RU2822203C2 - Inducing pancreatic beta cells by stem cell differentiation under action of rna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to cell biology and cell therapy. Disclosed is a method for inducing stem cell differentiation into glucose-sensitive insulin-secreting pancreatic beta cells. Method involves converting pluripotent stem cells into mesoendoderm, and then into endoderm, into pancreatic endocrine cells and finally into pancreatic beta cells using certain combinations of mRNA. Also disclosed is a method of producing induced glucose-sensitive insulin-secreting pancreatic beta cells and a method for eliminating or relieving symptoms of diabetes in an individual by administering the produced glucose-sensitive insulin-secreting pancreatic beta cells.

EFFECT: method enables to obtain functional pancreatic beta cells without using growth factors for their production.

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Description

Cross references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/423120, filed November 16, 2016, which is incorporated herein by reference.

Field to which the invention relates

[0002] The present invention relates to the regulation of the induction of pancreatic beta cells from pluripotent stem cells through kinetically regulated cell growth processes using specific combinations and different ranges of cell densities, reagent concentrations and specific combinations of mRNA.

Prerequisites for creating the invention

[0003] Recently, efforts to produce human stem cells and then differentiate them have redefined paradigms regarding variability in cell status, human disease models, and clinical therapies. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from somatic cells can be differentiated into a growing list of specific cell types that are indistinguishable from the corresponding primary cells. As a result, stem cells are very promising for the development of new cell therapies for human treatment. iPSCs offer great opportunities in the field of personalized medicine due to the unlimited availability of cells, non-invasive procedures for obtaining cells and the ability to provide immunocompatible therapy for each individual patient, which eliminates the need for immunosuppressive drugs.

[0004] Large amounts of research dollars are spent on the development of cell replacement therapies for the treatment or prevention of various human diseases. For example, autoimmune diseases cause patients with type 1 diabetes (T1D) to lose pancreatic beta cells and therefore their ability to respond to blood glucose levels and produce insulin in the body. Thus, patients with T1D may experience a noticeable benefit by replacing their insulin-producing beta cells with an external source. Transplantation of human islet cells containing insulin-producing beta cells as well as other endocrine cells has had some success, but the problem of finding a suitable pancreas donor remains unresolved. Currently, many theoretical and industrial engineering groups have developed ways to transform ESCs or iPSCs into pancreatic progenitor cells, and create insulin-secreting beta cells, with the hope of eventually providing stem cell therapies along with derivatives of insulin-producing and glucose-responsive cells. However, most of these methods, available in publicly available computer programs at the time of their writing, do not allow the production of fully functional or mature insulin-secreting pancreatic beta cells.

[0005] To reduce the cost burden and waste, those skilled in the art often look for small molecules that can interfere with signaling pathways as growth factor receptor agonists or antagonists, and thus can be used to replace growth factors. Small molecules are usually much cheaper than growth factors. However, one of the major disadvantages of small molecules is the non-specific effects that may affect off-target targets such as cell membrane-associated receptors, intracellular organelles or genomic components, etc.

[0006] Another key component of a typical differentiation protocol is the cell culture medium, which may consist of nutrients (lipids, amino acids, carbohydrates, vitamins, etc.), salts at appropriate concentrations, pH buffering agents, essential elements and common protein factors such as insulin or serum albumin. Different cell types have different nutrient and media requirements, which creates additional challenges when using cell type-specific growth factors and small molecules for signal transduction. Thus, a special "differentiation medium" is often carefully analyzed by removing or adding one component at a time.

[0007] In clinical applications of stem cell-derived tissue cells, most components of the established differentiation medium require individual certification in accordance with current manufacturing practices (cGMP) under the supervision of Regulatory Authorities; for example, growth factors must be obtained through special procedures and require individual certification. Likewise, some small molecules added to a specific differentiation medium are produced by specific chemical synthesis methods that vary in their assessment of purity, stability, and toxicity. Regarding stem cell cultures, some previously published protocols also rely on animal products such as serum or Matrigel.

The essence of the invention

[0008] Despite recent advances in generating iPSC-derived pancreatic beta cells, efficient methods for sequentially generating insulin-producing beta cells are still under development. Many current protocols require the use of a combination of growth factors, hormones, cytokines, signal peptides and other intercellular signaling molecules (herein, for simplicity, these molecules will be referred to as “growth factors”) at each stage of the differentiation cascade to produce insulin-producing beta -cells. Unfortunately, growth factors, when supplied as purified polypeptides, are typically expensive, unstable and vary from batch to batch, making them difficult to use. In addition, since growth factors act through a highly specific combinatorial mechanism, each stage of differentiation requires a different series of growth factors, which creates certain difficulties and increases the cost of optimization. One of the objectives of the present invention is to completely eliminate the need for the use of growth factors for the production of pancreatic beta cells.

[0009] The present invention relates to methods for inducing stem cell differentiation by modulating cell growth kinetics and associated parameters, and thereby the specific combination of cell density, reagent concentrations, and mRNA combinations used to regulate the direction of differentiation/induction.

