WO2023149566A1 - 多能性幹細胞の分化誘導制御剤および分化誘導安定化剤 - Google Patents
多能性幹細胞の分化誘導制御剤および分化誘導安定化剤 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention relates to an agent for controlling differentiation induction of pluripotent stem cells and an agent for stabilizing differentiation induction of pluripotent stem cells.
- pluripotent stem cells may be used as a cell source.
- this cell production (differentiation induction) process it is necessary to induce differentiation of pluripotent stem cells into target cells as uniformly as possible, mature them, and maintain the target differentiation state.
- non-target cells may be generated.
- the speed of differentiation induction may differ between individual cells, and it may not be possible to obtain uniformly differentiated cells.
- the quality of the obtained cells may differ depending on the procedure.
- Patent Documents 1 and 2 By the way, in the process of inducing the differentiation of pluripotent stem cells, there are attempts to increase the production of specific target differentiated cells or to improve the efficiency of differentiation induction using E-cadherin inhibitors (Patent Documents 1 and 2). .
- Patent Document 1 discloses a method for increasing the production of human embryonic stem (ES) cells by causing an E-cadherin inhibitor to act on them, improving the efficiency of differentiation into definitive endoderm cells using TGF ⁇ , and increasing the production of the cells.
- Patent Document 2 discloses that in a method for inducing differentiation of ES or induced pluripotent stem (iPS) cells, E-cadherin inhibitors delay differentiation into most cell lineages, but delay differentiation into neural progenitor cells. As a result, E-cadherin inhibitors contribute to the induction of differentiation into neural progenitor cells.
- ES human embryonic stem
- iPS induced pluripotent stem
- Patent Documents 3 and 4 disclose methods for removing cells deviating from an undifferentiated state that have occurred or can occur during culturing of pluripotent stem cells using hemagglutinin and its variants. Further, Patent Document 4 discloses a method of dividing cell clusters in suspension culture of iPS cells into small clusters using hemagglutinin. Furthermore, Patent Document 5 discloses a method that enables mass culture of pluripotent stem cells using hemagglutinin or a variant thereof.
- Patent Documents 1 and 2 there are no reports that E-cadherin inhibitors contribute to differentiation induction of pluripotent stem cells other than the induction of differentiation into definitive endoderm cells or neural progenitor cells as shown in Patent Documents 1 and 2.
- the inventions described in Patent Documents 3 to 5 are techniques for maintaining, expanding and culturing pluripotent stem cells, and are not techniques for induction of differentiation.
- An object of the present invention is to provide pluripotent stem cells that can provide any desired differentiated cells from pluripotent stem cells as a uniform and homogeneous cell population by suppressing the generation of unintended cells.
- the objective is to provide means for controlling differentiation.
- Another object of the present invention is to provide a method for stably inducing the differentiation of pluripotent stem cells into target cells using the differentiation control means.
- the present inventors have so far developed a technique for removing "cells that have deviated from an undifferentiated state" generated during the culture of pluripotent stem cells, and a technique for mass production of pluripotent stem cells, "cells that maintain an undifferentiated state”. has been involved in the development of technology for cultivating
- the technology is based on the mechanism that hemagglutinin, which is an inhibitor of E-cadherin, a cell adhesion molecule, specifically binds to E-cadherin to inhibit intercellular adhesion, thereby alleviating intercellular adhesion. (WO2014/104207, WO2015/199243 and WO2018/117110). It is thought that the relaxation of cell-to-cell adhesion makes the culture environment of each cell in the colony of pluripotent stem cells uniform and homogenous, which makes it difficult for deviating cells to occur and facilitates cell proliferation.
- the present inventors have developed the above-described findings regarding the maintenance of an undifferentiated state of pluripotent stem cells, and have found that in inducing differentiation from pluripotent stem cells to cells of interest in vitro, when a conventional differentiation induction method is used, cells
- the reason why pluripotent stem cells grow and differentiate in different states, resulting in the generation of unintended cells is that during the process of pluripotent stem cell proliferation and formation of clumps (colonies), cell-to-cell adhesion depends on the position within the clumps. I thought it might be because the degree is different.
- the cells are not so crowded and the cell-cell adhesion is not strong, but inside the colonies and clumps, the cells are crowded and the cells adhere strongly to each other.
- the differentiation-inducing environment of the pluripotent stem cells that make up the colonies and aggregates is different, so we hypothesized that cells deviating from the desired differentiation state are generated.
- the present inventors added hemagglutinin, which can inhibit E-cadherin, a protein that controls intercellular adhesion, to the differentiation-inducing system of pluripotent stem cells, thereby equalizing the intercellular adhesion force in aggregates. tried to As a result, surprisingly, by changing the concentration of hemagglutinin, it is possible to control the direction of differentiation induction from pluripotent stem cells to any of the three germ layer lineages. It was found that it is possible to direct the differentiation of Furthermore, it was found that administration of hemagglutinin can improve differentiation induction efficiency.
- the present invention is as follows.
- (Section 1) An agent for directional control of differentiation induction of pluripotent stem cells, containing an E-cadherin inhibitor.
- (Section 2) Item 2.
- the agent according to item 1, wherein the pluripotent stem cells are embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.
- (Section 3) Item 3.
- the agent according to Item 1 or 2 wherein the directional control of differentiation induction of pluripotent stem cells is a change in differentiation efficiency from pluripotent stem cells to any of the three germ layer lineages.
- (Section 5) Item 5.
- (Section 6) A method for controlling the direction of differentiation induction of pluripotent stem cells, comprising the step of culturing pluripotent stem cells in a medium containing an E-cadherin inhibitor.
- (Section 8) Item 8.
- the agent according to Item 11, wherein the stabilization of induction of differentiation of pluripotent stem cells is improvement in efficiency of differentiation from pluripotent stem cells to any of the three germ layer lineages.
- Item 13 Item 12.
- the agent according to Item 11, wherein the stabilization of induction of differentiation of pluripotent stem cells is the reduction or removal of cells deviating from the intended differentiated cells.
- Item 14 Item 14.
- the agent according to any one of Items 11 to 13, wherein the E-cadherin inhibitor is hemagglutinin.
- a method for producing desired differentiated cells from pluripotent stem cells comprising the following steps.
- the direction of differentiation induction of pluripotent stem cells can be controlled.
- differentiation induction from pluripotent stem cells to target cells can be stabilized. That is, according to the present invention, by changing the concentration of an E-cadherin inhibitor (preferably hemagglutinin), the environment in which cells are induced to differentiate can be changed. You can direct the induction. Then, by using an E-cadherin inhibitor (preferably, hemagglutinin), a uniform intercellular adhesion environment suitable for the target differentiated cells is constructed in the entire culture system, resulting in the differentiation induction efficiency into the target differentiated cells. can be significantly improved.
- an E-cadherin inhibitor preferably, hemagglutinin
- RPE cells retinal pigment epithelial cells
- known RPE cell differentiation induction basal medium RPE cell maintenance medium containing no hemagglutinin or containing hemagglutinin at various concentrations
- the lower part of the photo is an enlarged part of the upper part.
