, 40%-60%, 40%-50%, or 50%-60% of the ISLl-positive cells are NKX6.1-negative. In some embodiments, about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more of the ISLl-positive cells are NKX6.1-negative.

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 20% (e.g., at least 20%, 30%, 40%, 50% or 60%) of NXK6.1-positive, ISLl- positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, 40%-50%, 40%- 60%, or 50-60% of NXK6.1-positive, ISLl-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% of NXK6.1 -positive, ISLl-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises less than 25% (e.g., less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less) of NKX6.1-positive, ISLl-negative cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 2%-25%, 2%-20%, 2%-15%, 2%-10%, 2%-5%, 5%-25%, 5%-20%, 5%-15%, 5%-10%, 10%-25%, 10%-20%, 10%-15%, 15%-25%, 15%-20%, or 20%-25% of NKX6.1 -positive, ISLl-negative cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 2%- 10%, 2%-8%, 2%-6%, 2%-4%, 4%-10%, 4%-8%, 4%-6%, 6%-10%, 6%-8%, or 8%-10% of NKX6.1-positive, ISLl-negative cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 2%, 4%, 6%, 8%, or 10% of NKX6.1- positive, ISLl-negative cells.

In some embodiments, the disclosure provides for ISLl-positive; NKX6.1 -positive cells. In some embodiments, the disclosure provides for a composition comprising a plurality of ISLl- positive; NKX6.1 -positive cells. In some embodiments, 20%-50%, 20%-40%, 20%-30%, 30%- 50%, 30%-40%, 40%-50%, 40%-60%, or 50-60% of the cells in the composition are NXK6.1- positive, ISLl-positive cells. In some embodiments, the disclosure comprises a method of contacting (or culturing) a plurality of cells with any of the reagents disclosed herein, wherein 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, 40%-50%, 40%-60%, or 50-60% of the cells in the composition are NKX6.1 -positive; ISLl-positive cells.

In some embodiments, the disclosure provides for ISLl-positive; NKX6.1 -negative cells. In some embodiments, the disclosure provides for a composition comprising a plurality of ISLl- positive; NKX6.1 -negative cells. In some embodiments, 20%-50%, 20%-40%, 20%-30%, 30%- 50%, 30%-40%, 40%-50%, 40%-60%, or 50-60% of the cells in the composition are NXK6.1- negative, ISLl-positive cells. In some embodiments, the disclosure comprises a method of contacting (or culturing) a plurality of cells with any of the reagents disclosed herein, wherein 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, 40%-50%, 40%-60%, or 50-60% of the cells in the composition are NKX6.1 -negative; ISLl-positive cells.

In some embodiments, the disclosure provides for ISLl-positive cells. In some embodiments, the disclosure provides for a composition comprising or a method of contacting a plurality of ISLl-positive cells. In some embodiments, 50-60%, 50-90%, 60-70%, 60-90%, 70- 90%, 70-85%, 70-80%, 80-95%, or 90-95% of the cells in the composition express ISL1. In some embodiments, the disclosure comprises a method of contacting (or culturing) a plurality of cells with any of the reagents disclosed herein, wherein 50-60%, 50-90%, 60-70%, 60-90%, 70- 90%, 70-85%, 70-80%, 80-95%, or 90-95% of the cells in the composition express ISL1.

In some embodiments, the disclosure provides for CHGA-positive cells. In some embodiments, the disclosure provides for a composition comprising a plurality of CHGA- positive cells. In some embodiments, 70-100%, 70-95%, 70-90%, 70-85%, 70-80%, 85-100%, 85-95%, 90-100%, 90-99%, 90-98%, 90-97%, 90-96%, 90-95%, or 90-93% of the cells in the composition express chromogranin A (CHGA). In some embodiments, the disclosure comprises a method of contacting (or culturing) a plurality of cells with any of the reagents disclosed herein, 70-100%, 70-95%, 70-90%, 70-85%, 70-80%, 85-100%, 85-95%, 90-100%, 90-99%, 90- 98%, 90-97%, 90-96%, 90-95%, or 90-93% of the cells in the composition express chromogranin A (CHGA).

In some embodiments, 0-10%, 0-5%, 0-2%, 0.5-1%, 0.5-2%, 1-3%, 3-10%, or none of the cells in any of the compositions disclosed herein are ISL1 -negative; NKX6.1 -negative cells. In some embodiments, the disclosure comprises a method of contacting (or culturing) a plurality of cells with any of the reagents disclosed herein, wherein 0-10%, 0-5%, 0-2%, 0.5-1%, 0.5-2%, 1-3%, 3-10%, or none of the cells in the plurality of cells are ISLl-negative; NKX6.1 -negative cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises ghrelin-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises less than 5% (e.g., less than 5%, less than 3%, less than 2%, less than 1% or less than 0.5%) ghrelin-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises at least 0.05% (e.g., at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 3%, 4%, or 5%) ghrelin-positive cells. In some embodiments, a population of in vitro differentiated cells described herein comprises 1-5%, 2-5%, 3-5%, 0.1-5%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-5%, 0.5-3%, 0.5-2%, 0.5- 1%, 0.5-0.8%, 0.05-1%, 0.05-0.7%, or 0.05-2% ghrelin-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises ARX-positive cells. In some embodiments, at least 4% (e.g., at least 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 35%, 40%, or 50%) of the cells in the population are ARX-positive cells. In some embodiments, less than 50% (e.g., less than 50%, 40%, 35%, 30%, 28%, 26%, 24%, 22%, 20%, 18%, 16%, 14%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%) of the cells in the population are ARX-positive cells. In some embodiments, the population of cells comprises between 4-50%, 4-40%, 4-30%, 4-25%, 4-20%, 4-15%, 4-10%, 4-8%, 8-50%, 8-40%, 8-30%, 8-25%, 8-20%, 8-15%, 8-10%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, or 10-15% ARX-positive cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises cells that express insulin (e.g., cells that express insulin but not glucagon or somatostatin), cells that express glucagon (e.g., cells that express glucagon but not insulin or somatostatin), and cells that express somatostatin (e.g., cells that express somatostatin but not insulin or glucagon). In some embodiments, the expression of insulin in a cell of the compositions suggests that the cell is a SC-P cell. In some embodiments, the expression of glucagon and not expressing somatostatin in a cell of the composition suggests that the cell is a SC-a cell. In some embodiments, the expression of somatostatin and not expressing glucagon in a cell of the composition suggests that the cell is a SC-5 cell. In some embodiments, cells that express insulin are also glucose responsive insulin producing cells.

In some embodiments, cells that express insulin (i.e., SC-P cells) in a population of in vitro differentiated cells described herein exhibit glucose stimulated insulin secretion (GSIS). In some embodiments, cells that express insulin (i.e., SC-P cells) in a population of in vitro differentiated cells described herein further mature (e.g., further maturing in a subject after transplantation) into cells that exhibit glucose stimulated insulin secretion (GSIS).

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 5% or at least 10% (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more) of stem cell-derived alpha cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 5%-50%, 10%-50%, 5%-25%, 5%-20%, 5%-15%, 10%-40%, 10%-30%, 10%-20%, 20%-50%, 20%-40%, 20%-30%, 30%-50%, 30%-40%, or 40%-50% of stem cell-derived alpha cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of stem cell-derived alpha cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 30% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% or more) of stem cell-derived beta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 30%-60%, 30%-50%, 30%-40%, 35%-60%, 35%-55%, 35%-50%, 35%-45%, 35%-40%, 40%-60%, 40%-55%, 40%- 50%, 40%-45%, 45%-60%, 45%-55%, 45%-50%, 509%-60%, 50%-55%, or 55%-60% of stem cell-derived beta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 35%, 40%, 45%, 50%, 55%, or 60% of stem cell-derived beta cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 4% (e.g., at least 4%, at least 5%, at least 10%, at least 15%, at least 20% or more) of stem cell-derived delta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 5%-20%, 5%- 15%, 5%-10%, 10%-20%, 10%- 15%, or 15%-20% of stem cell-derived delta cells. In some embodiments, a population of in vitro differentiated cells described herein comprises about 4%, 5%, 10%, 15%, or 20% of stem cell-derived delta cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises less than 5% (e.g., less than 5%, less than 3%, less than 2%, less than 1% or less than 0.5%) stem cell-derived epsilon cells. In some embodiments, a population of in vitro differentiated cells described herein comprises at least 0.05% (e.g., at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 3%, 4%, or 5%) stem cell-derived epsilon cells. In some embodiments, a population of in vitro differentiated cells described herein comprises 1-5%, 2-5%, 3-5%, 0.1-5%, 0.1-3%, 0.1-2%, 0.1-1%, 0.5-5%, 0.5-3%, 0.5-2%, 0.5- 1%, 0.5-0.8%, 0.05-1%, 0.05-0.7%, or 0.05-2% stem cell-derived epsilon cells.

In some embodiments, a population of in vitro differentiated cells described herein comprises at least 5% of (e.g., about 5%, 6%, 7%, 8%, 9%, or 10%) cells that are glucagonpositive and somatostatin-negative. In some embodiments, a population of in vitro differentiated cells described herein comprises at least 5% (e.g., about 5%, 6%, 7%, 8%, 9%, or 10%) of cells that are glucagon-negative and somatostatin-positive.

In some embodiments, a population of in vitro differentiated cells described herein comprises: (a) 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%- 90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, 70%-90%, 70%-80%, or 80%-90% of the cells in the population of cells express insulin; (b) 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%- 20%, 5%-15%, 5%-10%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 10%-15%, 15%-40%, 15%-35%, 15%-30%, 15%-25%, 15%-20%, 20%-40%, 20%-35%, 20%-30%, 20%- 25%, 25%-40%, 25%-35%, 25%-30%, 30%-40%, 30%-35% or 35%-40% of the cells in the population of cells express glucagon but not somatostatin; and/or (c) 3%-20%, 3%- 15%, 3%- 12%, 3%-10%, 3%-8%, 3%-5%, 4%-20%, 4%-15%, 4%-12%, 4%-10%, 4%-8%, 4%-5%, 5%- 20%, 5%-15%, 5%-12%, 5%-10%, 5%-8%, 7%-20%, 7%-15%, 7%-12%, 7%-10%, 9%-20%, 9%-15%, 9%-12%, 8%-10%, 8%-12%, 8%-15%, 8%-20%, 10%-20%, 10%-12%, 10%-15%, 12%-20%, 12%- 15% or 15%-20% of the cells in the population of cells express somatostatin but not glucagon.

In some embodiments, a population of in vitro differentiated cells described herein comprises: (a) 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%- 90%, 60%-80%, 60%-70%, 70%-90%, 70%-80%, 70%-90%, 70%-80%, or 80%-90% of the cells in the population of cells express insulin; (b) 5%-40%, 5%-35%, 5%-30%, 5%-25%, 5%- 20%, 5%-15%, 5%-10%, 10%-40%, 10%-35%, 10%-30%, 10%-25%, 10%-20%, 10%-15%, 15%-40%, 15%-35%, 15%-30%, 15%-25%, 15%-20%, 20%-40%, 20%-35%, 20%-30%, 20%- 25%, 25%-40%, 25%-35%, 25%-30%, 30%-40%, 30%-35% or 35%-40% of the cells in the population of cells express glucagon but not somatostatin; and (c) 3%-20%, 3%- 15%, 3%-12%, 3%-10%, 3%-8%, 3%-5%, 4%-20%, 4%-15%, 4%-12%, 4%-10%, 4%-8%, 4%-5%, 5%-20%, 5%- 15%, 5%-12%, 5%-10%, 5%-8%, 7%-20%, 7%-15%, 7%-12%, 7%-10%, 9%-20%, 9%- 15%, 9%-12%, 8%-10%, 8%-12%, 8%-15%, 8%-20%, 10%-20%, 10%-12%, 10%-15%, 12%- 20%, 12%- 15% or 15%-20% of the cells in the population of cells express somatostatin but not glucagon.

In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1-positive, ISLl-positive cells that express lower levels of MAFA than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1-positive, ISLl-positive cells that express higher levels of MAFB than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1 -positive, ISLl- positive cells that express higher levels of SIX2, HOPX, IAPP and/or UCN3 than NKX6.1- positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1- positive, ISLl-positive cells that do not express MAFA. In some embodiments, a population of in vitro differentiated cells described herein comprises NKX6.1-positive, ISLl-positive cells that express MAFB. As defined herein, the healthy control adult subject is a non-diabetic subject with a healthy functioning pancreas.