[0010] To achieve this goal and in accordance with the purpose of the present invention, carried out and broadly disclosed in the present application, one aspect of the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, wherein the specified the method includes the steps of: (a) cultivating induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0011] In one embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells in differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said first mRNA combination includes FoxA2 mRNA.

[0012] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said first mRNA combination includes Sox17 mRNA.

[0013] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said first mRNA combination comprises FoxA2 and Sox17 mRNA.

[0014] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, wherein said first combination of mRNAs includes FoxA2, Sox17, GATA4 and GATA6 mRNAs.

[0015] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, wherein said second combination of mRNAs includes at least one of PDX1, Hlxb9, Ptf1a, ixl1 HNF1a and b, and Sox9.

[0016] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, wherein said first combination of mRNAs comprises at least one of PDX1, NKX6.1, NKX2.2 mRNAs , Pax6, Pax4, Hlxb9 and Ngn3.

[0017] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, wherein the mRNA affects pancreatic beta cell commitment and includes at least one of the NKX6.1 mRNAs, MAFA or MAFA.

[0018] In another embodiment, the present invention relates to a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; ((b) inducing said parental cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endoderm cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) additionally converting said endodermal cells into pancreatic progenitor cells by transfecting with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs and (f) collecting clusters enriched in pancreatic beta cells; responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said parent cells are collected from physiological fluid or tissue.

[0019] One aspect of the invention relates to a cell obtained by a method of inducing the differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under conditions differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0020] One aspect of the invention relates to a composition for the treatment of a disease, disorder or syndrome comprising a cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, wherein the method includes the steps of: (a) culturing induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0021] One aspect of the invention relates to a method of treating a disease, disorder or syndrome, comprising the step of administering to an individual in need thereof at least one cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells where the method includes the steps of: (a) cultivating induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, and a composition for treating a disease, disorder or syndrome containing a cell obtained by a method of inducing differentiation of stem cells into sensitive to glucose and insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) cultivating induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0022] In one embodiment, the present invention relates to a method of treating a disease, disorder or syndrome, comprising the step of administering to an individual in need thereof at least one cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting cells. pancreatic beta cells, where the method includes the steps of: (a) cultivating induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose; and a composition for treating a disease, disorder or syndrome, comprising a cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, wherein the method includes the steps of: (a) culturing induced pluripotent stem cells as parent cells under conditions of differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said cell is collected from a recipient individual.

[0023] In one embodiment, the present invention relates to a method of treating a disease, disorder or syndrome, comprising the step of administering to an individual in need thereof at least one cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting cells. pancreatic beta cells, where the method includes the steps of: (a) cultivating induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose; and a composition for treating a disease, disorder or syndrome, comprising a cell obtained by a method of inducing differentiation of stem cells into glucose-sensitive and insulin-secreting pancreatic beta cells, wherein the method includes the steps of: (a) culturing induced pluripotent stem cells as parent cells under conditions of differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose, wherein said parent cells are collected from the recipient.

[0024] A method for producing induced glucose-sensitive and insulin-secreting pancreatic beta cells includes the steps of: (a) culturing the induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and within specific time windows; (d) further converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs; (e) further maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs; and (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0025] Accordingly, the present invention also provides new methods for determining the status of cells without the use of small molecules or using small amounts of these molecules. The main advantage of the present invention is the ease of preparation of differentiation media using appropriately delivered differentiation regulatory gene mRNAs. However, the optimal combination of mRNA and appropriate medium can further improve this method and is an integral part of the present invention. In addition to the present invention, there is another incentive to develop a new method, which is based mainly on the use of a single type of molecule suitable for uniform certification and quality control.

[0026] New methods are disclosed herein to determine how cell density and cell division rates must be regulated to achieve desired differentiation outcomes. This application also describes the optimization of timing, addition order, RNA dosage and ratios between different RNAs in the RNA transfection process, as well as their duration and number of repetitions. In addition, the present invention relates to the selection of the surface of the culture vessels and environmental conditions such as oxygen concentration. The present invention also includes methods and methods for selecting desired cells or increasing their percentage in the general population, as well as methods for cryopreservation and re-culture of differentiated cells.

[0027] The present invention relates to methods for inducing and/or producing stem cells and their differentiation by modulating the kinetics of cell growth and associated parameters, and thereby the specific combination of cell density, concentration of reagents and combinations of mRNAs used to regulate the direction of differentiation/ induction. In one aspect, the present invention relates to a newly developed protocol for obtaining functional and mature beta cells that function in vivo , and methods of using these cells in therapies for the treatment of various diseases, including type I and type II diabetes.

[0028] In some embodiments of the invention, a representative protocol for inducing stem cells can be represented by the schemes and steps described and illustrated below in the examples. In some aspects of the invention, very high efficiency has been achieved at lower cost, without the use of large quantities of growth factors, by contacting the mRNA with stem cells at key moments of change in cell status in the desired doses and under appropriate delivery conditions. Since mRNAs are more specific for regulating cellular and developmental events by encoding functional proteins, the method disclosed herein has surprisingly been found to be more reliable than any known methods for obtaining functional pancreatic beta cells, thereby paving the way for the treatment of diabetes and other conditions associated with diabetes in humans or mammals.