- Human iPS cells were cultured in a medium containing various concentrations of hemagglutinin for 24 hours, and then further cultured in a medium for inducing differentiation into each of the three germ layers.
- Results of comparing the expression levels of target germ layer marker genes It is a figure which shows. The vertical axis indicates the relative expression level of each gene, with the expression level when cultured in a hemagglutinin-free medium being set to 1.
- FIG. 4 shows the results of comparing the expression levels of cardiomyocyte marker genes when human iPS cells were cultured in a medium containing various concentrations of hemagglutinin for 24 hours and then further cultured in a cardiomyocyte differentiation-inducing medium. .
- the vertical axis indicates the relative expression level of each gene, with the expression level when cultured in a hemagglutinin-free medium being set to 1.
- * p ⁇ 0.05, ** : p ⁇ 0.1
- IQR interquartile range
- Fig. 10 shows the results of comparing the expression levels of marker genes in various cells when human iPS cells were cultured in a medium containing hemagglutinin at various concentrations for 3 days and then further cultured in a medium for inducing differentiation into hepatocytes.
- (A) shows a comparison of expression levels of pluripotency marker genes, endoderm marker genes, and liver common marker genes 3 days after initiation of hepatocyte induction (after endoderm induction treatment).
- the present invention is at least partially based on the fact that in the maintenance and expansion culture of pluripotent stem cells, cells that deviate from an undifferentiated state are generated in the colony because the intercellular adhesion force due to E-cadherin has strength and weakness in the colony.
- the susceptibility of each pluripotent stem cell to a medium environment that supports maintenance of an undifferentiated state is due to heterogeneity.
- the present inventors focused on the inhibition of cell-cell adhesion by E-cadherin inhibitors based on the findings obtained from pluripotent stem cell maintenance and expansion techniques, It is speculated that this also contributes to the “homogenization and homogenization” of the intracellular environment in the pluripotent stem cells, resulting in the qualitative and quantitative improvement of the differentiated cells obtained in the differentiation induction system of pluripotent stem cells.
- This hypothesis may explain the seemingly contradictory phenomenon that E-cadherin contributes not only to the maintenance of the undifferentiated state of pluripotent stem cells but also to the induction of differentiation into specific differentiated cells.
- the degree of inhibition of intercellular adhesion by hemagglutinin, an E-cadherin inhibitor (specifically, differences in the concentration of hemagglutinin contained in the medium, the treatment time with hemagglutinin, the timing of hemagglutinin treatment, etc.) It was also found to affect phenotypic differences in the resulting differentiated cells. It is thought that this is because the difference in the strength of cell-to-cell adhesion affects the "direction of differentiation induction" in the early stages of differentiation induction. ), it is possible to efficiently (more uniformly and homogenously) induce differentiation into any desired cells.
- Control agent of the present invention provides an agent for directional control of differentiation induction of pluripotent stem cells, which contains an E-cadherin inhibitor. Hereinafter, it may be referred to as "the control agent of the present invention”.
- pluripotent stem cells are undifferentiated cells that have "self-renewal ability" that can proliferate while maintaining an undifferentiated state and "pluripotency” that can differentiate into all three germ layer lineages. Examples include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Pluripotent stem cells also include, for example, pluripotent stem cells derived from mammals. Pluripotent stem cells derived from mammals are not particularly limited, and examples thereof include human pluripotent stem cells. Human pluripotent stem cells are not particularly limited, and examples thereof include human iPS cells and human ES cells.
- differentiation induction in “directional control of differentiation induction of pluripotent stem cells” means placing pluripotent stem cells in an environment that does not support maintenance of an undifferentiated state. Not only are pluripotent stem cells placed in the presence of a differentiation-inducing agent that supports differentiation into predetermined target cells, but also substances that simply support maintenance of an undifferentiated state (e.g., bFGF, SCF, LIF, etc.) Absence is also included.
- pluripotent stem cells formation of embryoid bodies from pluripotent stem cells, differentiation from pluripotent stem cells to any of the three germ layers (differentiation from pluripotent stem cells to ectoderm, differentiation from pluripotent stem cells to mesoderm) or differentiation from pluripotent stem cells to endoderm), and differentiation from pluripotent stem cells to ectodermal cells, mesodermal cells, or endoderm cells.
- ectodermal cells are not particularly limited, and include, for example, neural cells, epidermal cells, and the like. Specific examples thereof include retinal pigment epithelial cells and the like.
- Mesodermal cells are not particularly limited, and examples include blood cells (including hematopoietic cells), vascular cells (vascular endothelial cells, etc.), cardiomyocytes (e.g., atrial myocytes, ventricular myocytes, etc.), and osteocytes. , chondrocytes, tendon cells, adipocytes, skeletal muscle cells, smooth muscle cells and the like.
- Endoderm cells are not particularly limited, and examples include digestive system cells, pancreatic cells, liver cells, respiratory system cells, thyroid cells, and the like.
- the “directional control of differentiation induction of pluripotent stem cells” performed using the control agent of the present invention refers to three germ layers (ectoderm, mesoderm, endoderm) in the differentiation induction of pluripotent stem cells. ) refers to changing the efficiency of differentiation into cells of either lineage.
- the E-cadherin inhibitor contained in the control agent of the present invention has the function of alleviating intercellular adhesion by E-cadherin. Therefore, by adding the control agent of the present invention to the culture system, it is possible to “equalize” the strength of the physical intercellular adhesion in the cell cluster, and by adjusting the amount added, “equalize” the strength. It is possible to "control" the "direction” of cell differentiation induction by adjusting the strength of intercellular adhesion.
- the cell culture environment is homogenized by homogenizing the physical adhesion between cells. This is presumed to reduce the number of cells that deviate from the intended differentiated cells when pluripotent stem cells are induced to differentiate. Furthermore, according to the studies of the present inventors, by controlling the physical intercellular adhesion force, in the initial stage of differentiation induction, "direction of differentiation induction" to one of the three germ layer lineages (herein, "differentiation It was found that it is possible to control the directional control of induction). This is because by changing the amount of the control agent of the present invention in the differentiation induction system, the intracellular environment is affected by changing the intercellular adhesion force, and the efficiency of differentiation into any of the desired three germ layer lineages changes.
- the E-cadherin inhibitory substance may be any substance capable of inhibiting intercellular adhesion by E-cadherin, such as hemagglutinin, nucleic acids capable of inhibiting expression of E-cadherin (e.g., antisense oligonucleotides, etc.), Examples thereof include antibodies against E-cadherin.
- hemagglutinin is used as E-cadherin inhibitor.
- Hemagglutinin (HA) is not particularly limited in its acquisition method or origin, as long as it can inhibit intercellular adhesion by E-cadherin.
- Clostridium botulinum can be any of A, B, C, and D types.
- Hemagglutinin refers to neurotoxin complexes (also referred to as hemagglutinin complexes) unless otherwise specified.
- the hemagglutinin may be a wild type, a variant of hemagglutinin, or a miniaturized hemagglutinin.
- hemagglutinin may be a variant of hemagglutinin to the extent that it has the effect of contributing to directional control of differentiation induction of pluripotent stem cells.
- One or more embodiments of the variant include, for example, those in which one, two or three amino acid sequences of the subcomponents have at least one amino acid mutation.