In some embodiments, the population comprises one or more NKX6.1-positive, ISLl- positive cells that express CHGA, MAFB, and/or ESRRG at a higher level (e.g., at least 10%, 30%, 50%, 70%, 100%, 125%, 150%, or 200% higher) than a NKX6.1 -positive, ISLl-positive cell from the pancreas of a healthy control adult subject. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISLl-positive cells in a population of cells express CHGA, MAFB, and/or ESRRG at a higher level than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises one or more NKX6.1-positive, ISLl-positive cells that express SIX3, MAFA, CHGB, RBP4 and/or FXYD2 at a lower level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% lower) than a NKX6.1-positive, ISLl-positive cell from the pancreas of a healthy control adult subject. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISLl-positive cells in a population of cells express SIX3, MAFA, CHGB, RBP4 and/or FXYD2 at a lower level than at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1- positive, ISLl-positive cells that express lower levels of MAFA than NKX6.1-positive, ISL1- positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISLl-positive cells that express lower levels of SIX3 than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISLl-positive cells that express lower levels of CHGB than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1 -positive, ISLl-positive cells that express lower levels of RBP4 than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject. In some embodiments, the population comprises NKX6.1-positive, ISLl-positive cells that express lower levels of FXYD2 than NKX6.1-positive, ISLl-positive cells from the pancreas of a healthy control adult subject.

In some embodiments, a population of in vitro differentiated cells described herein comprises a C-peptide content per 1,000 of the in vitro differentiated cells of at least 300 pM (e.g., at least 300 pM, at least 400 pm, at least 500 pm, 300pm-500pm, 300pm-400pm, or 400pm-500pm). In some embodiments, a population of in vitro differentiated cells described herein comprises a glucagon content per 1,000 of the in vitro differentiated cells of at least 100 pM (e.g., at least 100pm, at least 200pm, at least 300 pM, at least 400 pm, at least 500 pm, at least 600pm, at least 700pm, at least 800pm, 100pm-800pm, 100pm-700pm, 100pm-600pm, 100pm-500pm, 100pm-400pm, 100pm-300pm, 100pm- 200pm, 200pm-800pm, 200pm-700pm, 200pm-600pm, 200pm-500pm, 200pm-400pm, 200pm-300pm, 300pm-800pm, 300pm-700pm, 300pm-600pm, 300pm-500pm, 300pm-400pm, 400pm-800pm, 400pm-700pm, 400pm-600pm, 400pm-500pm, 500pm-800pm, 500pm-700pm, 500pm-600pm, 600pm-800pm, 600pm-700pm, or 700pm-800pm).

In some embodiments, the percentage of cells expressing a marker provided herein is measured by flow cytometry. The skilled worker is aware of representative methods for testing whether a cell or collection of cells is positive or negative for expression of a specific gene marker (e.g., NKX6.1, ISL1, INS, GCG, ARX, or ghrelin) by flow cytometry. In some embodiments, a cell is considered positive for expression of a particular gene (e.g., NKX6.1, ISL1, INS, GCG, ARX, or ghrelin) based on median fluorescence intensity (rMFI). As used herein, the term “rMFI” or relative median fluorescence intensity is the ratio between the fluorescence intensity measured by use of an antibody to a specific target (e.g., NKX6.1, ISL1, INS, GCG, ARX, or ghrelin) versus the intensity obtained from a control antibody (isotype control). In some embodiments, an anti-(human) NKX6.1, ISL1, INS, GCG, ARX or ghrelin antibody is used. Examples of suitable antibodies for use in flow cytometry are any of the antibodies disclosed in Table 8. An example of a suitable flow cytometer is the Accuri 6 flow cytometer. In some embodiments, the target-expressing cells (e.g., cells expressing NKX6.1 and/or ISL1), if tested, exhibit a target relative medium fluorescence intensity (rMFI) of at least 6, 6.5, 7, 8, 9 or 10 as measured by flow cytometry. In another embodiment, said rMFI is between 6.5 and 15, between 6.5 and 14, between 6.5 and 13, between 6.5 and 13, between 6.5 and 12, or between 6.5 and 10.

In some embodiments, the percentage of cells expressing a marker provided herein is measured by qRT-PCR. In some embodiments, the percentage of cells expressing a marker provided herein is measured by single cell RNA sequencing analysis. The skilled worker is aware of methods for testing whether a cell or collection of cells is positive for expression of a specific gene marker (e.g., NKX6.1, ISL1, INS, GCG, ARX, or ghrelin) by single cell RNA sequencing analysis.

In some embodiments, cells in a population of in vitro differentiated cells described herein form cell clusters. The terms "cluster" and "aggregate" can be used interchangeably, and refer to a group of cells that have close cell-to-cell contact, and in some embodiments, the cells in a cluster can be adhered to one another. A cell cluster comprises a plurality of cells. In some embodiments, a cell cluster comprises at least 10, at least 50, at least 200, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 20,000, at least 30,000, or at least 50,000 cells. In some embodiments, a cell cluster comprises between 10-10,000 cells, between 50-10,000, between 100-10,000, between 100-10,000, between 1,000-10,000, between 500 and 10,000, between 500 and 5,000, between 500 and 2,500, between 500 and 2,000, between 1,000 and 100,000, between 1,000 and 50,000, between 1,000 and 40,000, between 1,000 and 20,000, between 1,000 and 10,000, between 1,000 and 5,000 and between 1,000 and 3,000 cells. In some embodiments, a cell cluster comprises at least 500 cells. In some embodiments, a cell cluster comprises at least 1,000 cells. In some embodiments, a cell cluster comprises at least 2,000 cells. In some embodiments, a cell cluster comprises at least 5,000 cells. In some embodiments, a cell cluster comprises no more than 100,000, no more than 90,000, no more than 80,000, no more than 70,000, no more than 60,000, no more than 50,000, no more than 40,000, no more than 30,000, no more than 20,000, no more than 10,000, no more than 7,000, no more than 5,000, no more than 3,000, no more than 2,000 cells, or no more than 1,000 cells. In some embodiments, the cells in a cluster have not been previously subjected to a cell-sorting process (e.g., affinity binding purification or FACS). In some embodiments, a cell cluster comprises 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-900, 500-800, 500-700, 500-600, 600-900, 600-800, 600-700, 700-900, 700-800, or 800-900 stem cell-derived beta cells. In some embodiments, a cell cluster comprises 300-1000, 300-900, 300-800, 300- 700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500- 900, 500-800, 500-700, 500-600, 600-900, 600-800, 600-700, 700-900, 700-800, or 800-900 NKX6.1-positive, ISLl-positive cells.

The clusters described herein can be homo-cellular or hetero-cellular. The term “homo- cellular” cell clusters or equivalents thereof refers to a plurality of cell clusters in suspension, wherein each cell cluster comprises a plurality of living cells of substantially a single cell type. In some embodiments, the compositions, methods, and systems, disclosed herein can result in clusters that are substantially homo-cellular, consisting substantially of stem cells (e.g., hESCs), consisting of substantially of definitive endoderm cells, foregut endoderm cells, or consisting substantially of pancreatic endoderm cells. As used herein, the term “substantially” means either a de minimus or a reduced amount of a component or cell present in any cell aggregate suspension type. As used herein, “hetero-cellular” or equivalents thereof refers to cell clusters whereby each individual cell cluster comprises a plurality of cell types. In some embodiments a hetero-cellular cluster can include at least two, three, four, five, six, or more cell types. For example, a hetero-cellular cluster may comprise a plurality of NKX6.1-positive, ISLl-positive cells and a plurality of NKX6.1 -negative, ISLl-positive cells.

A cell cluster can be in a size similar to an endogenous pancreatic islet. For example, a cell cluster can have a diameter similar to an endogenous pancreatic islet. A diameter of a cell cluster can refer to the largest linear distance between two points on the surface of the cell cluster. In some embodiments, the diameter of a cell cluster is at most 300 pm, 200 pm, 150 pm, 100 pm, 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, or 40 pm. The diameter of a cell cluster can be from about 75 pm to about 250 pm. The diameter of a cell cluster can be at most 100 pm.

In some embodiments, a cell cluster is between about 80 and 270 microns in diameter. In some embodiments, a cell cluster is between about 100 and about 250 microns in diameter (e.g., about 125, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 200, about 210, about 215, about 220, or about 225, microns in diameter). For example, in some embodiments, the cell cluster is between about 125 and about 225, between about 130 and about 160, between about 170 and about 225, between about 140 and about 200, between about 140 and about 170, between about 160 and about 220, between about 170 and about 215, or between about 170 and about 200, microns in diameter.

In some embodiments, the disclosure provides for a composition comprising one or more cell clusters. In some embodiments, the composition comprises 500-20000, 500-15000, 500-10000, 500-5000, 500-2000, 500-1000, 1000-20000, 1000-15000, 1000-10000, 1000-5000, 1000-2000, 2000-20000, 2000-15000, 2000-10000, 2000-5000, 5000-20000, 5000-15000, 5000- 10000, 10000-20000, 10000-15000, 15000-20000, or 3000-9000 cell clusters. In some embodiments, any of the cells disclosed herein comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said cells comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, any of the cells disclosed herein (e.g., any of the SC-derived beta cells or cells in any of the clusters disclosed herein) comprise a genomic disruption in at least one gene sequence, wherein said disruption reduces or eliminates expression of a protein encoded by said gene sequence. In some embodiments, said at least one gene sequence is the ABO sequence, such that the disruption results in the cell being blood type O. In some embodiments, said at least one gene sequence encodes an MHC-Class I gene. In some embodiments, said MHC-Class I gene encodes beta-2 microglobulin (B2M), HLA-A, HLA-B, or HLA-C. In some embodiments, said at least one gene sequence encodes CIITA. In some embodiments, the cells comprise a genomic disruption in the genes encoding HLA-A and HLA-B, but do not comprise a genomic disruption in the gene encoding HLA-C. In some embodiments, the cells comprise a genomic disruption in the gene encoding CXCL10. In some embodiments, the cells comprise a genomic disruption in the gene encoding renalase. In some embodiments, said cells comprise a genomic disruption in a natural killer cell activating ligand gene. In some embodiments, said natural killer cell activating ligand gene encodes intercellular adhesion molecule 1 (ICAM1), CD58, CD 155, carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAM1), cell adhesion molecule 1 (CADM1), MHC-Class I polypeptide-related sequence A (MICA), or MHC-Class I polypeptide-related sequence B (MICB). In some embodiments, the cells have reduced expression of one or more of beta-2 microglobulin, CIITA, HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLADR, relative to stem cells that are not genetically modified. In some embodiments, the cells have increased expression of CD47, PDL1, HLA-G, CD46, CD55, CD59, CTLA, PDL2, HLA-C, HLA-E, HLA-G, Cl-inhibitor, IL-35, DUX4, IDO1, IL10, CCL21, CCL22, CD16, CD52, H2- M3, CD200, FASLG, MFGE8, and/or SERPINB9 relative to cells that are not genetically modified. In particular embodiments, the pancreatic islet cells disclosed herein (e.g., the SC-beta cells) have increased expression of PDL1 as compared to endogenous pancreatic islet cells from a healthy control subject. In particular embodiments, the pancreatic islet cells disclosed herein (e.g., the SC-beta cells) have increased expression of CD47 as compared to endogenous pancreatic islet cells from a healthy control subject. In some embodiments, the genomic disruption is induced by use of a gene editing system, e.g., CRISPR Cas technology. In some embodiments, any of the isolated cells (e.g., a stem cell or a NKX6.1-positive, ISLl-positive cell) described herein comprises a disruption (e.g., deletion, insertion, translocation, inversion, or substitution of one or more nucleotides) in any one or more of the genes encoding: B2M, CIITA, CXCL10, renalase, HLA-A, HLA-B, HLA-C, RFX-ANK, NFY-A, NLRC5, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAPI, GITR, 4-1BB, CD28, B7-1, CD47, B7- 2, 0X40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD 160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS, IDO1, TRAC, TRB, NFY-A, CCR5, F3, CD142, MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D, PDGFRa, OLIG2, and/or GFAP. In some embodiments, disruption of a gene results in an at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% decrease in expression of the gene as compared to the expression of the gene in the same type of cell without the disruption. In some embodiments, the gene is disrupted using CRISPR/Cas, piggybac transposon, TALEN, and/or zinc finger technology.