[0029] The present invention relates to differentiation methods that utilize highly efficient and well-regulated expression of key regulatory genes or key transcription factors for tissue-specific differentiation. More specifically, these factors are introduced into pluripotent stem cells in the form of appropriately modified and purified mRNA molecules, demonstrated by way of example.

[0030] In yet another aspect, the present invention relates to a method of inducing cell differentiation, comprising: the use of key cell status factors and hybrids between conventional transcription factors (TFs) with transactivating domains optimized for the transformation of stem cells into various cell types; introduction of these factors in the form of synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at a preferred density using methods that ensure appropriate levels of transgene expression; maintaining cells in optimal conditions to achieve high efficiency of specific differentiation and thereby inducing pluripotent stem cells or progenitor stem cells or progenitor cells to become cells of a specific lineage or cell tissue of that type.

[0031] In one aspect, the present invention relates to a method of producing pluripotent stem cells or progenitor stem cells or progenitor cells for the purpose of their transformation into cells of a specific lineage or cell tissue of that type using such a cell induction method, including : the use of key cell status factors and hybrids between conventional transcription factors (TFs) with transactivating domains optimized for the conversion of stem cells into various cell types; introducing these factors in the form of synthetic messenger RNA (mRNA) into cultured pluripotent stem cells having a preferred density, using methods that ensure appropriate levels of transgene expression; maintaining cells in optimal conditions to achieve high efficiency of specific differentiation.

[0032] In one aspect, the present invention relates to a method for producing induced glucose-sensitive and insulin-secreting pancreatic beta cells, wherein the method comprises: (a) culturing iPSCs as parent cells under the experimentally validated conditions described herein application, and obtaining cells as source cells for differentiation; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the differentiation of cells into endoderm cells using mRNAs of endoderm-specific genes by transfecting the cell culture at a described dose and within specific time windows; (d) further converting endodermal cells into pancreatic progenitor cells using additional mRNA gene molecules or combinations thereof through transfection; (e) additional maturation of pancreatic progenitor cells into pancreatic endocrine cells using yet another mRNA or gene combinations; and (f) collection of clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose.

[0033] In another aspect, the present invention relates to methods for converting pluripotent stem cells or progenitor stem cells or progenitor cells into cells of a specific lineage or cell tissue of that type, wherein the methods include at least one of the steps of: obtaining stem cells expressing key genes responsible for cell status (collectively referred to as "stem cells"), including key cell status factors and hybrids between conventional transcription factors (TFs) with transactivating domains optimized for the transformation of stem cells into various cell types; introducing these factors in the form of synthetic messenger RNA (mRNA) into cultured pluripotent stem cells having a preferred density, using methods that ensure appropriate levels of transgene expression; maintaining cells in optimal conditions to achieve high efficiency of specific differentiation.

[0034] Additional objects and advantages of the present invention will be set forth in part in the description of the invention, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and achieved using the elements and combinations specifically set forth in the appended claims.

[0035] It should be noted that the above general description and the following detailed description are provided for illustrative and explanatory purposes only, and should not be construed as a limitation of the claimed invention.

[0036] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the present invention.

Brief description of drawings

[0037] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be made available by the Office upon request and upon payment of the required fee.

[0038] The foregoing aspects and advantages of the present invention will be apparent from the following detailed description of the invention with reference to the accompanying drawings, in which:

[0039] In FIG. 1A shows endoderm cells derived from parent cells of varying densities and illustrates a representative embodiment of endoderm induction.

[0040] In FIG. 1B shows endoderm cells induced from iPSCs using Sox 17 mRNA at various densities (eg, low to high density as described in the Examples) and illustrates a representative embodiment of endoderm induction.

[0041] In FIG. 2 shows that pancreatic progenitor cells are derived from endodermal cells at varying densities and illustrates a representative embodiment of the induction of pancreatic progenitor cells. In fig. Figure 2A shows one image of a representative cell density during induction. In fig. Figure 2B shows one image of a representative cell density during induction. In fig. Figure 2C shows one image of a representative cell density during induction. In fig. Figure 2D shows one image of a representative cell density during the induction process.

[0042] In FIG. 3 shows pancreatic endocrine cells derived from endodermal cells at various densities forming clusters and illustrates a representative embodiment of pancreatic endocrine cell induction. In fig. Figure 3A shows one image of a representative cell density/clustering during the induction process. In fig. 3B shows one image of representative cell density/clustering during induction. In fig. Figure 3C shows one image of representative cell density/clustering during induction.

[0043] In FIG. 4 shows pancreatic endocrine cells matured in culture and forming clusters that have undergone flotation and illustrates a representative example of pancreatic endocrine cell maturation. In fig. 4A shows one image of a representative cell maturation during induction. In fig. 4B shows one image of a representative cell maturation during induction. In fig. Figure 4C shows one image of a representative cell maturation during induction.

[0044] In FIG. 5A shows the morphology of a beta cell cluster as determined by phase contrast staining and illustrates a representative example of pancreatic islet beta cell-like cluster formation.

[0045] In FIG. 5B shows insulin staining of beta cells (green) 10 days after differentiation (DAPI shown in blue) and illustrates a representative pattern of pancreatic islet beta cell-like cluster formation.