- Specific examples of the variants include mutant hemagglutinin (HA) complex proteins described in WO2015/199243.
- hemagglutinin may be miniaturized hemagglutinin as long as it has the effect of contributing to directional control of differentiation induction of pluripotent stem cells.
- Miniaturized hemagglutinins include, for example, HA complex proteins that retain cadherin function-inhibiting activity and lack all or part of subcomponents.
- Specific examples of miniaturized hemagglutinin include miniaturized hemagglutinin complex proteins having E-cadherin function-inhibiting activity described in WO2019/103111.
- hemagglutinin includes “variants of hemagglutinin” and “downsized hemagglutinin”.
- hemagglutinin variant and “miniaturized hemagglutinin” refer to those having the effect of contributing to directional control of differentiation induction of pluripotent stem cells.
- the control agent of the present invention may further contain a differentiation-inducing agent suitable for the target differentiated cells.
- the differentiation-inducing agent may be provided as a separate reagent from the control agent of the present invention and formed into a kit together with the control agent of the present invention.
- culturing pluripotent stem cells using the control agent of the present invention it is possible to control the direction of differentiation induction of pluripotent stem cells.
- “Directional control of differentiation induction of pluripotent stem cells” is as described above.
- a person skilled in the art can determine the differentiation induction efficiency to the obtained differentiated cells by appropriately determining the amount of the regulatory agent of the present invention that is suitable for the target differentiated cells and the differentiation-inducing system (differentiation-inducing agent) to be used. can be changed. For example, when hemagglutinin is used as an E-cadherin inhibitor, the hemagglutinin concentration is appropriately varied between 1 and 80 nM to test the efficiency of inducing differentiation into target cells.
- the amount of control agent of the invention can be determined.
- the amount of the control agent of the present invention used in differentiation induction varies depending on the scale, culture conditions, culture environment, etc. of the differentiation-inducing system to be applied. It's not something you can do.
- the control agent of the present invention is useful only in the sense that a person skilled in the art can change the differentiation induction efficiency by applying the control agent of the present invention to a differentiation-inducing system that he/she implements based on his/her own experience. It is something to have.
- control agent of the present invention can be used to reduce or eliminate cells that deviate from the intended differentiated cells in the differentiation-inducing system.
- the use of the control agent of the present invention homogenizes the physical intercellular adhesive force in the cell culture mass, thereby homogenizing the cell culture environment. This is presumed to reduce the number of cells that deviate from the intended differentiated cells in the differentiation-inducing system.
- “cells that deviate from the target differentiated cells” refer to cells that have been induced to become different cells from the target differentiated cells in an unspecified differentiation induction system, or intermediate cells that have not been sufficiently induced to differentiate. A cell that is mixed with a target differentiated cell.
- Such deviant cells can be identified by cell morphology and the expression of differentiation markers, and should be removed in the production of cell pharmaceuticals. has a very high value.
- Control method of the present invention also provides a method for directional control of differentiation induction of pluripotent stem cells, comprising the step of culturing pluripotent stem cells in a medium containing an E-cadherin inhibitor. .
- the control method of the present invention it may be referred to as "the control method of the present invention”.
- the medium used for culturing pluripotent stem cells is not particularly limited as long as it can maintain, proliferate and/or induce differentiation of pluripotent stem cells.
- the medium used for culturing pluripotent stem cells can be a medium that contains an E-cadherin inhibitor and supports maintenance of the undifferentiated state of pluripotent stem cells.
- Such a medium can be prepared by adding an E-cadherin inhibitor to a known pluripotent stem cell maintenance medium.
- the medium used for culturing pluripotent stem cells may be a medium that contains an E-cadherin inhibitor and does not support maintenance of the undifferentiated state of pluripotent stem cells.
- Such a medium is not particularly limited as long as it contains an E-cadherin inhibitor and does not support maintenance of the undifferentiated state of pluripotent stem cells.
- a medium containing a substance preferably hemagglutinin (especially hemagglutinin derived from Clostridium botulinum), and other components.
- the medium preferably does not contain substances (e.g., bFGF, SCF, LIF, etc.) that support the maintenance of the undifferentiated state, but if the medium as a whole does not support the maintenance of the undifferentiated state of the pluripotent stem cells, This is not the case.
- the basal medium one or a combination of a plurality of synthetic media commonly used for culturing mammalian cells can be used, such as Glasgow MEM (GMEM), IMDM (Iscove's Modified Dulbecco's Medium), DMEM.
- GMEM Glasgow MEM
- IMDM Iscove's Modified Dulbecco's Medium
- DMEM DMEM
- NeuroBasal medium NeuroBasal-A medium
- F12-Ham F12-Ham
- Serum derived from mammals such as bovines and humans can be used.
- a serum substitute is a low-protein substitute for serum such as FBS used in cell culture, and commercially available products include, for example, Knockout Serum Replacement (KSR) and Chemically-defined Lipid concentrated (Gibco). , Glutamax (manufactured by Gibco), and N2 and B27, which are serum substitutes for nerve cell culture.
- KSR Knockout Serum Replacement
- Glutamax manufactured by Gibco
- N2 and B27 which are serum substitutes for nerve cell culture.
- the concentration of serum or serum substitute can be appropriately set, for example, in the range of 0.5 to 30% (v/v), and the concentration may be constant or may be changed stepwise.
- the concentration of the E-cadherin inhibitor is not particularly limited, from the viewpoint of reducing the number of cells deviating from the target differentiated cells in the induction of differentiation from pluripotent stem cells to any of the three germ layers, and from the viewpoint of changing the efficiency of differentiation induction. It is possible for a person skilled in the art to set it as appropriate from the point of view. For example, when hemagglutinin is used as an E-cadherin inhibitor, the hemagglutinin suitable for the differentiation induction system to be used is determined by appropriately varying the concentration of hemagglutinin between 1 and 80 nM and assaying the efficiency of differentiation induction into the target cells. Concentration can be determined.
- the concentration is not particularly limited for the same reason as above. It can be set low overall.
- the concentration is not particularly limited for the same reason as above, but the miniaturized hemagglutinin generally tends to have a lower activity in the function of relieving intercellular adhesion. Therefore, the target concentration can be set to be high as a whole.
- the medium can contain other components commonly used for culturing mammalian cells.
- the medium that contains an E-cadherin inhibitor and does not support the maintenance of the undifferentiated state of pluripotent stem cells may further contain a differentiation inducer suitable for the target differentiated cells.
- an E-cadherin inhibitor may be added to (1) the medium during expansion culture in which pluripotent stem cells are maintained in an undifferentiated state, in order to achieve uniform and homogenous induction of differentiation.
- (2) may be added to the medium in the induction of differentiation from pluripotent stem cells to specific differentiated cells of interest, or (3) contains an E-cadherin inhibitor and does not induce differentiation into specific differentiated cells It may be added to a medium (without a differentiation inducer) that does not support maintenance of an undifferentiated state.
- the cells obtained by the culturing step can be further cultured in a medium (containing a differentiation-inducing agent) for inducing differentiation into specific target differentiated cells.
- the differentiation-inducing medium may or may not contain an E-cadherin inhibitor.