In some embodiments, a cell (e.g., an isolated stem cell or a NKX6.1-positive, ISLl- positive cell) described herein is negative for A antigen and negative for B antigen. In some embodiments, the cell described herein is negative for A antigen. In some embodiments, the cell described herein is negative for B antigen. In some embodiments, a cell (e.g., an isolated stem cell or a NKX6.1-positive, ISLl-positive cell) described herein is negative for Rh antigen. In some embodiments, a cell (e.g., an isolated stem cell or a NKX6.1-positive, ISLl-positive cell) described herein is negative for A antigen, negative for B antigen, and negative for Rh antigen. An “A antigen,” as used herein, refers to a histo-blood group antigen produced by 3a-N- acetylgalactosaminyltransferase and expressed as a cell-surface antigen. A “B antigen,” as used herein, refers to a histo-blood group antigen produced by 3a-galactosaminyltransferase and expressed as a cell-surface antigen. In some embodiments, the cell comprises a disruption in the ABO gene. In some embodiments, the cell comprises a disruption in the ABO gene such that the cell has reduced or absent levels of A and B antigens. In some embodiments, the cell comprises a disruption in the FUT1 gene. In some embodiments, the cell comprises a disruption in the FUT1 gene such that Galactoside 2-alpha-L-fucosyltransferase 1 expression is reduced or absent. An “Rh antigen,” as used herein, refers to a highly immunogenic antigen encoded by two highly polymorphic genes, RHD and RHCE. Rh antigen proteins are transmembrane proteins. In some embodiments, the cell comprises a disruption in the RHAG gene. In some embodiments, the cell comprises a disruption in the RHAG gene such that the cell has reduced or absent levels of Rh- associated glycoprotein. In some embodiments, the cell has a reduced or eliminated Rh protein antigen expression selected from the group consisting of Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNS antigen U, and MNS antigen S.

In some embodiments, any of the cells disclosed herein (e.g., any of the stem cells disclosed herein) comprises a “safety switch.” In some embodiments, the safety switches are nucleic acid constructs encoding a switch protein that inducibly causes cell death or stops cell proliferation. In some embodiments, the safety switch is inserted at a defined, specific target locus (e.g., a safe harbor locus) in the genome of an engineered cell, usually at both alleles of the target locus. In some embodiments, the target locus is a safe harbor locus, such as ActB or CEYBE. In some embodiments, the target locus is a gene targeted for disruption (e.g., B2M or OITA). In some embodiments, the switch protein is activated by contacting with an effective dose of a clinically acceptable orthologous small molecule. In some embodiments, when activated, the safety switch causes the cell to stop proliferation, in some embodiments by activating apoptosis of the cell. In some embodiments, the switch protein comprises herpes- simplex-thymidine-kinase. In some embodiments the switch protein comprises a human caspase protein, e.g. caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase

8, caspase 9, caspase 10, caspase 14, etc. In certain embodiments the protein is human caspase

9. In some embodiments, the caspase protein is fused to a sequence that provides for chemically induced dimerization (CID), in which dimerization occurs only in the presence of the orthologous activating agent. One or more CID domains may be fused to the caspase protein, e.g. two different CID domains may be fused to the caspase protein. In some embodiments the CID domain is a dimerization domain of FKBP or FRB (FKBP-rapamycin-binding) domain of mTOR, which are activated with rapamycin analogs. In some embodiments, the safety switch is any of the safety switches described in WO2021173449 and Jones et al., 2014, Frontiers in Pharmacology, 5(254): 1-8, each of which is incorporated herein in its entirety.

In some embodiments, a population or composition further includes a medium. In some embodiments, the medium comprises a sugar. In some embodiments, the sugar is sucrose or glucose. In some embodiments, the medium comprises the sugar at a concentration of between about 0.05% and about 1.5%. In some embodiments, the medium is a CMRL medium; or wherein the medium is HYPOTHERMOSOL® FRS Preservation Media.

Some embodiments of the present disclosure provide compositions including any of the cells, any of the cell populations of in vitro differentiated cells, any of the clusters, and/or any of the plurality of cell clusters described herein. In some embodiments, a composition comprising population of in vitro differentiated cells described herein are therapeutic compositions. The therapeutic compositions can further comprise a physiologically compatible solution including, for example, artificial cerebrospinal fluid or phosphate-buffered saline. The therapeutic composition can be used to treat, prevent, or stabilize a disease (e.g., diabetes).

In some embodiments, a therapeutic composition further comprises other active agents, such as anti-inflammatory agents, exogenous small molecule agonists, exogenous small molecule antagonists, anti-apoptotic agents, antioxidants, and/or growth factors known to a person having skill in the art.

In some embodiments, a therapeutic composition further comprises a pharmaceutically acceptable carrier (e.g., a medium or an excipient). The term pharmaceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, can refer to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication. Suitable pharmaceutically acceptable carriers can include water, salt solution (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical compositions comprising cellular components or products, but not live cells, can be formulated as liquids. Pharmaceutical compositions comprising living non-native pancreatic P cells can be formulated as liquids, semisolids (e.g., gels, gel capsules, or liposomes) or solids (e.g., matrices, scaffolds and the like).

In some embodiments, a therapeutic composition is formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

In some embodiments, a therapeutic composition is optionally manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

In some embodiments, a therapeutic composition comprises one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

In some embodiments, a therapeutic composition further comprises one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, a therapeutic composition is suitable for administration by any administration route, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra- articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration routes. In some embodiments, a therapeutic composition is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra- articular, intraperitoneal, or intracranial) administration.

In some embodiments, a therapeutic composition further comprises one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

In some embodiments, a therapeutic composition comprises a population of in vitro differentiated cells described herein in an amount that is effective to treat or prevent e.g., diabetes. In some embodiments, a therapeutic composition further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, a therapeutic composition comprising cells, cell components or cell products may be delivered to the kidney of a patient in one or more of several methods of delivery known in the art. In some embodiments, the compositions are delivered to the kidney (e.g., on the renal capsule and/or underneath the renal capsule). In another embodiment, the compositions may be delivered to various locations within the kidney via periodic intraperitoneal or intrarenal injection. Alternatively, the compositions may be applied in other dosage forms known to those skilled in the art, such as pre-formed or in situ-formed gels or liposomes.

In some embodiments, therapeutic compositions comprising live cells in a semi-solid or solid carrier may be formulated for surgical implantation on or beneath the renal capsule. It should be appreciated that liquid compositions also may be administered by surgical procedures. In particular cases, semi-solid or solid pharmaceutical compositions may comprise semi- permeable gels, lattices, cellular scaffolds and the like, which may be non-biodegradable or biodegradable. For example, in certain cases, it may be desirable or appropriate to sequester the exogenous cells from their surroundings, yet enable the cells to secrete and deliver biological molecules (e.g., insulin) to surrounding cells or the blood stream. In these cases, cells may be formulated as autonomous implants comprising living cells by a non-degradable, selectively permeable barrier that physically separates the transplanted cells from host tissue. Such implants are sometimes referred to as “immunoprotective,” as they have the capacity to prevent immune cells and macromolecules from killing the transplanted cells in the absence of pharmacologically induced immunosuppression. Various encapsulation devices, degradable gels and networks can be used for the pharmaceutical compositions of the present disclosure. For example, degradable materials particularly suitable for sustained release formulations include biocompatible polymers, such as poly(lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.

In some embodiments, it may be desirable or appropriate to deliver the cells on or in a biodegradable, preferably bioresorbable or bioabsorbable, scaffold or matrix. These typically three-dimensional biomaterials contain the living cells attached to the scaffold, dispersed within the scaffold, or incorporated in an extracellular matrix entrapped in the scaffold. Once implanted into the target region of the body, these implants become integrated with the host tissue, wherein the transplanted cells gradually become established. Examples of scaffold or matrix (sometimes referred to collectively as “framework”) material that may be used in the present disclosure include nonwoven mats, porous foams, or self-assembling peptides. Nonwoven mats, for example, may be formed using fibers comprising a synthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA), foams, and/or poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer. In some embodiments, the framework is a felt, which can be composed of a multifilament yam made from a bioabsorbable material, e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarn is made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling. In another embodiment, cells are seeded onto foam scaffolds that may be composite structures. In many of the abovementioned cases, the framework may be molded into a useful shape. Furthermore, it will be appreciated that nonnative pancreatic P cells may be cultured on pre-formed, non-degradable surgical or implantable devices.

In some embodiments, the matrix, scaffold or device may be treated prior to inoculation of cells in order to enhance cell attachment. For example, prior to inoculation, nylon matrices can be treated with 0.1 molar acetic acid and incubated in polylysine, PBS, and/or collagen to coat the nylon. Polystyrene can be similarly treated using sulfuric acid. The external surfaces of a framework may also be modified to improve the attachment or growth of cells and differentiation of tissue, such as by plasma coating the framework or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate, chondroitin-6- sulfate, dermatan sulfate, keratin sulfate), a cellular matrix, and/or other materials such as, but not limited to, gelatin, alginates, agar, agarose, and plant gums, among others.

In some embodiments, the present disclosure provided devices comprising a population of in vitro differentiated cells described herein. In some embodiments, the population of in vitro differentiated cells described herein form cell clusters. A device can be configured to house the cells described herein which, in particular embodiments, produce and release insulin when implanted into a subject. In some embodiment, a device can further comprise a semipermeable membrane. The semipermeable membrane can be configured to retain the cell cluster in the device and permit passage of insulin secreted by the cells. In some embodiments of the device, the cells can be encapsulated by the semipermeable membrane. The encapsulation can be performed by any technique available to one skilled in the art. The semipermeable membrane can also be made of any suitable material as one skilled in the art would appreciate and verify. For example, the semipermeable membrane can be made of polysaccharide or polycation. In some embodiments, the semipermeable membrane can be made of poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other poly hydroxy acids, poly (caprolactone), polycarbonates, polyamides, poly anhydrides, polyphosphazene, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates, biodegradable polyurethanes, albumin, collagen, fibrin, polyamino acids, prolamines, alginate, agarose, agarose with gelatin, dextran, poly acrylates, ethylene- vinyl acetate polymers and other acyl-substituted cellulose acetates and derivatives thereof, polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins, polyethylene oxide, or any combinations thereof. In some embodiments, the semipermeable membrane comprises alginate. In some embodiments, the cells are encapsulated in a microcapsule that comprises an alginate core surrounded by the semipermeable membrane. In some embodiments, the alginate core is modified, for example, to produce a scaffold comprising an alginate core having covalently conjugated oligopeptides with an RGD sequence (arginine, glycine, aspartic acid). In some embodiments, the alginate core is modified, for example, to produce a covalently reinforced microcapsule having a chemoenzymatically engineered alginate of enhanced stability. In some embodiments, the alginate core is modified, for example, to produce membrane-mimetic films assembled by in-situ polymerization of acrylate functionalized phospholipids. In some embodiments, microcapsules are composed of enzymatically modified alginates using epimerases. In some embodiments, microcapsules comprise covalent links between adjacent layers of the microcapsule membrane. In some embodiment, the microcapsule comprises a subsieve-size capsule comprising alginate coupled with phenol moieties. In some embodiments, the microcapsule comprises a scaffold comprising alginate-agarose. In some embodiments, the cells are modified with PEG before being encapsulated within alginate. In some embodiments, the cells are encapsulated in photoreactive liposomes and alginate. It should be appreciated that the alginate employed in the microcapsules can be replaced with other suitable biomaterials, including, without limitation, polyethylene glycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronic acid, dextran with ROD, BHD and polyethylene glycol-diacrylate (PEGDA), poly(MPC-co-n-butyl methacrylate-co-4-vinylphenyl boronic acid) (PMBV) and poly(vinyl alcohol) (PVA), agarose, agarose with gelatin, and multilayer cases of these. In some embodiments, the device provided herein comprise extracorporeal segment, e.g., part of the device can be outside a subject’s body when the device is implanted in the subject. The extracorporeal segment can comprise any functional component of the device, with or without the cells or cell cluster provided herein.

Further provided herein are methods for treating or preventing a disease in a subject. A composition comprising a population of in vitro differentiated cells described herein can be administered into a subject to restore a degree of pancreatic function in the subject. In some embodiments, such composition is transplanted in a subject. The term “transplant” can refer to the placement of cells or cell clusters, any portion of the cells or cell clusters thereof, any compositions comprising cells, cell clusters or any portion thereof, into a subject, by a method or route which results in at least partial localization of the introduced cells or cell clusters at a desired site. In some embodiments, the desired site is the pancreas. In some embodiments, the desired site is a non-pancreatic location, such as in the liver or subcutaneously, for example, in a capsule (e.g., microcapsule) to maintain the implanted cells at the implant location and avoid migration. In some embodiments, the transplanted cells release insulin in an amount sufficient for a reduction of blood glucose levels in the subject.