[0046] In FIG. Figure 6 shows endodermal cells and beta cells derived from human iPSCs, as indicated by specific cellular markers. In fig. 6A shows endodermal cells stained with green: CXCR4, and blue: DAPI, a reference nuclear dye. In fig. 6B shows endoderm cells stained green: FOXA1; blue: DAPI; red: phalloidin cell membrane. In fig. 6C shows beta cell differentiation at day 10, green: insulin; blue: nuclear DAPI. In fig. 6D shows beta cell differentiation at day 8, green: nuclear NKX6.1: red: phalloidin cell membrane.

[0047] In FIG. Figure 7 shows glucose-stimulated insulin production by chemical synthesis beta cells (according to the Milton protocol) compared to mRNA-derived beta cells (according to the allelic protocol) (cells were stimulated with batches of glucose every hour) and illustrates pancreatic beta -islets obtained in vitro and secreting insulin in response to glucose.

[0048] In FIG. Figure 8 shows two million iPSCs in the starting population that were transfected using a MaxCyte STX device set to Optimize mode 2 on an OC-100 processing unit. Photographs were taken 24 hours after transfection using the EVOS imaging system at 10× magnification. In fig. Figure 8A shows a representative image of iPSCs during a representative transfection protocol. In fig. 8B shows a representative image of iPSCs undergoing a representative transfection protocol. In fig. Figure 8C shows a representative image of iPSCs during a representative transfection protocol. In fig. 8D shows a representative image of iPSCs during a representative transfection protocol. In fig. 8E shows a representative image of iPSCs during a representative transfection protocol. In fig. 8F shows a representative image of iPSCs during a representative transfection protocol.

[0049] In FIG. Figure 9 shows large islets of beta cells transformed into 3D from 2D culture at the end of mRNA-mediated differentiation of pancreatic beta cells. In fig. 9A shows a representative image of beta cells associated with differentiation. In fig. 9B shows a representative image of beta cells associated with differentiation.

Detailed Description of the Invention

[0050] When describing the present invention, all terms not defined in this application have their generally accepted meanings known to those skilled in the art. The following description, to the extent that it relates to a particular embodiment of the invention or to a particular application of the present invention, is provided for illustrative purposes only and does not limit the scope of the claimed invention. The following description covers all alternatives, modifications and equivalents that fall within the spirit and scope of the invention.

[0051] The concept of a “master regulatory gene,” that is, one key gene (usually a transcription factor gene, sometimes referred to generically as a “small number of worker genes”) that can determine the status of cells and tissues and, ultimately, the formation of an entire organ during development was essentially based on studies of muscles (MyoD), eyes (Pax6), and other areas of the body in accordance with developmental biology. Shinya Yamanaka's discovery that differentiated cells can be returned to a pluripotent state through the expression of a selected group of transcription factors expressed in stem cells, and demonstrated the effectiveness of a small number of key transcription factors in inducing cell development through a long-term, multistep change in their status. Research by other iPSC generation groups has expanded the choice of reprogramming factors and has shown that some changes may be acceptable when selecting a transcription factor for reprogramming purposes. In Yamanaka's original work, expression of reprogramming factors was achieved through the use of viral vectors that integrate into the genome, since long-term expression of these factors is necessary for effective cell transformation. Concomitant genome modification represents an important barrier to the therapeutic application of iPSCs, and the possibility of reactivating expression from integrated viral clusters is a challenge even for in vitro studies. The use of mRNA transfection for reprogramming, most recently described by the present team, is particularly attractive because this system allows the expression of mixtures of reprogramming factors and even their individual components, which will be modulated in a short time simply by the changes that the transcripts added to the cell make. cultural environment. Once transfection of a particular factor is complete, ectopic expression in target cells quickly ceases due to rapid degradation of the mRNA in the cytoplasm. Even though the mRNA is not retained in the target cell, it is able to be directly translated into the cytoplasm without any rate-limiting translocation into the nucleus, as is the case with transfected DNA and integrating viral vectors, which greatly compensates for the short half-life of mRNA and results in highly efficient expression and also within a small time window, which is important for determining the status of the cell.

[0052] To reduce the risk of accidental genomic integration, long-lasting DNA vectors such as episomal plasmids should be avoided if they are used to alter cellular status. RNA viruses or their derivatives, such as Sendai virus or Venezuelan equine encephalitis (VEE) virus, even after being stripped to form a modified non-infectious RNA replicon, still carry viral elements that tend to recombine with latent viral elements in the host genome. It is never entirely certain that cells will be deprived of viral vectors without laborious searching for evidence in the form of negative data. The present invention discloses many inventive steps to take advantage of mRNA-based cellular status detection for targeted differentiation. In summary, the present invention discloses one or multiple rounds of ectopic expression of a transcription factor in a primary mode of regulating cell differentiation.