- an E-cadherin inhibitor by contacting pluripotent stem cells with an E-cadherin inhibitor at an early stage of differentiation induction (for example, at a stage where differentiation into specific differentiated cells such as embryoid body formation is not induced), any three germ layers It is possible to direct the cells to this lineage, and the subsequent culture using a differentiation inducer can improve the homogeneity and homogeneity of the desired differentiated cells obtained, and also improve the yield. can. That is, the E-cadherin inhibitor can be applied to the "stabilization method of the present invention" described later from the same viewpoint.
- the E-cadherin inhibitor achieves uniform intercellular adhesion and direction of differentiation induction into the desired differentiated cells, and E-cadherin It can be appropriately set within a range in which long-term contact with the inhibitor does not adversely affect cells.
- E-cadherin inhibitor was added from day 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50 days or more) can continue to be added.
- an E-cadherin inhibitor is added, for example, for several hours to several days, preferably can be cultured for 1 to several days (eg, 5, 4, 3, 2) and then further cultured in medium B, which may or may not contain an E-cadherin inhibitor.
- the adherent culture method includes, for example, a method using an adherent cell culture vessel, a method in which pluripotent stem cells are cultured while adhered to feeder cells (for example, WO2001/088100), and a culture medium coated with a laminin E8 fragment.
- a method using a material for example, WO2015/053375 is known, but it is not limited to these.
- a group of pluripotent stem cells that aggregate to form a clump is cultured in a suspended state in a culture medium (for example, WO2008/087917, Nature. 2011 Apr 7;472( 7341):51-6), but are not limited to these.
- the density of the pluripotent stem cells used for culture is not particularly limited as long as the pluripotent stem cells can be uniformly seeded and adherently cultured. It is desirable that the cell density is such that the degree of cell-to-cell adhesion is homogenized and the state of differentiation induction is appropriately controlled, and such a cell density can be set as appropriate. Other culture conditions such as culture temperature and CO 2 concentration can also be set appropriately.
- the culture period is not particularly limited as long as the induction of differentiation of pluripotent stem cells can be controlled, but the culture period is such that the E-cadherin inhibitor maintains the uniforming effect of intercellular adhesion and the induction of differentiation can be controlled. It is desirable to have This culture period can be appropriately changed depending on the number of pluripotent stem cells used for culture, culture equipment, culture substrate, culture method, and the like.
- the culture substrate used in the adherent culture method is not particularly limited, and includes known culture substrates such as culture substrates coated with laminin E8 fragments.
- the culture may be suspension culture in addition to the adhesion culture described above.
- the number of pluripotent stem cells forming aggregates used for suspension culture is such that the degree of cell-to-cell adhesion is homogenized by administering an E-cadherin inhibitor, and the state of differentiation induction can be appropriately controlled. , and can be appropriately set depending on the target differentiated cells and the differentiation induction method to be applied. Other culture conditions such as culture temperature and CO 2 concentration can also be set appropriately.
- the culture period is not particularly limited as long as the induction of differentiation of pluripotent stem cells can be controlled, but the culture period is such that the E-cadherin inhibitor maintains the uniforming effect of intercellular adhesion and the induction of differentiation can be controlled. It is desirable to have This culture period can be appropriately changed depending on the number of pluripotent stem cells used for culture, culture equipment, culture substrate, culture method, and the like.
- the incubator used in the suspension culture method is not particularly limited as long as it allows cells to be cultured in suspension.
- Such incubators include, for example, flasks, tissue culture flasks, petri dishes, tissue culture dishes, multidishes, microplates, microwell plates, micropores, multiplates, multiwell plates, chamber slides, tubes, Examples include trays, culture bags, roller bottles, and the like.
- the differentiation-inducing agent used in the control method of the present invention is not particularly limited, and a known differentiation-inducing agent suitable for the target differentiated cells can be used as appropriate. Examples include Nodal signal inhibitors, Wnt signal inhibitors, Sonic hedgehog signal inhibitors, Activin signal promoters, and the like, but these are illustrative and not limiting.
- Stabilizing agent of the present invention also provides an agent for stabilizing differentiation induction from pluripotent stem cells to target differentiated cells, which contains an E-cadherin inhibitor.
- the stabilizer of the present invention it may be referred to as "the stabilizer of the present invention”.
- the E-cadherin inhibitor, pluripotent stem cells, and directional control of differentiation induction of pluripotent stem cells in the stabilizing agent of the present invention refer to the contents described in "1. Regulator of the present invention" as appropriate.
- stabilizing agent of the present invention “differentiation induction” in “stabilization of differentiation induction of pluripotent stem cells” refers to the content described in "1. Regulator of the present invention”.
- stabilization of differentiation induction of pluripotent stem cells” performed using the stabilizer of the present invention means that in the above-mentioned induction of differentiation of pluripotent stem cells, the efficiency of differentiation induction into target cells is improved. and/or the ability to reproducibly induce differentiation into target cells.
- the stabilizing agent of the present invention By adding the stabilizing agent of the present invention to a culture system, the strength of physical intercellular adhesion in cell clusters is homogenized to control the direction of differentiation induction of cells, and the addition amount is adjusted. By adjusting the strength of the intercellular adhesion that has been homogenized by , it is possible to stabilize the induction of differentiation into the desired differentiated cells.
- the above-mentioned "directional control of differentiation induction" As described, it is possible to increase the efficiency of induction of differentiation into any lineage of the desired three germ layers and the reproducibility thereof.
- Those skilled in the art can appropriately determine the amount of the stabilizing agent of the present invention that is suitable for the target differentiated cells, thereby improving the efficiency of induction of differentiation into differentiated cells and the reproducibility thereof.
- the hemagglutinin concentration of the present invention suitable for the differentiation-inducing system to be used is determined by appropriately varying the concentration between 1 and 80 nM and assaying the efficiency of differentiation into target cells. can be determined.
- the amount of the stabilizing agent of the present invention used in the induction of differentiation varies depending on the scale, culture conditions, culture environment, etc. of the differentiation-inducing system to be applied.
- the stabilizing agent of the present invention is useful in the sense that a person skilled in the art can improve the differentiation induction efficiency by applying the stabilizing agent of the present invention to a differentiation-inducing system that he or she implements based on his/her own experience. It has a nature.
- the stabilizing agent of the present invention can be used to reduce or eliminate cells that deviate from the target cells in the differentiation-inducing system.
- the use of the stabilizing agent of the present invention homogenizes the physical intercellular adhesive force in the cell culture mass, thereby homogenizing the cell culture environment. This is presumed to reduce the number of cells deviating from target cells in the differentiation-inducing system.
- the “cells deviating from the target cells” are as described above.
- the present invention also provides a method for efficiently producing target differentiated cells from pluripotent stem cells, in other words, a method for stabilizing differentiation induction into target cells.
- this method may be referred to as the "stabilization method of the present invention”.
- the method includes (1) the steps of forming aggregates of pluripotent stem cells, and (2) culturing the aggregates obtained in step (1) in a medium containing an E-cadherin inhibitor, It is not particularly limited as long as it includes a step of stabilizing the induction of differentiation into the target cells.