In some embodiments, a composition comprising a population of in vitro differentiated cells described herein are housed in a device that is implanted in a subject. In some embodiments, a composition comprising a population of in vitro differentiated cells described herein are housed in a device suitable for implantation into a subject. In some embodiments, the device upon implantation in a subject releases insulin while retaining the cells in the device, and facilitates tissue vascularization in and around the device. Exemplary devices are described, for example in WO2018232180, W02019068059, WO2019178134, W02020/206150, and W02020/206157, each of which is incorporated-by-reference in its entirety. In some embodiments, a subject is not administered an immune suppression agent during the implantation or vascularization of the device. In some embodiments, the device has a thickness of at least about 300 pm. In some embodiments, the device comprises a membrane comprising a plurality of nodes interconnected by a plurality of fibrils.

In some embodiments, the device comprises a first membrane having a first surface comprising a plurality of channels, and a plurality of second surfaces opposing the first surface; and a second membrane opposite and attached to the plurality of the second surfaces of the first membrane; wherein the first membrane and the second membrane form an enclosed compartment having a surface area to volume ratio of at least about 40 cm-1, and wherein the enclosed compartment provides a volume for housing a cell within the device.

In some embodiments, the enclosed compartment comprises a single continuous open chamber. In some embodiments, the volume is about 8 pL to about 1,000 pL. In some embodiments, the device has at least one of a length and a width of about 0.25 cm to about 3 cm. In some embodiments, the device has a thickness of at least about 300 pm.

In some embodiments, the plurality of channels is generally perpendicular with respect to the first membrane. In some embodiments, the plurality of channels is arranged in a rectilinear array. In some embodiments, the plurality of channels is arranged in a polar array. In some embodiments, the channel has an average diameter of about 400 pm to about 3,000 pm. In some embodiments, the diameter is measured at a narrowest point in the channel. In some embodiments, a center of each channel is separated from the center of another channel by a distance of about 75 pm to about 500 pm. In some embodiments, the channel has a height to diameter ratio of at least about 0.2. In some embodiments, the device has a number of channels per area along a transverse plane, and In some embodiments the number is greater than about 50/cm2.

In some embodiments, at least one of the first membrane and the second membrane comprise a plurality of nodes interconnected by a plurality of fibrils. In some embodiments, at least one of the first membrane and the second membrane comprise PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, PLLA, or any combination thereof. In some embodiments, the device further comprises an opening through the first membrane and/or the second membrane within the channel. In some embodiments, the opening has a concentricity with respect to the channel of at most about 25% the diameter of the channel. In some embodiments is a frame configured to receive the device described herein. In some embodiments, the frame is configured to receive a plurality of cell housing devices. In some embodiments, the frame comprises a flexing mechanism configured to prevent buckling of the cell housing device.

In some embodiments, an implantable encapsulation device comprises an internal volume comprising, disposed therein, a population of in vitro differentiated cells or a composition comprising a population of in vitro differentiated cells described herein described herein. In some embodiments, the implantable encapsulation device comprises at least one membrane that at least partially defines the internal volume. In some embodiments, the at least one membrane includes a first membrane and a second membrane, wherein the first membrane and the second membrane are bonded together to form a seal extending at least partially around the internal volume disposed between the first membrane and the second membrane. In some embodiments, the at least one membrane comprises at least one selected from PVDF, PTFE, ePTFE, PCL, PE/PES, PP, PS, PMMA, PLGA, and PLLA. In some embodiments, the at least one membrane comprises ePTFE.

In some embodiments, a method described herein comprises transplanting a population of in vitro differentiated cells described herein to a subject using any means in the art. For example the methods can comprise transplanting the cell cluster via the intraperitoneal space, portal vein, renal subcapsule, renal capsule, omentum, subcutaneous space, or via pancreatic bed infusion. For example, transplanting can be subcapsular transplanting, intramuscular transplanting, or intraportal transplanting, e.g., intraportal infusion. Immunoprotective encapsulation can be implemented to provide immunoprotection to the cell clusters. In some embodiments, the methods of treatment provided herein can comprise administering one or more immune response modulators for modulating or reducing transplant rejection response or other immune response against the implant e.g., the cells or the device). Examples of immune response modulator that can be used in the methods can include purine synthesis inhibitors like Azathioprine and Mycophenolic acid, pyrimidine synthesis inhibitors like Leflunomide and Teriflunomide, antifolate like Methotrexate, Tacrolimus, Ciclosporin, Pimecrolimus, Abetimus, Gusperimus, Lenalidomide, Pomalidomide, Thalidomide, PDE4 inhibitor, Apremilast, Anakinra, Sirolimus, Everolimus, Ridaforolimus, Temsirolimus, Umirolimus, Zotarolimus, Anti-thymocyte globulin antibodies, Anti-lymphocyte globulin antibodies, CTLA-4, fragment thereof, and fusion proteins thereof like Abatacept and Belatacept, TNF inhibitor like Etanercept and Pegsunercept, Aflibercept, Alefacept, Rilonacept, antibodies against complement component 5 like Eculizumab, anti-TNF antibodies like Adalimumab, Afelimomab, Certolizumab pegol, Golimumab, Infliximab, and Nerelimomab, antibodies against Interleukin 5 like Mepolizumab, anti-Ig E antibodies like Omalizumab, anti-Interferon antibodies like Faralimomab, anti-IL-6 antibodies like Elsilimomab, antibodies against IL- 12 and IL-23 like Lebrikizumab and Ustekinumab, anti-IL-17A antibodies like Secukinumab, anti-CD3 antibodies like Muromonab- CD3, Otelixizumab, Teplizumab, and Visilizumab, anti-CD4 antibodies like Clenoliximab, Keliximab, and Zanolimumab, anti-CDlla antibodies like Efalizumab, anti-CD18 antibodies like Erlizumab, anti-CD20 antibodies like Obinutuzumab, Rituximab, Ocrelizumab and Pascolizumab, anti-CD23 antibodies like Gomiliximab and Lumiliximab, anti-CD40 antibodies like Teneliximab and Toralizumab, antibodies against CD62L/L- selectin like Aselizumab, anti- CD80 antibodies like Galiximab, anti-CD147/Basigin antibodies like Gavilimomab, anti-CD154 antibodies like Ruplizumab, anti-BLyS antibodies like Belimumab and Blisibimod, anti-CTLA-4 antibodies like Ipilimumab and Tremelimumab, anti-CAT antibodies like Bertilimumab, Lerdelimumab, and Metelimumab, anti-Integrin antibodies like Natalizumab, antibodies against Interleukin-6 receptor like Tocilizumab, anti-LFA-1 antibodies like Odulimomab, antibodies against IL-2 receptor/CD25 like Basiliximab, Daclizumab, and Inolimomab, antibodies against T-lymphocyte (Zolimomab aritox) like Atorolimumab, Cedelizumab, Fontolizumab, Maslimomab, Morolimumab, Pexelizumab, Reslizumab, Rovelizumab, Siplizumab, Talizumab, Telimomab aritox, Vapaliximab, and Vepalimomab.

As used herein, the term “treating” and “treatment” can refer to administering to a subject an effective amount of a composition (e.g., cell clusters or a portion thereof) so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (e.g., partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term “treatment” includes prophylaxis.

Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

By “treatment,” “prevention” or “amelioration” of a disease or disorder is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing down or stopping the progression, aggravation or deterioration the progression or severity of a condition associated with such a disease or disorder. In one embodiment, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% in comparison to a nontreated subject.

Treatment of Diabetes is determined by standard medical methods. A goal of Diabetes treatment is to bring sugar levels down to as close to normal as is safely possible. Commonly set goals are 80-120 milligrams per deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. A particular physician may set different targets for the patent, depending on other factors, such as how often the patient has low blood sugar reactions. Useful medical tests include tests on the patient's blood and urine to determine blood sugar level, tests for glycosylated hemoglobin level (HbAlc; a measure of average blood glucose levels over the past 2-3 months, normal range being 4-6%), tests for cholesterol and fat levels, and tests for urine protein level. Such tests are standard tests known to those of skill in the art (see, for example, American Diabetes Association, 1998). A successful treatment program can also be determined by having fewer patients in the program with complications relating to Diabetes, such as diseases of the eye, kidney disease, or nerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoin sulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, and can include the entire lifespan of the subject. In some embodiments, the reduction of blood glucose levels in the subject, as induced by the transplantation of the cell, or the composition or device provided herein, results in an amount of glucose which is lower than the diabetes threshold. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is human. In some embodiments, the amount of glucose is reduced to lower than the diabetes threshold in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the implanting.

A subject that can be treated by the methods herein can be a human or a non-human animal. In some embodiments, a subject can be a mammal. Examples of a subject include but are not limited to primates, e.g., a monkey, a chimpanzee, a bamboo, or a human. In some embodiments, a subject is a human. A subject can be non-primate animals, including, but not limited to, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, and the like. In some embodiments, a subject receiving the treatment is a subject in need thereof, e.g., a human in need thereof.

The terms, “patient” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of Type 1 diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having Diabetes (e.g., Type 1 or Type 2), one or more complications related to Diabetes, or a pre-diabetic condition, and optionally, but need not have already undergone treatment for the Diabetes, the one or more complications related to Diabetes, or the pre-diabetic condition. A subject can also be one who is not suffering from Diabetes or a pre-diabetic condition. A subject can also be one who has been diagnosed with or identified as suffering from Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition, but who show improvements in known Diabetes risk factors as a result of receiving one or more treatments for Diabetes, one or more complications related to Diabetes, or the pre-diabetic condition. Alternatively, a subject can also be one who has not been previously diagnosed as having Diabetes, one or more complications related to Diabetes, or a pre-diabetic condition. For example, a subject can be one who exhibits one or more risk factors for Diabetes, complications related to Diabetes, or a pre- diabetic condition, or a subject who does not exhibit Diabetes risk factors, or a subject who is asymptomatic for Diabetes, one or more Diabetes-related complications, or a pre-diabetic condition. A subject can also be one who is suffering from or at risk of developing Diabetes or a pre-diabetic condition. A subject can also be one who has been diagnosed with or identified as having one or more complications related to Diabetes or a pre-diabetic condition as defined herein, or alternatively, a subject can be one who has not been previously diagnosed with or identified as having one or more complications related to Diabetes or a pre-diabetic condition.

The above-described embodiments of the technology described herein can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computing device or distributed among multiple computing devices. Such processors may be implemented as integrated circuits, with one or more processors in an integrated circuit component, including commercially available integrated circuit components known in the art by names such as CPU chips, GPU chips, microprocessor, microcontroller, or co-processor. Alternatively, a processor may be implemented in custom circuitry, such as an ASIC, or semicustom circuitry resulting from configuring a programmable logic device. As yet a further alternative, a processor may be a portion of a larger circuit or semiconductor device, whether commercially available, semi-custom or custom. As a specific example, some commercially available microprocessors have multiple cores such that one or a subset of those cores may constitute a processor. Though, a processor may be implemented using circuitry in any suitable format.

Further, it should be appreciated that a computing device may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computing device may be embedded in a device not generally regarded as a computing device but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone, tablet, or any other suitable portable or fixed electronic device.

Also, a computing device may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, individual buttons, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computing device may receive input information through speech recognition or in other audible format.

Such computing devices may be interconnected by one or more networks in any suitable form, including as a local area network or a wide area network, such as an enterprise network or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

In this respect, the embodiments described herein may be embodied as a computer readable storage medium (or multiple computer readable media) (e.g., a computer memory, one or more floppy discs, compact discs (CD), optical discs, digital video disks (DVD), magnetic tapes, flash memories, RAM, ROM, EEPROM, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments discussed above. As is apparent from the foregoing examples, a computer readable storage medium may retain information for a sufficient time to provide computer-executable instructions in a non-transitory form. Such a computer readable storage medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computing devices or other processors to implement various aspects of the present disclosure as discussed above. As used herein, the term "computer-readable storage medium" encompasses only a non-transitory computer-readable medium that can be considered to be a manufacture (i.e., article of manufacture) or a machine. Alternatively or additionally, the disclosure may be embodied as a computer readable medium other than a computer-readable storage medium, such as a propagating signal.

The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computing device or other processor to implement various aspects of the present disclosure as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computing device or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present disclosure.

Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments.

The embodiments described herein may be embodied as a method, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

EXAMPLES

These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1: Expansion of hESCs using Continuous Perfusion

This example illustrates a medium exchange suitable for the expansion of embryonic stem cells. Continuous perfusion is a medium refresh method where cell clusters are retained with a separation device while medium continuously flows in and out of the reactor. Media exchange using an exemplary tangential flow filtration system, i.e., alternating tangential flow (ATF), was evaluated herein.