[0053] However, there are technical barriers to mRNA-based stem cell differentiation. Not all types of stem cells and culture media are equally conducive to efficient delivery of mRNA and are currently a barrier to mRNA-based differentiation. It is also widely known that stem cells, and in particular most human stem cell lines, are quite difficult to culture without developing transfection-resistant patches. It is part of the present invention that pluripotent stem cells can be cultured under conditions in which most cells can be transfected with modified mRNAs. In another embodiment of the invention, a dose of RNA and transfection reagent (which have associated toxicity) is introduced into cells at levels capable of influencing the effects of essential regulatory genes that maintain the viability of target cells in the face of proapoptotic and cytostatic forces that arise during the change. cellular status.

[0054] Accordingly, taking into account the problems associated with previously known stem cell differentiation procedures, new methods, materials and protocols described herein have been developed to produce various cell types from iPSCs or ESCs with increased efficiency and quality of the resulting cells. The present invention achieves significant improvement by potentiating TF mRNA introduced into target stem cells. The present invention also provides novel protocols for achieving the production of non-footprinted tissue cells from human stem cells without the use of feeder cells or any other potential xenocontaminated reagents. New protocols expand the benefits of modified mRNA and help remove remaining barriers to the therapeutic application of stem cell derivatization technology.

[0055] Considering that differentiation from pluripotent cells to end-stage cells often requires multiple steps and takes weeks or even months, a growth factor-based stepwise strategy is inherently inefficient and time-consuming. Accordingly, embodiments of the present invention completely eliminate the need for growth factors to regulate pancreatic beta cell production.

[0056] More specifically, the present invention relates to the conversion of pluripotent stem cells or progenitor stem cells or progenitor cells into cells of a specific lineage or cell tissue of that type by expressing key genes responsible for the status of cells (referred to generically as “stem cells”) "), including key cell status factors and hybrids between conventional transcription factors (TFs) with transactivating domains optimized for the conversion of stem cells into various cell types; introducing these factors in the form of synthetic messenger RNA (mRNA) into cultured pluripotent stem cells having a preferred density, using methods that ensure appropriate levels of transgene expression; maintaining cells in optimal conditions to achieve specific differentiation efficiency that could not be achieved previously. Factors expressed through the introduction of mRNA may also be growth factors, cytokines, hormones, signal peptides and other secreted factors or modifying enzymes that affect the status of cells. Using a similar procedure, microRNAs (miRNAs) or other non-protein-coding RNAs can be introduced into transitional cells to regulate differentiation. The methods according to the invention, in comparison with other known methods, can significantly reduce the time, cost and effort spent on differentiating stem cells into beta cells.

[0057] The present invention describes a method for converting pluripotent stem cells or progenitor stem cells or progenitor cells into cells of a specific lineage or cell tissue of that type, wherein the methods include at least one of the steps of: expression of key genes responsible for cell status, including key cell status factors optimized for the development of stem cells into different cell types; introducing these factors in the form of synthetic messenger RNA (mRNA) into cultured pluripotent stem cells having a preferred density, using methods that ensure appropriate levels of transgene expression; maintaining cells in optimal conditions to achieve high efficiency of specific differentiation.

[0058] In some embodiments, the present invention relates to fully stabilized expanded iPSCs.

[0059] Some embodiments do not require removal of episomes or RNA viruses (eg, Sendai virus) that may appear 10 passages after iPSC isolation.

[0060] In some embodiments of the invention, this method is carried out without feeders.

[0061] In some embodiments of the invention, this method is non-xenogeneic and includes the introduction of all synthetic or human reagents, but does not include any components of animal origin.

[0062] In some embodiments of the invention, the method is carried out without footprinting, which eliminates random integration of DNA into the genome (as is often the case with episomes).

[0063] In some embodiments, this method produces a completely corrected genetic background through the use of patient-specific source tissue and/or genome editing.

[0064] In another experiment, as an alternative to the method described in Table 1, iPSC cells cultured as spheres in suspension were directly transfected by electroporation (eg, using a MaxCyte STX electroporator) without coating the plate surface. In one embodiment, 2 million original iPSC beads were transfected into suspension with other mRNAs, such as Sox 17 or Pax6, or mock transfected. The amount of mRNA tested, as shown in Figure 8, was 2500 ng. Cells were then cultured in NBM in the case of Soxl7 transfection, or MEMalpha with KSR in the case of Pax6 transfection. Transfection can be repeated 1, 2, 3, 4, 5 or even more times if the transition takes a longer period of time. As a result, after the first transfection of Sox 17 mRNA, cell clusters became significantly smaller and less compact spheres, losing a defined “edge” or outer boundary. In contrast, mock-transfected spheres retained well-defined, and easily visible outer “edges” in 2D photographs. Smaller spheres of untransfected or mock-transfected iPSCs had a transparent appearance, whereas larger spheres appeared less transparent and had thick cell layers. In comparison, iPSC spheres transfected with Pax6 mRNA (neural differentiation TF) moved toward the ectoderm, i.e., neural progenitor cells, causing the spheres to become darker and have a less sharp “edge” than the mock-transfected spheres, but were larger and larger. sharper boundaries than Sox 17-transfected spheres.

[0065] Germ layer-specific intermediate cells, such as endodermal cells, and subsequent intermediate cells, such as hepatocyte progenitor cells, pancreatic progenitor cells, and the like, can also be transfected with additional TF mRNAs in spheres along the same lines principle and by the same methods. Cells transfected in this manner are more resistant to toxicity caused by small molecules, growth factors, or other elements in cell cultures, and generally should differentiate more efficiently than cells 2D transfected using chemical reagents. This observation, which went unnoticed in scientific publication, was made accidentally during testing of electroporation equipment, and served as a stimulating method during the disclosure of the present invention.