- the stabilization method of the present invention uses pluripotent stem cells as starting cells and stabilizes the induction of differentiation into target differentiated cells. is a method of manufacturing Since pluripotent stem cells are preferably in a state where cell-to-cell adhesion exists, the induction method of the present invention includes the step of (1) forming aggregates of pluripotent stem cells.
- “Aggregate” in step (1) refers to a mass formed by aggregating cells dispersed in the medium and in a state in which the cells adhere to each other. Therefore, colonies, aggregates, cell clusters, embryoid bodies, spheres, and spheroids are also included in aggregates, and in a cell culture system, cells are in a confluent state to the extent that they adhere to each other. If it is, that state is also included. Aggregates of pluripotent stem cells are formed, for example, by bringing cells to a confluent state by an adherent culture method, or by culturing pluripotent stem cells by a suspension culture method. Such media and culture methods include those described above.
- the medium and culture method used in step (2) include those described in "2. Control method of the present invention”. That is, in the same manner as in the aspect described in "2. Control method of the present invention", the stabilization method of the present invention also uses an E-cadherin inhibitor in each step of inducing differentiation into the target differentiated cells. , can stabilize induction of differentiation into target differentiated cells.
- Target differentiated cells include, for example, ectodermal cells, mesoderm cells, and endoderm cells.
- Ectodermal cells are not particularly limited, and include, for example, nervous system cells and epidermal cells. Specific examples thereof include retinal pigment epithelial cells and the like.
- Mesodermal cells are not particularly limited, and examples include blood cells (including hematopoietic cells), vascular cells (vascular endothelial cells, etc.), cardiomyocytes (e.g., atrial myocytes, ventricular myocytes, etc.), and osteocytes. , chondrocytes, tendon cells, adipocytes, skeletal muscle cells, smooth muscle cells and the like.
- Endoderm cells are not particularly limited, and examples include digestive system cells, pancreatic cells, liver cells, respiratory system cells, thyroid cells, and the like.
- the concentration of the E-cadherin inhibitor is not particularly limited, and from the viewpoint of reducing the number of cells deviating from the target cells in the induction of differentiation from pluripotent stem cells to any of the three germ layers, the differentiation induction efficiency and its reproducibility are improved. That is, from the viewpoint of stabilizing the induction of differentiation into the desired differentiated cells, a person skilled in the art can set it as appropriate. For example, when hemagglutinin is used as an E-cadherin inhibitor, the concentration of hemagglutinin is appropriately varied between 1 and 80 nM, and the efficiency of induction of differentiation into the target cells and its variation are tested. A suitable hemagglutinin concentration can be determined.
- the concentration is not particularly limited for the same reason as above. It can be set low overall.
- the concentration is not particularly limited for the same reason as above, but the miniaturized hemagglutinin generally tends to have a lower activity in the function of relieving intercellular adhesion. Therefore, the target concentration can be set to be high as a whole.
- Example 1 Direction of differentiation induction using hemagglutinin
- a basal medium for inducing differentiation into retinal pigment epithelial cells RPE cells
- Induction of retinal pigment epithelial cells was promoted without using a differentiation-inducing agent (for example, SB431542, CKI-7, etc.) that promotes differentiation induction of retinal pigment epithelial cells.
- a differentiation-inducing agent for example, SB431542, CKI-7, etc.
- botulinum-derived hemagglutinin was added at different concentrations to observe the effect on induction of differentiation. Specific operations are as follows.
- the reagents used are as follows. ⁇ Differentiation-inducing basal medium (Glasgow's MEM (GMEM) medium (Invitrogen), KnockOut TM Serum Replacement (KSR) (Invitrogen), 0.1 mM MEM non-essential amino acid solution (Invitrogen), 1 mM sodium pyruvate (SIGMA), StemSure (registered Trademark) 10 mmol/l 2-mercaptoethanol ( ⁇ 100) (Wako Pure Chemical), 100 U/ml penicillin-100 ⁇ g/ml streptomycin (Invitrogen)) ⁇ First differentiation-inducing medium (differentiation-inducing basal medium containing 20% KSR, 10 ⁇ M Y-27632 (Wako Pure Chemical), predetermined concentration of hemagglutinin (Table 1)) ⁇ Second differentiation induction medium (differentiation induction basal medium containing 15% KSR, 10 ⁇ M Y-27632 (Wako Pure Chemical), pre
- Human skin-derived iPS cells (253G1, provided by Kyoto University) were seeded on a laminin-coated culture dish (manufactured by Sumika Bakelite Co., Ltd.) at 9 ⁇ 10 6 cells/9 cm dish.
- a laminin-coated culture dish was prepared by coating a 9 cm culture dish (BD FALCON) with a 0.5 ⁇ g/cm 2 solution of laminin 511E8 fragment (protein disclosed in Example (3) of WO2011043405, provided by Osaka University) at 37° C. for 1 hour. Created by coating above. The iPS cells quickly adhered to the culture dish, and formation of floating aggregates was not confirmed.
- the first day of culture was Day 0, and the entire amount of medium was changed every day from the start of culture (Day 1) to around Day 40, when melanocytes were confirmed.
- the composition of the medium was changed stepwise as shown below. That is, Day 0 is a differentiation-inducing basal medium, Days 1-4 is a primary differentiation-inducing medium (20% KSR), Days 5-8 is a secondary differentiation-inducing medium (15% KSR), and Days 9-12 is a tertiary differentiation-inducing medium. (10% KSR), and from Day 13 to Day 40, a quaternary differentiation induction medium (10% KSR) was used. By adding different concentrations of hemagglutinin to each medium, the effect on differentiation induction was observed. Experimental conditions (Conditions 1 to 3) are shown in Table 1, and the results are shown in Table 1 and FIG.
- retinal pigment epithelial cells which are cells with a gradually darker pigment, were gradually induced from cell aggregates of pluripotent stem cells (Table 1 (condition 1), left column in FIG. 1). ).
- hepatocyte-like cells were formed at a concentration of 10 nM (Table 1 (condition 2), center column in FIG. 1), and at a concentration of 30 nM.
- muscle cells and cardiomyocytes (Table 1 (condition 3), Fig. 1 right column) were induced, respectively.
- Example 2 Examination of direct differentiation of pluripotent stem cells using hemagglutinin Next, cell aggregates of pluripotent stem cells were treated with hemagglutinin at different concentrations for 24 hours, and then treated with the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies). Co.) was used to induce differentiation into each of the three germ layers, and the differentiation ability was examined by RT-PCR. 1383D2 human iPS cells were seeded on an Elplasia round-bottom plate with an ultra-low adhesion surface (manufactured by Corning) at 200 cells/dimple, and cultured in StemFit AK02N medium containing ROCK inhibitor for 1 day.
- STEMdiff Trilineage Differentiation Kit STEMdiff Trilineage Differentiation Kit
- the cells were cultured in ROCK inhibitor-free StemFit AK02N medium for 5 days, and then cultured in StemFit AK02N medium containing varying concentrations of hemagglutinin for 24 hours. After that, they were differentiated into each germ layer using the STEMdiff Trilineage Differentiation Kit, and gene analysis was performed by RT-PCR 6 days later. The result is shown in figure 2.