Briefly, human embryonic stem cells (hESCs) were expanded in 2D cultures (T-flasks and cell stacks). The hESCs were adapted from the 2D flasks into stirred tank bioreactors (STRs) R01-R06. The adaptation process involved dissociating the monolayer cultures into single cells using lx Accutase, which were inoculated into bioreactors comprising Nutristem Media and Y-27632 and allowed to form aggregates (also referred to as clusters). Cells were adapted into the 3D aggregate culture at 0.5 viable cells (vc)/mL. The conditions evaluated for expanding stem cell aggregates are summarized in Table 1. In R05, cells were spun down, old media was removed and the cell culture concentrated, and added back to the reactor. Fresh media was then added to the concentrated cell culture.

For R06, hESCs expanded in 2D culture were adapted into 3D aggregate culture in a stirred-tank reactor as described above. Two days post-inoculation, a medium exchange was performed by halting agitation, settling the clusters to the bottom of the vessel, removing the spent medium supernatant, and adding fresh medium. This medium exchange method removed 93% of the total spent media volume. See, FIG. ID for simplified schematic of the settling approach. In R01-R04, cell clusters were retained using an ATF system. See, FIG. 1A for simplified schematic of ATF system. The bioreactor/ ATF system operated as follows. Each STR comprised a cell culture (comprising adapted cell clusters and liquid media) and was connected to an ATF system by means of a tube. The ATF system comprised a column having a plurality of hollow fibers, where the hollow fibers each had a lumen diameter of 1mm, and where the hollow fibers comprised PES filters having pores 0.2 pm in diameter. The cell culture was pumped out of the bioreactor by means of the diaphragm pump into the ATF system, where liquid media was removed through the pores of the hollow fibers as permeate (and ultimately discarded) and cell clusters and residual media were retained as retentate within the hollow fibers. The retentate was then returned back to the bioreactor by means of the diaphragm pump. Media that was lost from the bioreactor (as a result of the permeate removed by the ATF system) was replenished in the bioreactor as fresh media. Several tangential flow rates were tested: 0.3 Liters per minute (LPM) for R01, 0.5 LPM for R02, and 0.7 LPM for R03. The perfusion rate for R01-R03 was set so that 2.7 volumes of media (in relation to the original volume in the bioreactor) were exchanged in a 24-hour period, resulting in 93% medium exchange over 24 hours (an exchange of 2.7 vessel volumes per day (VVD)). This perfusion rate resulted in shear rates in the ATF system of 637 s’¹, 1061 s’¹, and 1485 s’¹ for R01, R02, and R03, respectively. R5 and R6 used less media (one exchange a day). For R01-R03, perfusion was initiated 1-day post adaptation. To determine whether there was an impact from gradual medium exchange, a combined perfusion and centrifugation was evaluated (R04). For R04, perfusion was started 1- day post adaptation and used for 2 full days at 1 bioreactor volume exchange, before centrifugation was performed. Perfusion was resumed immediately following centrifugation.

Table 1: Bioreactor conditions of Example 1.

Medium

Bioreactor Exchange Retention System Conditions

Method

R01 Perfusion ATF rate=0.3 1pm, 2.7 VVD

R02 Perfusion ATF rate=0.5 1pm, 2.7 VVD

R03 Perfusion ATF rate=0.71pm, 2.7 VVD

Perfusion and

R04 ATF 0.5 1pm, 1 VVD + Minifuge 3 cycles

Centrifugation

R05 Minifuge 3 cycles (93% exchange) by Minifuge

R06 Settling N/A Three days post-adaptation, an in-vessel-passage (IVP) was performed. Clusters were dissociated in the bioreactor using lx Accutase, transferred into a new bioreactor, and perfusion was restarted in the new bioreactor 24-hours after the transfer. IVP was performed on all reactors, and all reactors continued with the same medium exchange method (perfusion or centrifugation) and cell culture parameters as prior to the IVP.

Cell counts in each bioreactor were assessed 3-days post adaptation and 3-days post IVP for each reactor condition. Perfusion-based expansion of stem cells led to improved growth compared to the conditions with discrete medium exchange (settling or centrifugation). Results demonstrated that, of the perfusion conditions tested, the highest flow rate (0.7 LPM) led to the largest fold expansion (R03), performing equivalently to the centrifugation conditions (FIG. 4A). In all reactors tested, >90% of cells expressed the pluripotency marker OCT4 and did not express a marker for definitive endoderm (SOX17), as assessed by flow cytometry (FIG. 4B).

Example 2: Differentiation of Beta Cells using Continuous Perfusion

This example illustrates the use of a medium exchange for differentiation of NKX6.1- positive, ISLl-positive cells. The results evidence that stem cell clusters produced using continuous perfusion can be successfully differentiated. Furthermore, continuous perfusion can be used for the differentiation of stem cells into beta cells. Additional approaches to improve aggregation and survival post adaptation are described herein.

ATF, settling or centrifugation conditions were tested in a manner similar to that described in Example 1, but with several conditions evaluated for further improving aggregation and survival post adaptation as summarized in Table 2.

To alleviate potential shear damage from the ATF, the shear protectants PVA-80 and Pluronic F-68 were investigated. 0.1% PVA-80 was used in culture R09. 2 g/L Pluronic F-68 were used in culture RIO. Perfusion was initiated 24 hours post-seeding for R08-R10 and R12- R14. To minimize shear damage during aggregate formation (first two days), perfusion was not started until 48h post inoculation in R11. To improve aggregation R13 was inoculated at half the total working volume but with the same total number of cells (0.76E6 vc/mL). To achieve the growth demonstrated, R14 was extended (in perfusion mode) for up to 6 days without performing IVP 3-days post adaptation. Table 2: Bioreactor conditions for evaluating shear protectants and process parameters to improve perfusion-based expansion of stem cell aggregates

Media Exchange Shear

Bioreactor Process Parameters Method Protectant

R08 Settling None

R09 Perfusion 0.1% PVA

RIO Perfusion 2 g/L Pluronic

Perfusion for

Rll expansion, Settling for None Start ATF 48h post inoculation differentiation

Perfusion for

R12 expansion, Settling for None 1 VVD differentiation

Perfusion

R13 None Inoculate at half volume

Extend perfusion in adaptation to 6

R14 Perfusion None days

The extended growth condition R14 led to a final density of 10 million cells per mL, equivalent to a 20-fold expansion over 6 days. However, the aggregate sizes were much larger than those of other reactors (FIG. 5A-5B).

Differentiation of Expanded hESCs from R08-R14

At 4-6 days post adaptation, 30 ml was taken from R14 on days 4, 5 and 6 (R14-1, R14- 2, and R14-3 respectively) and re- seeded in Biotts. R14-1, 14-2 and 14-3 were differentiated under batch conditions.

The reactors that expanded as stem cells were differentiated to determine their capability to convert into NKX6.1-positive, ISLl-positive cells. NKX6.1-positive, ISLl-positive cells were produced in 3D aggregate cultures via a 5-stage differentiation protocol, which is laid out in Table 3.

Table 3: Differentiation Media Stages 1-5 (S1-S5)

Small molecule and protein factors were introduced and removed at fixed points during the process to induce cells into each developmental stage as indicated in Table 3. These factors were added and removed via the rapid medium exchange methods (settling or centrifugation). Given the success of continuous perfusion for stem cell expansion (see Example 1), a continuous method of factor introduction and removal was investigated. The factors were added to the medium at the same concentration as used for discrete medium exchanges. The medium and factors were perfused through the reactor at 2.7 VVD. At later stages (4 and 5), the medium was not exchanged daily in the discrete cases, and the perfusion rate was dropped to 1 VVD on those days.

Part-way through differentiation, the cells of the high-density reactor R14 began dying for an unknown reason and was terminated. However, reactors R09, RIO, and R13 running the perfusion-based differentiation process enabled production of ISL1+/NKX6.1+ cells (expression as assessed using flow cytometry) (FIG. 6). Notably, the Biotts passaged from the extended growth reactor produced similar percentages of on-target ISLl-positive, NKX6.1-postive cells as the discrete medium exchange conditions.

Satellite cell cultures were taken at consecutive days from R14 and an in-vessel-passage was performed on them in a small vessel (Biott) prior to the attempted differentiation of R14. These satellite cell cultures were healthy, and able to be put through the standard differentiation process. The successful differentiation of the Biotts inoculated from the high-density reactor R14 indicates that a high-density perfusion culture can be used to inoculate several differentiation reactors. These reactors can be operated in perfusion mode for differentiation. Example 3: Differentiation of Beta Cells using Continuous Perfusion and Centrifugation

Stem cells were differentiated to determine their capability to convert into NKX6.1- positive, ISLl-positive cells. NKX6.1-positive, ISLl-positive cells were produced in 3D aggregate cultures via a 5-stage differentiation protocol, which is laid out in Table 4.

Table 4: Differentiation Media Stages 1-5 (S1-S5)

Small molecule and protein factors were introduced and removed at fixed points during the process to induce cells into each developmental stage as indicated in Table 4. The cells were differentiated by one of three different protocols: a) exchanging media (including reagents listed in Table 4) using rapid medium exchange by means of centrifugation; b) exchanging media (including reagents listed in Table 4) using continuous perfusion throughout the entirety of the differentiation protocol (see Example 2); or c) exchanging media (including reagents listed in Table 4) using continuous perfusion when no reagents are being newly introduced to or completely removed from the media, but using centrifugation when transitioning to media that newly introduces or completely removes a Table 4 reagent (i.e., at the start of a new row of Table 4). For example, in protocol c), media (including reagents listed in Table 4) were exchanged using continuous perfusion S4D5-6, followed by rapid medium exchange using centrifugation on S5D1, followed by continuous perfusion on S5D1-3, followed by rapid medium exchange using centrifugation on S5D4, followed by continuous perfusion on S5D4-7. However, if reagents are only being newly introduced and not completely removed (e.g., the transition between S4D1-4 and S4D5-6), rapid medium medium exchange may be skipped and the newly introduced reagent may be introduced via continuous perfusion (e.g., S4D1-6 are performed solely by continuous perfusion without any rapid medium exchange). For protocols b) and c), the medium and factors were perfused through the reactor at 1.5-2.7 VVD. At later stages (4 and 5), the medium was not exchanged daily in the discrete cases, and the perfusion rate was dropped to 1 VVD on those days.

Cells differentiated using protocols a)-c) were capable of forming cell clusters. However, the clusters generated using only continuous perfusion (protocol b)) were atypical in size and morphology as compared to the clusters generated using only rapid media exchange (protocol a)). The clusters generated using the hybrid perfusion/centrifugation protocol (protocol c)) appeared to have similar size and morphology as those generated using only rapid media exchange (protocol a)).

Example 3: Rapid Filtration-Based Exchange

This example describes the successful implementation and use of a rapid media exchange using a system provided herein. In this method, the removal of spent medium and replacement of new medium are performed were performed as discrete steps, rather than as part of a simultaneous process of removal and addition of new media (continuous perfusion). The cells were retained in the bioreactor via a TFF system comprising a hollow-fiber filter membrane, while the spent medium was removed through the permeate side of the membrane. During spent medium removal, the vessel volume was reduced and the culture concentration was increased. After spent medium removal, fresh medium was added to return the volume to the initial working volume of the vessel. This process was usually completed over the course of between 30 min to several hours. In the example below, 70% to 90% exchanges were performed during a differentiation protocol according to Table 4, using a hollow-fiber membrane with pore sizes of 0.2 microns (Reactor R87) and 50 microns (Reactor R84). In reactors R84 and R87, the following parameters were used for filtration: the membrane/filter material was PES, the lumen size was 1 mm, the shear rate was 1400 s’¹, and the the Reynolds number (Re) was 250. The permeate fluxes (LMH) used for the rapid exchange were 11.5 liters/m²/h for the 0.2 um pore size (Reactor R87) and 500 liters/ m²/h for the 50 um pore size (Reactor R84).

As a comparison, the differentiation protocol according to Table 4 was performed where centrifugation was used for rapid media exchange (Reactor R83). FIGS. 7-8 present the results, which demonstrate that both methods produced equivalent suitable cell yield and composition.

FIG. 7 shows the yield of NKX6.1-positive/ISEl-positive cells produced in each reactor. As shown, the rapid media exchange in reactors R84 and R87 both produced a yield exceeding 25%, demonstrating improved performance over the centrifugation method used in reactor R83. FIG. 8, meanwhile, shows the growth curves throughout the differentiation process, which was measured in reactor R83 and reactor R87. The X axis shows each culture stage (SO, SI, S2, S3, S4, and S5), and demarcates the duration of the stages in terms of days from the start of each stage. The Y axis shows viable cell density in the vessel. As shown, both centrifugation and rapid media exchange produced suitable densities of viable cells that changed comparably over time, demonstrating that the methods can be used to achieve comparable results.