Definitions

[0066] To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have generally accepted meanings commonly understood by one skilled in the art to which the invention relates. Function words such as "a", "an" and "the" can be used with a noun that defines not only any individual object, but also with a noun that refers to an entire class of these objects, a specific example of which can be used to illustrations. The terminology used in the present invention is used to describe specific embodiments of the invention, but its use does not limit the present invention unless it goes beyond the scope of the stated claims.

[0067] The term "beta cell-like cells" means cells that share common characteristics with pancreatic beta cells. Beta cell-like cells are further defined by their morphological properties as well as specific marker properties. Because induced pluripotent stem cell-derived beta cell-like cells share properties (including marker and hormonal properties) with pancreatic beta cells, induced pluripotent stem cell-derived beta cell-like cells can be used as synonymous with the term "beta cells derived from induced pluripotent stem cells".

[0068] The term "embryoid body" means an aggregate of cells derived from pluripotent cells, where cell aggregation can be initiated by any method that prevents cells from adhering to a surface to form typical colony growth. As used herein, the term “embryoid body” means a three-dimensional spheroidal aggregate of pluripotent stem cells, including, but not limited to, embryonic stem cells formed after the blastocyst stage of mammalian embryos. The embryoid body may be formed from embryonic stem cells obtained using any technology generally known to those skilled in the art, including, but not limited to, somatic cell nuclear transfer or somatic cell reprogramming to produce induced pluripotent stem cells.

[0069] As used herein, the term “induced pluripotent stem cells” means pluripotent stem cells derived from somatic cells (eg, adult somatic cells). Induced pluripotent stem cells are similar to embryonic stem cells in their ability to differentiate into any type of adult cell that is not embryonic.

[0070] As used herein, the terms “cell,” “cell line,” and “cell culture” include progeny. It should also be noted that all offspring may not be completely identical in DNA content due to intentional or accidental mutations. The term also includes a progeny variant that has the same function or biological property as the original transformed cell as assessed by screening.

[0071] As used herein, the term “composition” means a combination of an active agent and at least one other compound or molecule that is inert (eg, a detectable agent or label) or active, such as an adjuvant.

[0072] As used herein, the term “culture” means maintaining cells under conditions in which they can proliferate and avoid aging as a group of cells. "Culturing" may also include conditions under which cells similarly or alternatively differentiate.

[0073] As used herein, the term "differentially expressed" refers to the differential production of RNA, including, but not limited to, mRNA, tRNA, miRNA, siRNA, snRNA and piRNA, transcribed from a gene or regulatory region of the genome or the protein product encoded by the gene, according to compared with the level of RNA production by the same gene or regulatory region in a normal or control cell. In another context, the term "differentially expressed" also refers to nucleotide sequences or proteins in a cell or tissue that have different temporal and/or spatial expression profiles compared to a normal or control cell.

[0074] As used herein, the term “overexpressed” or “overexpression” refers to an increase in the level of expression of the RNA product or protein encoded by a gene compared to the level of expression of the RNA product or protein in a normal or control cell.

[0075] As used herein, the term "underexpressed" or "underexpression" refers to a decrease in the level of expression of the RNA product or protein encoded by a gene compared to the level of expression of the RNA product or protein in a normal or control cell.

[0076] As used herein, the term “differentiate” or “differentiation” means the process by which progenitors or progenitor cells (ie, pancreatic progenitor cells) differentiate into specific cell types, for example, pancreatic beta cells.

[0077] As used herein, the term “effective amount” means an amount sufficient to produce a beneficial or desired biological, emotional, therapeutic or clinical response of a cell, tissue, system, animal or human. An effective amount may be administered in one or more divided doses, one or more applications, or in one or more doses. The term also includes, within its scope, an amount effective to enhance normal physiological function.

[0078] As used herein, the term “proliferation” or “expanded”, when referring to cells, indicates an increase in the number of characteristic cell types or cell types derived from the original cell population, which may or may not be identical. The original cells used for propagation do not have to be the same as the cells obtained after propagation. For example, expanded cells can be obtained by culturing ex vivo or in vitro and differentiating the original cell population.

[0079] As used herein, the term “expression” refers to the process of transcribing polynucleotides into RNA transcripts. When describing mRNA and other translated RNA molecules, the term "expression" also means the process or processes of sequential translation of transcribed RNA into peptides, polypeptides or proteins.

[0080] As used herein, the terms “induced pluripotent stem cell” or “iPS cell” or “iPSC” mean cells capable of differentiating into multiple cell types that have been artificially derived (non-naturally occurring) from non-pluripotent cells.

[0081] As used herein, the term “non-integrating iPS cell” means iPS cells that do not contain an exogenous transgene integrated into the genome of non-pluripotent cells.

[0082] As used herein, the term “isolated” refers to components separated from other components of cells and the like with which a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof are commonly associated in nature. Non-natural polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof do not require “isolation” in order to distinguish them from their natural counterpart.