- Example 3 Stabilization of cardiomyocyte differentiation of pluripotent stem cells using hemagglutinin (1)
- Hemagglutinin treatment test before differentiation induction test of culture system suitable for mass culture
- 1383D2 human iPS cells were seeded onto Elplasia round-bottom plates with an ultra-low adhesion surface at 200 cells/dimple, and cultured in a ROCK inhibitor-containing medium for 1 day. Thereafter, the cells were cultured in ROCK inhibitor-free StemFit AK02N medium for 2 days, and then cultured in StemFit AK02N medium containing varying concentrations of hemagglutinin for 24 hours.
- the cells were cultured in a known cardiomyocyte differentiation-inducing medium for 14 days to induce differentiation into cardiomyocytes. Differentiation was performed with reference to the description of Sougawa et al., Methods Mol Biol. 2320: 23-27 (2021). Genetic analysis of cardiomyocyte markers was performed by RT-PCR 18 days after iPS cell seeding. The results are shown in Figure 3.
- cardiomyocyte differentiation induction system by introducing the hemagglutinin treatment step, cardiomyocyte populations can be efficiently obtained, and it has the effect of reducing the variation in the results from culture to culture (stabilization of cardiomyocyte differentiation induction). It was suggested that
- Example 4 Stabilization of hepatocyte differentiation of pluripotent stem cells using hemagglutinin (test of culture system suitable for mass culture) 1383D2 human iPS cells were seeded onto Elplasia round-bottom plates with an ultra-low adhesion surface at 200 cells/dimple, and cultured in a ROCK inhibitor-containing medium for 1 day. Thereafter, the cells were cultured in ROCK inhibitor-free StemFit AK02N medium for 1 day, and then cultured in hemagglutinin-containing StemFit AK02N medium with varying concentrations of 0 nM, 1 nM, 5 nM and 10 nM for 3 days.
- the effect of loosening intercellular adhesion by adding hemagglutinin has the effect of changing differentiation induction to retinal pigment epithelial cells to hepatocytes and muscle cells, and furthermore, induction of cardiomyocytes and hepatocytes It was also shown to have the effect of stabilizing the process and efficiently obtaining cardiomyocytes and hepatocytes. From this, when pluripotent stem cells are induced to differentiate into target cells under arbitrary differentiation conditions, it is possible to efficiently induce differentiation into target cells by adding hemagglutinin at an appropriate concentration. can be done.
- compositions according to the present disclosure are useful, for example, in the field of regenerative medicine.
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Abstract
Description
本発明者らはこれらの知見に基づき、さらに鋭意研究を進めることで本発明を完成させた。
(項1)
Eカドヘリン阻害物質を含有する、多能性幹細胞の分化誘導の方向性制御のための剤。
(項2)
多能性幹細胞が胚性幹(ES)細胞または人工多能性幹(iPS)細胞である、項1に記載の剤。
(項3)
多能性幹細胞の分化誘導の方向性制御が、多能性幹細胞から三胚葉いずれかの系統への分化効率の変化である、項1または2に記載の剤。
(項4)
多能性幹細胞の分化誘導の方向性制御が、目的とする分化細胞から逸脱する細胞の減少もしくは除去である、項1または2に記載の剤。
(項5)
Eカドヘリン阻害物質がヘマグルチニンである、項1~4のいずれか1項に記載の剤。
(項6)
Eカドヘリン阻害物質を含有する培地中で多能性幹細胞を培養する工程を含む、多能性幹細胞の分化誘導の方向性を制御する方法。
(項7)
培養が分化誘導剤の存在下で行われる、項6に記載の方法。
(項8)
Eカドヘリン阻害物質を含有する培地中で多能性幹細胞を培養する工程において、該阻害物質の濃度を調整することを特徴とする、項6または7に記載の方法。
(項9)
多能性幹細胞の分化誘導の方向性制御が、多能性幹細胞から三胚葉いずれかの系統への分化効率の変化である、項6~8のいずれか1項に記載の方法。
(項10)
Eカドヘリン阻害物質がヘマグルチニンである、項6~9のいずれか1項に記載の方法。
(項11)
Eカドヘリン阻害物質を含有する、多能性幹細胞の分化誘導を安定化させるための剤。
(項12)
多能性幹細胞の分化誘導の安定化が、多能性幹細胞から三胚葉いずれかの系統への分化効率の向上である、項11に記載の剤。
(項13)
多能性幹細胞の分化誘導の安定化が、目的とする分化細胞から逸脱する細胞の減少もしくは除去である、項11に記載の剤。
(項14)
Eカドヘリン阻害物質がヘマグルチニンである、項11~13のいずれか1項に記載の剤。
(項15)
以下の工程を含む、多能性幹細胞から目的とする分化細胞を製造する方法。