The present application also discloses the subject matter as described in the following numbered paragraphs:

Item 1: A method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; and e) replacing the removed portion of the liquid media with a new portion of liquid media.

Item 2: The method of item 1, wherein the plurality of cell clusters comprises stem cells.

Item 3: The method of item 2, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells. Item 4: The method of any one of items 2 or 3, wherein the stem cells are embryonic stem cells.

Item 5: The method of any one of items 2 or 3, wherein the stem cells are induced pluripotent stem cells.

Item 6: The method of any one of items 1-5, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are OCT4-negative and SOX 17 -positive.

Item 7: The method of any one of items 1-6, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are FOXA2 -positive, PDX1 -negative.

Item 8: The method of any one of items 1-7, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1 -positive, NKX6.1 -negative.

Item 9: The method of any one of items 1-8, wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are PDX1 -positive, NKX6.1-positive.

Item 10: The method of any one of items 1-9, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are ISL1 -positive.

Item 11: The method of any one of items 1-10, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are chromogranin-positive.

Item 12: The method of any one of items 1-11, wherein 50-100%, 50-90%, 50-75%, 70- 100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75- 500, 75-400, or 75-300 pm in diameter.

Item 13: The method of any one of items 1-12, wherein the plurality of cell clusters are generated from a plurality of dissociated cells.

Item 14: The method of item 13, wherein prior to step a), the method comprises the steps of seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters.

Item 15: The method of item 14, wherein the dissociated cells are cultured until 50- 100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75-500, 75-400, or 75-300 pm in diameter. Item 16: The method of item 14, wherein the culturing step is 12-72, 12-60, 12-50, 12-36, 12-36, 18-60, 18-50, 18-36, 18-26, 26-60, 26-50, 26-36, 36-60, 36-50, or 44-52 hours in length before the step of transporting the portion of the cell culture from the bioreactor into the TFF.

Item 17: The method of any one of items 1-16, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

Item 18: The method of item 17, wherein 0.3-1, 0.3-0.8, 1-5, 1-4, 1-3, 2-5, 2-4, 2-3, 2.5- 3.0, or 2.5-3.5 volumes of media are exchanged in a 24-hour period.

Item 19: The method of any one of items 1-18, wherein the cell culture is transported from the bioreactor through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450-1550 sec’¹.

Item 20: The method of any one of items 1-19, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75- 100 microns.

Item 21: The method of any one of items 1-20, wherein the TFF system comprises a filter made of Poly ether sulfone (PES).

Item 22: The method of any one of items 1-21, wherein a shear protectant is present in the cell culture in the TFF.

Item 23: The method of item 22, wherein the shear protectant is polaxamer, polyvinyl alcohol (PVA) or pluronic.

Item 24: The method of item 23, wherein the shear protectant is PVA, and the PVA is PVA80 or PVA87-89.

Item 25: The method of item 22, wherein the shear protectant is pluronic, and the pluronic is P188 or PF68.

Item 26: The method of any one of items 1-25, wherein the TFF system comprises one or more cassette membranes.

Item 27: The method of any one of items 1-25, wherein the TFF system comprises one or more hollow fiber membrane.

Item 28: The method of item 27, wherein the hollow fiber membrane has a radius of 0.5- 10, 5-10, 2-5, 0.5-7 mm, 0.5-5, 0.5-3, 0.5-2, 0.5-1.2, 0.8-1.2, or 0.9-1.1 mm.

Item 29: The method of any one of items 27 or 28, wherein the TFF system comprises a plurality of hollow fiber membranes. Item 30: The method of any one of items 27-29, wherein the hollow fiber membrane comprises a plurality of pores, wherein the pore sizes are 0.15-0.2, 0.2-100, 0.2-75, 0.2-50, 0.2- 25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75-100 microns.

Item 31: The method of any one of items 27-30, wherein the hollow fiber membrane comprises polyethersulfone (PES).

Item 32: The method of any one of items 1-27, wherein the method comprises the steps of seeding the bioreactor with 0.01 x 10⁶-10 x 10⁶, 0.01 x 10⁶-5 x 10⁶, 0.01 x 10⁶- 1 x 10⁶ , 0.01 x 10⁶-0.5 x 10⁶ , 0.01 x 10⁶-0.05 X 10⁶, 0.1 x 10⁶- 1 x 10⁶ , or 0.3 x 10⁶-0.8 x 10⁶ viable cells/ml and culturing the viable cells to generate the plurality of cell clusters.

Item 33: The method of item 32, wherein the viable cells are dissociated cells.

Item 34: The method of any one of items 32 or 33, wherein 50-100%, 50-90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the viable cells are dissociated cells.

Item 35: The method of any one of items 1-34, wherein the method is repeatedly performed over a period of 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 2-12, 8-12, 3-8, 4-7, or 4-6 days.

Item 36: The method of item 35, wherein at the end of the period, the method comprises the step of dissociating the cell clusters.

Item 37: The method of item 36, wherein the cell clusters are dissociated by treating the cell clusters with one or more proteolytic and collagenolytic enzymes.

Item 38: The method of item 37, wherein the one or more proteolytic and collagenolytic enzymes comprise any one or more of trypsin, collagenase, trypsin-like protease XIV, or thermolysin.

Item 39: The method of item 36, wherein the cell clusters are dissociated by treating the cell clusters with Accutase cell dissociation reagent.

Item 40: The method of any one of items 37-38, wherein the dissociated cells are centrifuged and the one or more proteolytic or collagenolytic enzymes are removed.

Item 41: The method of any one of items 1-40, wherein the bioreactor holds a volume of 1-250, 1-200, 1-150, 1-100, 1-50, 1-25, 1-10, 1-5, 200-250, 150-200, 100-150, 50-100, 45-55, 190-210 liters of media.

Item 42: The method of any one of items 1-41, wherein the bioreactor is a stirred tank reactor.

Item 43: The method of any one of items 1-42, wherein the bioreactor comprises stem cells and a stem cell media.

Item 44: The method of any one of items 1-43, wherein the bioreactor comprises a Rho- associated, coiled-coil containing protein kinase (ROCK) inhibitor. Item 45: The method of item 44, wherein the ROCK inhibitor is selected from the group consisting of thiazovivin, fasudil, Y-27632, and HA1077.

Item 46: The method of any one of items 1-45, wherein the bioreactor comprises basic fibroblast growth factor (bFGF).

Item 47: The method of any one of items 1-46, wherein the portion of the cell culture is transported from the bioreactor into the TFF system by means of a pump.

Item 48: The method of item 47, wherein the pump is in the TFF system.

Item 49: The method of item 48, wherein the pump is a 4-piston diaphragm pump, a peristaltic pump, or a magnetic levitation pump.

Item 50: The method of any one of items 1-49, wherein the cell culture is agitated in the bioreactor to prevent settling of the cell clusters in the bioreactor.

Item 51: The method of item 50, wherein the agitation is performed using a wave reactor, a continuous stirred tank reactor or vertical wheel reactor.

Item 52: The method of any one of items 1-51, wherein the new portion of liquid media comprises one or more cell differentiation or survival factors.

Item 53: The method of item 52, wherein the method further comprises step f), wherein step f) comprises removing and replacing media in the cell culture, wherein step f) is performed for 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours.

Item 54: The method of item 53, wherein step f) comprises exchanging media in the cell culture by means of a centrifugation, tangential flow filtration, alternating tangential flow filtration (ATF) system exchange, or settling.

Item 55: The method of item 53 or 54, wherein the method comprises performing steps a)-e) for 12 hours to 9 days, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 3 days, 12 hours to 1 day, 2-4 days, 4-6 days, or 7-9 days before step f) is performed.

Item 56: The method of any one of items 53-55, wherein during the media exchange step f), the replacement media comprises one or more differentiation or survival factors.

Item 57: The method of any one of items 53-56, wherein 70-90%, 80-90%, 70-100%, 80- 100%, 90-100%, 90-95%, 95-99%, 95-98%, 95-97%, 91-96%, 92-95%, or 93-94% of the media in the reactor is removed and replaced in step f).

Item 58: The method of any one of items 53-57, wherein the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f). Item 59: The method of any one of items 53-58, wherein the replacement media in step f) does not comprise one or more differentiation or survival factors that were present in the media removed during step f).

Item 60: The method of any one of items 53-59, wherein the media exchange is completed within 1-30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7-8 hours.

Item 61: The method of any one of items 53-60, wherein dead or dying cells are removed during step f).

Item 62: The method of any one of items 53-60, wherein steps a)-e) are repeated following the completion of step f), optionally using media with one or more differentiation or survival factors that are not present in the first round of steps a)-e).

Item 63: The method of any one of items 53-62, wherein step f) is performed to remove one or more cell differentiation or survival factors from the cell culture.

Item 64: The method of any one of items 53-63, wherein step f) is performed to add one or more new cell differentiation or survival factors to the cell culture.

Item 65: The method of any one of items 53-64, wherein the one or more cell differentiation or survival factors comprise any one or more of: a Rho-associated protein kinase (ROCK) inhibitor, a transforming growth factor (TGF)-beta receptor agonist/ligand, Wnt activator, a fibroblast growth factor, a retinoic acid receptor activator, a sonic hedgehog inhibitor, a bone morphogenic protein (BMP) inhibitor, a protein kinase C activator, a FOXO1 inhibitor, a gamma-secretase inhibitor, a thyroid receptor activator, a TGF-P signaling pathway inhibitor, an epidermal growth factor (EGF) family member, a protein kinase inhibitor, an epigenetic modifying compound, or a Wnt inhibitor.

Item 66: The method of item 65, wherein the Rho-associated protein kinase (ROCK) inhibitor is Y-27632 or thiazovivin; the transforming growth factor (TGF)-beta receptor agonist/ligand is activin A, GDF8, or GDF11; the Wnt activator is CHIR99021; the fibroblast growth factor is KGF or FGF10; the a retinoic acid receptor activator is retinoic acid, the sonic hedgehog inhibitor is Santl; the bone morphogenic protein (BMP) inhibitor is DMH1, LDN193189, or dorsomorphin; the protein kinase C activator is PDBU or TPPB; the FOXO1 inhibitor is AS1842856; the gamma-secretase inhibitor is XX, XXI or DAPT; the thyroid receptor activator is T3 or GC-1; the TGF-P signaling pathway inhibitor is Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, or SD-208; the epidermal growth factor (EGF) family member is EGF or betacellulin; the protein kinase inhibitor is staurosporine; the epigenetic modifying compound is DZNEP; or the Wnt inhibitor is NVPTNKS656. Item 67: A tangential flow filtration (TFF) system comprising a cell culture, wherein the cell culture comprises a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor.

Item 68: The TFF system of item 67, wherein the plurality of cell clusters comprises stem cells.

Item 69: The TFF system of item 68, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells.

Item 70: The TFF system of any one of items 67-69, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

Item 71: The TFF system of any one of items 67-70, wherein the cell culture flows through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450-1550 sec'¹.

Item 72: The TFF system of any one of items 67-71, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75-100 microns.

Item 73: The TFF system of any one of items 67-72, wherein the TFF system comprises a filter made of polyethersulfone (PES).

Item 74: The TFF system of any one of items 67-73, wherein a shear protectant is present in the cell culture.

Item 75: The TFF system of item 74, wherein the shear protectant is polyvinyl alcohol (PVA) or pluronic.

Item 76: The TFF system of item 75, wherein the shear protectant is PVA, and the PVA is PVA80 or PVA87-89.

Item 77: The TFF system of item 74, wherein the shear protectant is pluronic, and the pluronic is P188 or PF68.

Item 78: The TFF system of any one of items 67-77, wherein the TFF system comprises one or more cassette membranes.

Item 79: The TFF system of any one of items 67-78, wherein the TFF system comprises one or more hollow fiber membrane.

Item 80: The TFF system of item 79, wherein the hollow fiber membrane has a radius of at 0.5-10, 5-10, 2-5, 0.5-7 mm, 0.5-5, 0.5-3, 0.5-2, 0.5-1.2, 0.8-1.2, or 0.9-1.1 mm. Item 81: The TFF system of any one of items 79 or 80, wherein the TFF system comprises a plurality of hollow fiber membranes.