[0083] As used herein, the term "concentrated" refers to a molecule, including, but not limited to, a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that differs from its natural counterpart in that the concentration or number of molecules in a given volume is greater than its natural counterpart.

[0084] As used herein, the term "dilute" refers to a molecule, including, but not limited to, a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that differs from its natural counterpart in that the concentration or number of molecules in a given volume is less than its natural counterpart.

[0085] As used herein, the term “separated” refers to the state of an entity being physically separated from the original source or population such that the separated compounds, agents, particles or molecules can no longer be considered part of the original source or population.

[0086] As used herein, the term "mammal" being treated means any animal belonging to the class of mammals, including humans, domestic and farm animals, non-human primates, as well as animals kept in zoos, animals participating in sports competitions or pets such as, but not limited to, dogs, horses, cats and cows.

[0087] As used herein, the term “stem cells” means any self-renewing totipotent, pluripotent or multipotent cells or their progenitor cells or progenitor cells that are capable of differentiating into various cell types.

[0088] As used herein, the term “totipotent” refers to cells that can differentiate and become all cell types in the body, as well as extraembryonic or placental cells.

[0089] As used herein, the term “pluripotent” refers to cells that can differentiate and become all cell types that make up an organism, including any fetal or adult cells, excluding extraembryonic or placental cells.

[0090] As used herein, the term "multipotent" refers to cells that can develop into more than one type of cell, but, unlike pluripotent cells, they are more limited in the types into which they can develop.

[0091] As used herein, the terms “subject,” “individual,” or “patient” are synonymous and refer to a vertebrate organism.

[0092] As used herein, the term “substantially pure cell population” means a population of cells having specific cellular marker properties and differentiation capacity, wherein said population is about 50%, preferably about 75-80%, more preferably about 85-90% and most preferably at least about 95% of the cells constituting the total cell population. Thus, a “substantially pure cell population” means a population of cells that contains less than about 50%, preferably less than about 20-25%, more preferably less than about 10-15%, and most preferably less than approximately 5% of cells that have specific cellular marker properties and the ability to differentiate under specified assay conditions.

[0093] As used herein, the term “predifferentiation” means the process by which progenitor or precursor cells (eg, pluripotent stem cells) differentiate into intermediate cell types, such as pancreatic progenitor cells, that have the ability to further differentiate into final effector cells. cells (eg beta cells).

[0094] As used herein, the term “therapeutic” refers to the treatment, therapy, and/or alleviation of symptoms of a disease, disorder, condition, or side effect, or the reduction in the rate of progression of a disease, disorder, condition, or side effect. The term also includes improvement of normal physiological function, palliation and partial relief of a disease, disorder or condition or side effect.

[0095] As used herein, the terms “treatment” and “therapy” refer generally to achieving a desired pharmacological and/or physiological effect. This effect may be prophylactic, that is, aimed at preventing or partially preventing a disease, symptom or condition, and/or may be therapeutic, aimed at partially or completely curing a disease, condition, symptom or side effect associated with such disease. As used herein, the term “treatment” includes any treatment of a mammal, and in particular a human, and includes: (a) prevention of a disease in an individual who may be predisposed to the disease but has not yet been diagnosed with it; (b) suppression of the disease, that is, stopping its development; or (c) alleviation of the disease, that is, mitigation or alleviation of the disease and/or its symptoms or conditions. As used herein, the term “treatment” means therapeutic treatment and prophylactic or preventive measures. Individuals in need of treatment are those already suffering from such a disorder, as well as individuals in need of its prevention.

[0096] As used herein, the term “preventive” refers to slowing or stopping the progression of a disease or condition before it occurs, even if it has not been diagnosed, or if the disease or condition is in a subclinical phase.

[0097] As used herein, the term "active agent" means a substance, compound or molecule that is biologically active or otherwise induces a biological or physiological effect in the individual to whom it is administered.

[0098] As used herein, the term "pharmaceutically acceptable carrier" means a diluent, adjuvant, excipient or carrier with which the active agent, chondrocytes of the invention, or a composition containing chondrocytes of the invention are administered in combinations where such administration is approved by Federal Government Regulatory Agencies or State governments, or where the specified carriers are listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias as approved for use in animals and/or humans.

[0099] Unless otherwise specified, all technical and scientific terms used herein have their generally accepted meanings known to one skilled in the art.

Cell types

[00100] Representative cell types may include, for example, endodermal cells, pancreatic progenitor cells, pancreatic endocrine cells, and beta cells.

[00101] Examples of suitable surfaces for culture vessels include, but are not limited to, vitronectin, E-cadherin, Corning® Synthemax® II or Matrigel for iPSC, which includes, but is not limited to, Matrigel for endoderm, which in turn includes, but is not limited to, Matrigel or collagen for pancreatic progenitor cells and pancreatic endocrine cells.

[00102] In one aspect of the invention, an exemplary method of blocking the differentiation or reprogramming of somatic cells may include the use of any one or more synthetic mRNA reprogramming factors selected from Oct4, Sox2, Klf4, CMYC, Nanog and Lin28 and transactivation domains for the purpose of reprogramming or blocking the differentiation of somatic cells. Methods and compositions for IPSC modulation are described in US Pat. Nos. 13/893166 and 14/292317, the contents of which are incorporated herein by reference.