(1)多能性幹細胞の凝集体を形成させる工程、および
(2)工程(1)で得られた凝集体を、Eカドヘリン阻害物質を含有する培地中で培養することにより、目的とする分化細胞への分化誘導を安定化させる工程
(項16)
培養が分化誘導剤の存在下で行われる、項15に記載の方法。
(項17)
多能性幹細胞がES細胞またはiPS細胞である、項15または16に記載の方法。
(項18)
Eカドヘリン阻害物質がヘマグルチニンである、項15~17のいずれか1項に記載の方法。
本発明は、Eカドヘリン阻害物質を含有する、多能性幹細胞の分化誘導の方向性制御のための剤を提供する。以下、「本発明の制御剤」と称する場合がある。
本明細書において、多能性(pluripotent)幹細胞は、未分化状態を保持したまま増殖できる「自己再生能」と三胚葉系列すべてに分化できる「分化多能性」とを有する未分化細胞であれば特に限定されず、例えば、人工多能性幹細胞(iPS細胞)、胚性幹細胞(ES細胞)等が挙げられる。また、多能性幹細胞としては、例えば、哺乳動物に由来する多能性幹細胞が挙げられる。哺乳動物に由来する多能性幹細胞は特に限定されず、例えば、ヒ卜多能性幹細胞が挙げられる。ヒ卜多能性幹細胞は特に限定されず、例えば、ヒ卜iPS細胞またはヒ卜ES細胞が挙げられる。
本明細書において、Eカドヘリン阻害物質は、Eカドヘリンによる細胞間接着を阻害可能な物質であればよく、例えば、ヘマグルチニン、Eカドヘリンの発現を阻害し得る核酸(例:アンチセンスオリゴヌクレオチド等)、Eカドヘリンに対する抗体等が挙げられる。好ましくは、Eカドヘリン阻害物質としてヘマグルチニンが用いられる。
本明細書において、ヘマグルチニンは、Eカドヘリンによる細胞間接着を阻害し得る限り、その取得方法や由来等については、特に限定されないが、例えば、ボツリヌス菌(Clostridium botulinum)またはその類縁菌由来のヘマグルチニンが挙げられる。ボツリヌス菌は、A型、B型、C型、およびD型のいずれであってもよい。ヘマグルチニンは、特に言及がない場合、神経毒素複合体(ヘマグルチニン複合体ともいう)をいう。
本明細書において、ヘマグルチニンは、多能性幹細胞の分化誘導の方向性制御に寄与する効果を有する範囲で、ヘマグルチニンの改変体であってもよい。該改変体の一または複数の実施形態として、例えば、サブコンポーネントの1つ、2つまたは3つのアミノ酸配列が、少なくとも1つのアミノ酸変異を有するものが挙げられる。該改変体の具体例としては、WO2015/199243に記載の変異型ヘマグルチニン(HA)複合体タンパク質等が挙げられる。
また、本明細書において、ヘマグルチニンは、多能性幹細胞の分化誘導の方向性制御に寄与する効果を有する範囲で、小型化されたヘマグルチニンであってもよい。小型化されたヘマグルチニンとしては、例えば、カドヘリン機能阻害活性を保持し、かつサブコンポーネントの全部または一部を欠失したHA複合体タンパク質等が挙げられる。小型化されたヘマグルチニンの具体例としては、WO2019/103111に記載のE-カドヘリンの機能阻害活性を有する小型化されたヘマグルチニン複合体タンパク質等が挙げられる。
本発明は、また、Eカドヘリン阻害物質を含有する培地中で多能性幹細胞を培養する工程を含む、多能性幹細胞の分化誘導の方向性制御のための方法を提供する。以下、「本発明の制御方法」と称する場合がある。
培養に用いられる多能性幹細胞の密度は、多能性幹細胞を均一に播種でき、接着培養可能な密度であれば特に限定されないが、均一に播種され接着培養した際にEカドヘリン阻害物質を投与することで細胞間接着の程度を均一化し、かつ分化誘導状態を適切に制御させられる程度の細胞密度であることが望ましく、そのような細胞密度は、適宜設定することが可能である。また培養温度、CO2濃度等の他の培養条件も適宜設定できる。培養の期間は、多能性幹細胞の分化誘導を制御しうる限り特に限定されないが、Eカドヘリン阻害物質による細胞間接着の均一化効果を保ち、分化誘導を制御することができる程度の培養期間であることが望ましい。この培養期間は、培養に用いられる多能性幹細胞の細胞数、培養器具、培養基材、培養方法などにより適宜変更することが可能である。
培養は、前述した接着培養のほかに浮遊培養であってもよい。浮遊培養に用いられる凝集体を形成する多能性幹細胞の数は、Eカドヘリン阻害物質を投与することで細胞間接着の程度を均一化し、かつ分化誘導状態を適切に制御させられる程度の細胞数であることが望ましく、目的とする分化細胞や適用する分化誘導法によって適宜設定することができる。また培養温度、CO2濃度等の他の培養条件も適宜設定できる。培養の期間は、多能性幹細胞の分化誘導を制御しうる限り特に限定されないが、Eカドヘリン阻害物質による細胞間接着の均一化効果を保ち、分化誘導を制御することができる程度の培養期間であることが望ましい。この培養期間は、培養に用いられる多能性幹細胞の細胞数、培養器具、培養基材、培養方法などにより適宜変更することが可能である。
本発明はまた、Eカドヘリン阻害物質を含有する、多能性幹細胞から目的とする分化細胞への分化誘導を安定化させるための剤を提供する。以下、「本発明の安定化剤」と称する場合がある。
本発明は、また、多能性幹細胞から目的の分化細胞を効率よく製造する方法、言い換えれば、目的の細胞への分化誘導を安定化する方法を提供する。以下、「本発明の安定化方法」と称する場合がある。当該方法は、(1)多能性幹細胞の凝集体を形成させる工程、および(2)工程(1)で得られた凝集体を、Eカドヘリン阻害物質を含有する培地中で培養することにより、目的の細胞への分化誘導を安定化させる工程を含む限り、特に限定されない。
本発明の安定化方法は、多能性幹細胞を出発細胞として、目的とする分化細胞への分化誘導を安定化することにより、最終的に効率よく(より均一かつ均質な)目的の分化細胞集団を製造する方法である。多能性幹細胞は、細胞どうしの細胞間接着が存在する状態であることが好ましいため、本発明の誘導方法は(1)多能性幹細胞の凝集体を形成させる工程を含む。
ヘマグルチニンを用いて分化誘導の方向付けがなされるかを検討するために、網膜色素上皮細胞(RPE細胞)への分化誘導基本培地を用い、網膜色素上皮細胞の分化誘導を促進する分化誘導剤(例えば、SB431542およびCKI-7等)を用いることなく、網膜色素上皮細胞の誘導を促した。この際、ボツリヌス菌由来ヘマグルチニンを濃度を変えて添加することで、その分化誘導に対する影響を観察した。具体的な操作は以下の通りである。
・分化誘導基本培地(Glasgow's MEM (GMEM) 培地(Invitrogen)、KnockOutTMSerum Replacement (KSR) (Invitrogen)、0.1 mM MEM非必須アミノ酸溶液(Invitrogen)、1 mM ピルビン酸ナトリウム(SIGMA)、StemSure (登録商標) 10 mmol/l 2-メルカプトエタノール (×100) (和光純薬)、100 U/ml ペニシリン-100μg/ml ストレプトマイシン(Invitrogen))
・第1分化誘導培地(KSRを20%含む分化誘導基本培地、10μM Y-27632(和光純薬)、所定濃度のヘマグルチニン(表1))
・第2分化誘導培地(KSRを15%含む分化誘導基本培地、10μM Y-27632(和光純薬)、所定濃度のヘマグルチニン(表1))
・第3分化誘導培地(KSRを10%含む分化誘導基本培地、10μM Y-27632(和光純薬)、所定濃度のヘマグルチニン(表1))
・第4分化誘導培地(KSRを10%含む分化誘導基本培地、所定濃度のヘマグルチニン(表1))
・RPE維持培地 (67% DMEM low glucose (SIGMA)、29% F12 (SIGMA)、1.9 mM L-glutamine(Invitrogen)、1.9% B-27 supplement (Invitrogen)、96 U/mL ペニシリンナトリウム、96μg/mL 硫酸ストレプトマイシン)
次に、多能性幹細胞の細胞凝集体に対し24時間濃度を変えたヘマグルチニンで処理したのち、STEMdiff Trilineage Differentiation Kit(STEMCELL Technologies 社)を用いて三胚葉由来のそれぞれに分化誘導しRT-PCRにより分化能を検討した。