Item 82. The TFF system of 81, wherein the hollow fiber membranes of the plurality of hollow fiber membranes comprise a plurality of pores, wherein the pore sizes are 0.15-0.2, 0.2- 100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75-100 microns.

Item 83: The TFF system of any one of items 81-82, wherein the hollow fiber membranes of the plurality of hollow fiber membranes comprise polyethersulfone (PES).

Item 84: The TFF system of any one of items 67-83, wherein the cell culture comprises a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor.

Item 85: The TFF system of item 84, wherein the ROCK inhibitor is selected from the group consisting of thiazovivin, fasudil, Y-27632, and HA1077.

Item 86: The TFF system of any one of items 67-85, wherein the bioreactor comprises basic fibroblast growth factor (bFGF).

Item 87: The TFF system of any one of items 67-86, wherein the TFF system comprises a pump.

Item 88: The TFF system of item 87, wherein the pump is a 4-piston diaphragm pump, a peristaltic pump, or a magnetic levitation pump.

Item 89: The TFF system of any one of items 67-88, wherein the cell culture comprises one or more cell differentiation or survival factors.

Item 90: The TFF system of item 89, wherein the one or more cell differentiation or survival factors comprise any one or more of: a Rho-associated protein kinase (ROCK) inhibitor, a transforming growth factor (TGF)-beta receptor agonist/ligand, Wnt activator, a fibroblast growth factor, a retinoic acid receptor activator, a sonic hedgehog inhibitor, a bone morphogenic protein (BMP) inhibitor, a protein kinase C activator, a FOXO1 inhibitor, a gamma-secretase inhibitor, a thyroid receptor activator, a TGF-P signaling pathway inhibitor, an epidermal growth factor (EGF) family member, a protein kinase inhibitor, an epigenetic modifying compound, or a Wnt inhibitor.

Item 91: The TFF system of item 90, wherein the Rho-associated protein kinase (ROCK) inhibitor is Y-27632 or thiazovivin; the transforming growth factor (TGF)-beta receptor agonist/ligand is activin A, GDF8, or GDF11; the Wnt activator is CHIR99021; the fibroblast growth factor is KGF or FGF10; the a retinoic acid receptor activator is retinoic acid, the sonic hedgehog inhibitor is Santl; the bone morphogenic protein (BMP) inhibitor is DMH1, LDN193189, or dorsomorphin; the protein kinase C activator is PDBU or TPPB; the FOXO1 inhibitor is AS1842856; the gamma-secretase inhibitor is XX, XXI or DAPT; the thyroid receptor activator is T3 or GC-1; the TGF-P signaling pathway inhibitor is Alk5i II, A83-01, SB431542, D4476, GW788388, LY364947, LY580276, SB505124, GW6604, SB- 525334, or SD-208; the epidermal growth factor (EGF) family member is EGF or betacellulin; the protein kinase inhibitor is staurosporine; the epigenetic modifying compound is DZNEP; or the Wnt inhibitor is NVPTNKS656.

Item 92: A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 400.

Item 93: A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a shear rate of greater than or equal to 400 s'¹.

Item 94: A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; and controlling the flow of the portion of the cell culture through the TFF system to shear the plurality of cell clusters to provide an average maximum transverse dimension of the plurality of cell clusters that is between or equal to 75 pm and 600 pm.

Item 95: A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a Reynold’s number (Re) of less than or equal to 400 to the portion of the portion of the cell culture pumped to the TFF system.

Item 96: A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a shear rate of greater than or equal to 400 s'¹ to the portion of the portion of the cell culture pumped to the TFF system. Item 97: The system of item 95, wherein the first port is in fluid communication with an inlet of the pump and further comprising a second port in fluid communication with an outlet of the pump.

Item 98: The system of item 95, further comprising the cell culture disposed in the bioreactor.

Item 99: The method of any one of items 92-94, wherein the plurality of cell clusters has an average maximum transverse dimension greater than or equal to 75 pm.

Item 100: The method of any one of items 92-94 and 99, wherein the plurality of cell clusters has an average maximum transverse dimension less than or equal to 600 pm.

Item 101: The method of any one of items 92-94 and 99-100, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 2000.

Item 102: The method of any one of items 92-94 and 99-101, further comprising returning a retentate of the portion of the cell culture to the bioreactor from the TFF system.

Item 103: The method of any one of items 92-94 and 99-102, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed continuously.

Item 104: The method of any one of items 92-94 and 99-103, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed periodically.

Item 105: The system of any one of items 95-98, wherein the pump is a positive displacement, diaphragm pump, or peristaltic pump.

Item 106: The method of any one of items 92-94 and 99-104, further comprising removing a retentate of the portion of the cell culture from the TFF system.

Item 107: The method of any one of items 92-94, 99-104, and 106, further comprising adding fresh liquid media to the bioreactor.

Item 108: The method of any one of items 92-94, 99-104, and 106-107, wherein the plurality of cell clusters has an average diameter of greater than or equal to 100 pm, 120 pm, 150 pm, 170 pm.

Item 109: The method of any one of items 92-94, 99-104, and 106-108, wherein the plurality of cell clusters has an average diameter of less than or equal to 500 pm, 300 pm, 250 pm.

Item 110: The method of any one of items 92-94, 99-104, and 106-109, wherein the greater than or equal to 50% of the cell clusters of the plurality of cell clusters have an average diameter of greater than or equal to 100 pm, 120 pm, 150 pm, 170 pm. Item 111: The method of any one of items 92-94, 99-104, and 106-110, wherein the less than or equal to 50% of the cell clusters of the plurality of cell clusters have an average diameter of greater than or equal to 500 pm, 300 pm, 250 pm.

Item 112: The method of any one of items 92-94, 99-104, and 106-111, wherein the cell culture is transported from the bioreactor through the TFF system under laminar flow conditions.

Item 113: The method of any one of items 92-94, 99-104, and 106-112, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of greater than or equal to 50.

Item 114: The method of any one of items 92-94, 99-104, and 106-113, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of less than or equal to 300.

Item 115: The method or system of any one of items 92-114, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

Item 116: The method or system of any one of items 92-115, wherein the TFF system comprises a plurality of hollow fibers, and wherein a ratio of an average lumen radius of the plurality of hollow fibers to the average maximum transverse dimension of the cell clusters is greater than or equal to 2 but less than or equal to 10.

Item 117: The method or system of item 116, wherein the plurality of hollow fibers have an average lumen radius between or equal to 0.1 mm and 10 mm.

Item 118: The method or system of any one of items 116-117, wherein the plurality of hollow fibers has an average length of greater than or equal to 1 cm and less than or equal to 1 m.

Item 119: The method of any one of items 92-94, 99-104, and 106-118, wherein the cell culture is transported from the bioreactor through the TFF system with a volumetric flow rate of greater than or equal to 0.3 L/min.

Item 120: The method of any one of items 92-94, 99-104, and 106-119, wherein at least 50% of the cells in the cell clusters are stem cells.

Item 121: The method of item 120, wherein the stem cells are embryonic stem cells.

Item 122: The method of any one of items 120-121, wherein the stem cells are induced pluripotent stem cells.

Item 123: The method of any one of items 92-94, 99-104, and 106-122, wherein at least 50% of the cells in the cell clusters are OCT4-negative and SOX 17 -positive.

Item 124: The method of any one of items 92-94, 99-104, and 106-123, wherein at least 50% of the cells in the cell clusters are FOXA2-positive, PDX1 -negative.

Item 125: The method of any one of items 92-94, 99-104, and 106-124, wherein at least 50% of the cells in the cell clusters are PDX1 -positive, NKX6.1-negative. Item 126: The method of any one of items 92-94, 99-104, and 106-125, wherein at least 20% of the cells in the cell clusters are PDX1 -positive, NKX6.1-positive.

Item 127: The method of any one of items 92-94, 99-104, and 106-126, wherein at least 50% of the cells in the cell clusters are ISL1 -positive.

Item 128: The method of any one of items 92-94, 99-104, and 106-127, wherein the shear rate is greater than or equal to 600 s'¹.

Item 129: The method of any one of items 92-94, 99-104, and 106-127, wherein the shear rate is greater than or equal to 800 s'¹.

Item 130: The method of any one of items 92-94, 99-104, and 106-127, wherein the shear rate is greater than or equal to 1000 s’¹.

Item 131: The system of any one of items 95-98, 105, or 115-118, wherein the shear rate is greater than or equal to 600 s’¹.

Item 132: The system of any one of items 95-98, 105, or 115-118, wherein the shear rate is greater than or equal to 800 s’¹.

Item 133: The system of any one of items 95-98, 105, or 115-118, wherein the shear rate is greater than or equal to 1000 s’¹.

Item 134: A system for culturing cells comprising: a bioreactor configured to contain a cell culture, the bioreactor comprising: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to altematingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

Item 135: The system of any one of items 67-91, 95-98, 105, 115-118, or 131-133, wherein the bioreactor comprises: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to altematingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

Item 136: The system of any one of items 134-135, wherein the first port is disposed in the bottom portion of the bioreactor when a base of the bioreactor is disposed on a level surface.

Item 137: The system of any one of items 134-136, further comprising a gas filter separating the conduit from an external atmosphere. Item 138: The system of item 137, further comprising a relief valve configured to limit the pressure differential between the fluid conduit and the external atmosphere by passing fluid through the gas filter.

Item 139. The system of any one of items 134-138, further comprising a controllable valve configured to change the pressure of the fluid conduit.

Item 140: The system of any one of items 134-139, further comprising a mixer configured to mix the cell culture within the bioreactor, and wherein the port is configured to be disposed below the mixer relative to the direction of gravity during operation.

Item 141: The system of any one of items 138-140, wherein the relief valve is the gas filter.

Item 142: The system of any one of items 138-141, wherein the relief valve is fluidically connected to the gas filter such that fluid passing through the relief valve passes through the gas filter to enter the conduit.

Item 143: The system of any one of items 137-142, wherein the gas filter comprises one or more hollow fiber membranes.

Item 144: The system of any one of items 134-143, wherein the system further comprises a perfusion tangential flow filtration (TFF) system configured for perfusion culture of cells cultivated within the bioreactor, and wherein the bioreactor further comprises a second port that fluidically connects the bioreactor to the perfusion TFF system.

Item 145: The system of item 144, wherein the gas filter comprises a membrane substantially similar to a membrane of the perfusion TFF system.

Item 146: The system of any one of items 137-145, wherein the system comprises an agitation TFF system fluidically coupled to the fluid conduit, wherein the hollow fiber membranes of the agitation TFF system act as the gas filter.

Item 147: The system of item 146, wherein the pressure source is fluidically coupled to the fluid conduit via the agitation TFF system.

Item 148: The system of any one of items 146-147, wherein the system is configured to prevent liquid from entering the agitation TFF system.

Item 149: The system of any one of items 137-148, wherein the system is configured to prevent contact of cell culture media with the gas filter.

Item 150: The system of any one of items 134-149, further comprising a volume displacement controller configured to control the flow of the cell culture media into and/or out of the fluid conduit.

Item 151: The system of any one of items 134-150, further comprising a sensor configured to sense one or more properties related to the portion of the cell culture media. Item 152: The system of item 151, wherein the system is configured to use the one or more properties sensed by the sensor to control the flow of cell culture media in the system.

Item 153: The system of any one of items 151-152, wherein the sensor is a bubble sensor.

Item 154: The system of any one of items 134-153, configured for culturing cell clusters.

Item 155: A method for culturing cells, the method comprising: alternatingly performing the steps of: drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

Item 156: The method of any one of items 1-66, 92-94, 99-104, 106-130, wherein the method further comprises: alternatingly performing the steps of: drawing a portion of the cell culture media out of the bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

Item 157: The method of any one of items 155-156, further comprising dispersing cells adjacent to the port in the bottom portion of the bioreactor into the cell culture media in response to alternatingly drawing a portion of cell culture media out of the bioreactor and returning the portion of cell culture media to the bioreactor.

Item 158: The method of any one of items 155-157, wherein the portion of the cell culture media is drawn out of the bioreactor and/or returned to the bioreactor by changing the pressure of a gas in the fluid conduit.

Item 159: The method of item 158, further comprising passing at least a portion of the gas through a gas filter.

Item 160: The method of any one of items 158-159, wherein the pressure of the fluid conduit is moderated, at least in part, by using a relief valve to limit the pressure of the gas.

Item 161: The method of any one of items 155-160, wherein the method of culturing cells is a method of culturing cell clusters.

Item 162: The method of any one of items 159-161, further comprising preventing contact between the cell culture media and the gas filter. Item 163: The method of any one of items 155-162, further comprising moderating the volume of the portion of the cell culture media using a volume displacement control.