ヒトiPS細胞である1383D2を超低接着表面のElplasiaラウンドボトムプレート(Corning社製)へ、200 cells/dimpleになるように播種し、ROCKインヒビター含有StemFit AK02N培地で1日間培養した。その後ROCKインヒビター不含StemFit AK02N培地で5日間培養したのち、濃度を変えたヘマグルチニン含有StemFit AK02N培地中で24時間培養した。その後、STEMdiff Trilineage Differentiation Kitで各胚葉へ分化させ、6日後にRT-PCRで遺伝子解析を行った。結果を図2に示す。
以上の結果から、ヘマグルチニンの添加濃度を調整することにより、三胚葉のいずれかへの分化誘導の方向性を制御し得ることが明らかとなった。
(1)分化誘導前にヘマグルチニン処理した試験(大量培養に適した培養システムの試験)
ヒトiPS細胞である1383D2を超低接着表面のElplasiaラウンドボトムプレートへ、200 cells/dimple になるように播種し、ROCKインヒビター含有培地で1日間培養した。その後ROCKインヒビター不含StemFit AK02N培地で2日間培養したのち、濃度を変えたヘマグルチニン含有StemFit AK02N培地中で24時間培養した。その後、既知の心筋細胞への分化誘導培地において14日間培養することで心筋細胞へ分化誘導した。分化誘導は、Sougawa et al., Methods Mol Biol. 2320: 23-27 (2021)の記載を参考に実施した。iPS細胞播種後18日後にRT-PCRで心筋細胞マーカーの遺伝子解析を行った。結果を図3に示す。
・第1分化誘導培地(C液不含StemFit AK02N培地、Y-27632、BMP-4)
・第2分化誘導培地(C液不含StemFit AK02N培地、BMP-4、ActivinA、bFGF)
・第3分化誘導培地(C液不含StemFit AK02N培地、IWP-3, SB431542, Dorsomorphin, VEGF)
・第4分化誘導培地(C液不含StemFit AK02N培地、bFGF, VEGF)
ヘマグルチニン濃度を高くするにつれて、心筋細胞特有の遺伝子発現量が向上していることが分かった。すなわちこのことにより、細胞集塊であっても安定に分化誘導することができ、大量培養システムにおいてもヘマグルチニンを添加することで分化誘導工程を安定化できることが示唆された。
心筋細胞への分化誘導前にヘマグルチニンで処理することなく、既知の心筋細胞への分化誘導培地にヘマグルチニンを添加することを除いて、上記(1)と同様に試験し、心筋細胞特有の遺伝子であるcTnTの発現細胞の比率を測定し(n=9)、その結果を箱ひげ図で示した(図4)。
心筋分化誘導マーカーcTnTを指標とした場合、ヘマグルチニンを添加しない従来の分化誘導に比べ、ヘマグルチニンを添加した分化誘導系では、cTnTマーカー陽性細胞の割合が増加傾向を示し、かつその際の分散も少なくなった。すなわち心筋細胞への分化誘導系において、ヘマグルチニン処理工程を導入することによって効率よく心筋細胞集団が得られるとともに、培養ごとの結果のばらつきを小さくする作用がある(心筋細胞への分化誘導を安定化させ得る)ことが示唆された。
ヒトiPS細胞である1383D2を超低接着表面のElplasiaラウンドボトムプレートへ、200 cells/dimple になるように播種し、ROCKインヒビター含有培地で1日間培養した。その後ROCKインヒビター不含StemFit AK02N培地で1日間培養したのち、0 nM、1 nM、5 nM、10 nMと濃度を変えたヘマグルチニン含有StemFit AK02N培地中で3日間培養した。次いで培地を洗浄したのち肝細胞への分化誘導を開始した。分化誘導は、Kajiwara et al., Proc Natl Acad Sci USA. 109(31): 12538-43の記載を参考に実施した。具体的には、まず1 x B27 supplement(Gibco)、100 ng/mL アクチビンA(R&D systems)、50 ng/mL Wnt3a(R&D systems)および0.5 mM NaB(Sigma)入りRPMI1640培地(Nacalai)中で3日間培養して内胚葉誘導を実施した。培地を洗浄したのち、次に20% (v/v) KSR(Gibco)、1 mM L-グルタミン、1% (v/v) NEAA、0.1 mM 2-メルカプトエタノールおよび1% (v/v) DMSO入り Knockout-DMEM(Gibco)中で7日間培養して肝細胞誘導を実施した。培地を洗浄したのち、最後に20 ng/mL HGF(PeproTech)、20 ng/mL オンコスタチンM(PeproTech)入りHepatocyte Culture Medium BulletKit(Lonza社製)中で7日間培養して肝細胞を成熟させた。
肝細胞誘導3日後(内胚葉誘導後)さらに17日後(肝細胞成熟後)にRT-PCRで肝細胞マーカーの遺伝子解析を行った。結果を図5に示す。
3日後(A)では、ヘマグルチニン濃度を高くするにつれて、Oct3/4の発現量が低下する一方で、内胚葉特有の遺伝子(Sox17)発現量が増加していることが分かった。さらに17日後(B)では、ヘマグルチニン濃度を高くするにつれて、肝前駆細胞マーカー(CK19)の発現量が低下する一方で、成熟した肝細胞に特有の遺伝子群(ALB、AAT、CYP34)の発現量が向上していることがわかった。すなわち肝細胞への分化誘導系において、ヘマグルチニン処理工程を導入することによって効率よく肝細胞集団が得られる(肝細胞への分化誘導を安定化させ得る)ことが示唆された。
Claims (18)
- Eカドヘリン阻害物質を含有する、多能性幹細胞の分化誘導の方向性制御のための剤。
- 多能性幹細胞が胚性幹(ES)細胞または人工多能性幹(iPS)細胞である、請求項1に記載の剤。
- 多能性幹細胞の分化誘導の方向性制御が、多能性幹細胞から三胚葉いずれかの系統への分化効率の変化である、請求項1または2に記載の剤。
- 多能性幹細胞の分化誘導の方向性制御が、目的とする分化細胞から逸脱する細胞の減少もしくは除去である、請求項1または2に記載の剤。
- Eカドヘリン阻害物質がヘマグルチニンである、請求項1または2に記載の剤。
- Eカドヘリン阻害物質を含有する培地中で多能性幹細胞を培養する工程を含む、多能性幹細胞の分化誘導の方向性を制御する方法。
- 培養が分化誘導剤の存在下で行われる、請求項6に記載の方法。
- Eカドヘリン阻害物質を含有する培地中で多能性幹細胞を培養する工程において、該阻害物質の濃度を調整することを特徴とする、請求項6または7に記載の方法。
- 多能性幹細胞の分化誘導の方向性制御が、多能性幹細胞から三胚葉いずれかの系統への分化効率の変化である、請求項6または7に記載の方法。
- Eカドヘリン阻害物質がヘマグルチニンである、請求項6または7に記載の方法。
- Eカドヘリン阻害物質を含有する、多能性幹細胞の分化誘導を安定化させるための剤。
- 多能性幹細胞の分化誘導の安定化が、多能性幹細胞から三胚葉いずれかの系統への分化効率の向上である、請求項11に記載の剤。
- 多能性幹細胞の分化誘導の安定化が、目的とする分化細胞から逸脱する細胞の減少もしくは除去である、請求項11に記載の剤。
- Eカドヘリン阻害物質がヘマグルチニンである、請求項11~13のいずれか1項に記載の剤。
- 以下の工程を含む、多能性幹細胞から目的とする分化細胞を製造する方法。
(1)多能性幹細胞の凝集体を形成させる工程、および
(2)工程(1)で得られた凝集体を、Eカドヘリン阻害物質を含有する培地中で培養することにより、目的とする分化細胞への分化誘導を安定化させる工程 - 培養が分化誘導剤の存在下で行われる、請求項15に記載の方法。
- 多能性幹細胞がES細胞またはiPS細胞である、請求項15または16に記載の方法。
- Eカドヘリン阻害物質がヘマグルチニンである、請求項15または16に記載の方法。
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