Item 164: The method of any one of items 155-163, further comprising moderating the volume of the portion of the cell culture media using a sensor. Item 165: The method of item 164, wherein the sensor is a bubble sensor.

Item 166: The method of item 54, wherein step f) is performed for 30 minutes to 2.5 hours.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described items and embodiments of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

CLAIMS We claim:

1. A method comprising the steps of: a) culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; b) transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; c) removing a portion of the liquid media from the cell culture in the TFF system while retaining a portion of the liquid media and cell clusters in the TFF system; d) returning the retained portion of the liquid media and cell clusters from the TFF system to the bioreactor; and e) replacing the removed portion of the liquid media with a new portion of liquid media.

2. The method of claim 1, wherein the plurality of cell clusters comprises stem cells.

3. The method of claim 2, wherein at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,

94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the cell clusters are stem cells.

4. The method of any one of claims 1-3, wherein 50-100%, 50-90%, 50-75%, 70-100%, 70- 85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75-500, 75- 400, or 75-300 pm in diameter.

5. The method of any one of claims 1-4, wherein the plurality of cell clusters are generated from a plurality of dissociated cells.

6. The method of claim 5, wherein prior to step a), the method comprises the steps of seeding the bioreactor with dissociated cells and culturing the dissociated cells to generate the plurality of cell clusters.

7. The method of claim 6, wherein the dissociated cells are cultured until 50-100%, 50- 90%, 50-75%, 70-100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the clusters are between 75-600, 75-500, 75-400, or 75-300 pm in diameter.

8. The method of claim 6, wherein the culturing step is 12-72, 12-60, 12-50, 12-36, 12-36, 18-60, 18-50, 18-36, 18-26, 26-60, 26-50, 26-36, 36-60, 36-50, or 44-52 hours in length before the step of transporting the portion of the cell culture from the bioreactor into the TFF.

9. The method of any one of claims 1-8, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

10. The method of claim 9, wherein 0.3-1, 0.3-0.8, 1-5, 1-4, 1-3, 2-5, 2-4, 2-3, 2.5-3.0, or 2.5-3.5 volumes of media are exchanged in a 24-hour period.

11. The method of any one of claims 1-10, wherein the cell culture is transported from the bioreactor through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450-1550 sec’¹.

12. The method of any one of claims 1-11, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75-100 microns in diameter.

13. The method of any one of claims 1-12, wherein the TFF system comprises one or more cassette membranes.

14. The method of any one of claims 1-12, wherein the TFF system comprises one or more hollow fiber membrane.

15. The method of claim 14, wherein the hollow fiber membrane has a radius of 0.5-10, 5-10, 2-5, 0.5-7 mm, 0.5-5, 0.5-3, 0.5-2, 0.5-1.2, 0.8-1.2, or 0.9-1.1 mm.

16. The method of any one of claims 6-14, wherein the method comprises the steps of seeding the bioreactor with 0.01 x 10⁶-10 x 10⁶, 0.01 x 10⁶-5 x 10⁶, 0.01 x 10⁶- 1 x 10⁶ , 0.01 x 10⁶-0.5 x 10⁶ , 0.01 x 10⁶-0.05 X 10⁶, 0.1 x 10⁶- 1 x 10⁶ , or 0.3 x 10⁶-0.8 x 10⁶ viable cells/ml and culturing the viable cells to generate the plurality of cell clusters.

17. The method of claim 16, wherein the viable cells are dissociated cells.

18. The method of any one of claims 16 or 17, wherein 50-100%, 50-90%, 50-75%, 70- 100%, 70-85%, 80-100%, 80-90%, 90-100%, or 90-95% of the viable cells are dissociated cells.

19. The method of any one of claims 1-18, wherein the method is repeatedly performed over a period of 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 2-12, 8-12, 3-8, 4-7, or 4-6 days.

20. The method of claim 19, wherein at the end of the period, the method comprises the step of dissociating the cell clusters.

21. The method of any one of claims 1-20, wherein the portion of the cell culture is transported from the bioreactor into the TFF system by means of a pump.

22. The method of claim 21, wherein the pump is in the TFF system.

23. The method of claim 22, wherein the method further comprises step f), wherein step f) comprises removing and replacing media in the cell culture, wherein step f) is performed for 1- 30 minutes, 30-60 minutes, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, or 7- 8 hours.

24. The method of claim 23, wherein step f) comprises exchanging media in the cell culture by means of a centrifugation, tangential flow filtration, alternating tangential flow filtration (ATF) system exchange, or settling.

25. The method of any one of claims 23 or 24, wherein the method comprises performing steps a)-e) for 12 hours to 9 days, 12 hours to 7 days, 12 hours to 5 days, 12 hours to 3 days, 12 hours to 1 day, 2-4 days, 4-6 days, or 7-9 days before step f) is performed.

26. The method of any one of claims 23-25, wherein during the media exchange step f), the replacement media comprises one or more differentiation or survival factors.

27. The method of any one of claims 23-26, wherein 70-90%, 80-90%, 70-100%, 80-100%, 90-100%, 90-95%, 95-99%, 95-98%, 95-97%, 91-96%, 92-95%, or 93-94% of the media in the reactor is removed and replaced in step f).

28. The method of any one of claims 23-27, wherein the replacement media in step f) comprises one or more differentiation or survival factors that were not present in the media removed during step f).

29. The method of any one of claims 23-28, wherein the replacement media in step f) does not comprise one or more differentiation or survival factors that were present in the media removed during step f).

30. A tangential flow filtration (TFF) system comprising a cell culture, wherein the cell culture comprises a liquid media and a plurality of cell clusters, and wherein the TFF system is in fluid communication with a bioreactor.

31. The TFF system of claim 30, wherein the plurality of cell clusters comprises stem cells.

32. The TFF system of any one of claims 30-31, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

33. The TFF system of any one of claims 30-32, wherein the cell culture flows through the TFF system at a shear rate of 400-800, 400-3500, 400-3000, 400-2500, 400-2000, 400-1500, 1000-3500, 1000-3000, 1000-2000, 2000-3500, 2000-3000, 1200-1800, 1400-1600, or 1450- 1550 sec'¹.

34. The TFF system of any one of claims 30-33, wherein the TFF system comprises one or more filters, wherein the one or more filters comprise a plurality of pores, wherein from the pores are 0.2-100, 0.2-75, 0.2-50, 0.2-25, 0.2-10, 0.2-5, 0.2-1, 1-10, 5-10, 25-50, 50-75, or 75- 100 microns.

35. The TFF system of any one of claims 30-34, wherein the TFF system comprises one or more cassette membranes.

36. The TFF system of any one of claims 30-35, wherein the TFF system comprises a pump.

37. A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 400.

38. A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; and transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; wherein: the cell culture is transported from the bioreactor through the TFF system with a shear rate of greater than or equal to 400 s'¹.

39. A method for culturing cells, the method comprising: culturing a cell culture in a bioreactor; wherein the cell culture comprises a liquid media and a plurality of cell clusters; transporting a portion of the cell culture from the bioreactor into a tangential flow filtration (TFF) system; and controlling the flow of the portion of the cell culture through the TFF system to shear the plurality of cell clusters to provide an average maximum transverse dimension of the plurality of cell clusters that is between or equal to 75 pm and 600 pm.

40. A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a Reynold’s number (Re) of less than or equal to 400 to the portion of the portion of the cell culture pumped to the TFF system.

41. A system for culturing cells comprising: a bioreactor configured to contain a cell culture; a tangential flow filtration (TFF) system including a first port in fluid communication with the cell culture and a waste port; and a pump configured to pump a portion of the cell culture to the tangential flow filtration system, wherein the pump is configured to return a retentate to the bioreactor, and wherein the TFF system and the pump are configured to apply a shear rate of greater than or equal to 400 s'¹ to the portion of the portion of the cell culture pumped to the TFF system.

42. The system of claim 40, wherein the first port is in fluid communication with an inlet of the pump and further comprising a second port in fluid communication with an outlet of the pump.

43. The system of claim 40, further comprising the cell culture disposed in the bioreactor.

44. The method of any one of claims 37-39, wherein the plurality of cell clusters has an average maximum transverse dimension greater than or equal to 75 pm.

45. The method of any one of claims 37-39 and 44, wherein the plurality of cell clusters has an average maximum transverse dimension less than or equal to 600 pm.

46. The method of any one of claims 37-39 and 44-45, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number (Re) of less than or equal to 2000.

47. The method of any one of claims 37-39 and 44-46, further comprising returning a retentate of the portion of the cell culture to the bioreactor from the TFF system.

48. The method of any one of claims 37-39 and 44-47, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed continuously.

49. The method of any one of claims 37-39 and 44-48, wherein transporting the portion of the cell culture to the TFF system and returning the portion of the cell culture to the bioreactor are performed periodically.

50. The system of any one of claims 40-43, wherein the pump is a positive displacement, diaphragm pump, or peristaltic pump.

51. The method of any one of claims 37-39 and 44-49, further comprising removing a retentate of the portion of the cell culture from the TFF system.

52. The method of any one of claims 37-39, 44-49, and 51, further comprising adding fresh liquid media to the bioreactor.

53. The method of any one of claims 37-39, 44-49, and 51-52, wherein the plurality of cell clusters has an average diameter of greater than or equal to 100 pm, 120 pm, 150 pm, 170 pm.

54. The method of any one of claims 37-39, 44-49, and 51-53, wherein the plurality of cell clusters has an average diameter of less than or equal to 500 pm, 300 pm, 250 pm.

55. The method of any one of claims 37-39, 44-49, and 51-54, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of greater than or equal to 50.

56. The method of any one of claims 37-39, 44-49, and 51-55, wherein the cell culture is transported from the bioreactor through the TFF system with a Reynold’s number of less than or equal to 300.

57. The method or system of any one of claims 37-56, wherein the TFF system is an alternating tangential flow filtration (ATF) system.

58. The method or system of any one of claims 37-57, wherein the TFF system comprises a plurality of hollow fibers, and wherein a ratio of an average lumen radius of the plurality of hollow fibers to the average maximum transverse dimension of the cell clusters is greater than or equal to 2 but less than or equal to 10.

59. The method or system of claim 58, wherein the plurality of hollow fibers have an average lumen radius between or equal to 0.1 mm and 10 mm.

60. The method or system of any one of claims 58-59, wherein the plurality of hollow fibers has an average length of greater than or equal to 1 cm and less than or equal to 1 m.

61. The method of any one of claims 37-39, 44-49, and 51-60, wherein the cell culture is transported from the bioreactor through the TFF system with a volumetric flow rate of greater than or equal to 0.3 L/min.

62. The method of any one of claims 37-39, 44-49, and 51-61, wherein at least 50% of the cells in the cell clusters are stem cells.

63. A system for culturing cells comprising: a bioreactor configured to contain a cell culture, the bioreactor comprising: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

64. The system of any one of claims 30-36, 41-43, 50, or 57-60, wherein the bioreactor comprises: a first port disposed in a bottom portion of the bioreactor relative to a direction of gravity; a fluid conduit connected to the first port; and a pressure source connected to the first port via the fluid conduit, wherein the pressure source is configured to alternatingly draw a portion of a cell culture media through the port and into a fluid conduit and return the cell culture media from the fluid conduit to the bioreactor through the port to agitate cells in the bottom portion of the bioreactor.

65. The system of any one of claims 63-64, further comprising a mixer configured to mix the cell culture within the bioreactor, and wherein the port is configured to be disposed below the mixer relative to the direction of gravity during operation.

66. A method for culturing cells, the method comprising: altematingly performing the steps of: drawing a portion of a cell culture media out of a bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

67. The method of any one of claims 1-29, 37-39, 44-49, 51-62, wherein the method further comprises: altematingly performing the steps of: drawing a portion of the cell culture media out of the bioreactor and into a fluid conduit through a port in a bottom portion of the bioreactor relative to a local direction of gravity; and returning the portion of the cell culture media from the fluid conduit to the bioreactor through the port; wherein the drawing the portion of the cell culture media from the bioreactor and the returning the portion of the cell culture media to the bioreactor are alternated to agitate cells in the bottom portion of the bioreactor.

68. The method of any one of claims 66-67, further comprising dispersing cells adjacent to the port in the bottom portion of the bioreactor into the cell culture media in response to altematingly drawing a portion of cell culture media out of the bioreactor and returning the portion of cell culture media to the bioreactor.

69. The method of any one of claims 66-68, wherein the portion of the cell culture media is drawn out of the bioreactor and/or returned to the bioreactor by changing the pressure of a gas in the fluid conduit.