[00103] In some embodiments, the present invention relates to protocols for the use of suspension cell cultures and culture plates and vessels with low cell adhesion for the preparation of such suspension cultures.

[00104] In some embodiments, environmental conditions, such as oxygen concentration, can be modulated to create optimal induction conditions.

[00105] In some embodiments, the present invention relates to methods and methods for selecting desired cells or increasing the percentage of their confluence or density in the general population of cell cultures.

[00106] In some embodiments, the present invention relates to methods for producing cryopreserved and differentiated beta cell-like cells. In some embodiments, differentiated cells are cryopreserved to achieve optimal cell viability during or after storage, for example, by providing a culture medium containing HSA and/or DMSA. In some embodiments, a culture medium may be prepared containing, for example, 2.5% HSA and 10% DMSO in the culture medium. In some embodiments of the invention, the number of cells can be optimized to further improve viability during or after storage.

[00107] The present invention also relates to methods for re-culturing differentiated cells. Cells can be recultured in most culture vessels: for example, T75 flask, T25 flask, 6-well plate, 96-well plate. Cells can be recultured at different densities for different applications.

[00108] In some embodiments, the present invention also provides methods for reducing the physical stress on cells during processing throughout the differentiation process, and thereby increasing their viability. Certain cell types are very small during differentiation, similar to iPSCs. These small cells are very sensitive to centrifugal force. iPSCs are very sensitive to excess centrifugal force. Some cell types are very sticky during differentiation, like iPSCs and endodermal stage cells. These cells are very sensitive to shear forces. When processing these cells, use a 10 ml pipette without using any small tips to repeatedly pipet up and down. For storage, these cells can be cultured as colonies and then dissociated into clusters rather than individual cells. For differentiation, if single cells are to be used, dissociation can be completed before cell detachment, followed by removal of the dissociating solution and subsequent dissociation of the cells in the residual dissociating solution. This protocol is commonly used for cell culture.

[00109] The description of the invention will be better understood with reference to the documents cited in this application. Embodiments of the invention are provided for purposes of illustration only and should not be construed as limiting the scope of the invention. It is well known to those skilled in the art that many other embodiments are included within the scope of the present invention. All publications and patents cited in this application are incorporated herein by reference in their entirety. If material incorporated by reference is inconsistent with or inconsistent with the description of this application, then such material may be replaced by any other material. The citation of any documents in this application should in no way imply that such documents are prior art.

[00110] Unless otherwise specified, all numbers expressing quantities of ingredients, reaction conditions, etc., used in the description, including the claims, in all cases should be understood as numbers having deviations from the specified value and defined by the term " approximately". Accordingly, unless otherwise stated, numerical parameters are approximate and may vary depending on the desired properties to be achieved in the present invention. In no way, and without any attempt to limit the application of the doctrine of equivalents to the scope of the claims, it should be noted that each numerical parameter must be presented as a number of significant figures, rounded according to ordinary rounding rules.

[00111] The use of the articles “a” or “an”, when used in combination with the word “comprising” in the claims and/or description of the invention, can mean “one”, but the meaning “one or more” is also allowed ", "at least one" and "one or more than one". The use of the word "or" in a claim means "and/or" unless it is expressly indicated only as an alternative meaning, or when the alternative meanings are mutually exclusive, although the present disclosure supports a definition that refers only to the alternatives and "and/or" "

[00112] Unless otherwise specified, the phrase “at least”, when used before listing a series of elements, should be understood to refer to each element in that series. Those skilled in the art will recognize or can independently determine, by no more than routine experimentation, many equivalents to specific embodiments of the invention described herein. Such equivalents are included within the scope of the following claims.

[00113] Unless otherwise specified, all technical and scientific terms used herein have their generally accepted meanings known to one skilled in the art to which the invention relates. Although any methods and materials similar or equivalent to those described herein may be used to practice the invention or perform tests in accordance with the present invention, preferred methods and materials are provided herein.

[00114] Other embodiments of the invention will be apparent to one skilled in the art from the description given herein and the practice of the invention disclosed herein. It should be noted that the description and examples are provided for illustrative purposes only, however, the true scope and essence of the invention are formulated only in the accompanying claims.

Examples

[00115] The present invention is described with reference to the following examples. These examples are provided for purposes of illustration, however, the present invention is not limited to these examples, but rather covers all variations that will be apparent from the description given herein.

Example 1: Production of endodermal cells from iPSCs

[00116] iPSCs were seeded into standard size 6-well cell culture plates (culture area approximately 9.5 cm ² /well) or standard size 12-well cell culture plates (culture area approximately 3.8 cm ² /well) to initiate differentiation. Likewise, but not necessarily, other sized culture vessels may be used, and plates having more wells than 6- or 12-well plates may sometimes be preferred because they provide greater reagent efficiency and reduced time. Representative conditions for a highly efficient protocol for obtaining functional differentiated beta cells are presented below in the Beta Cell Differentiation Table, which includes initial cell stage, culture vessels, coatings, dissociating agents, media name and main components, seeding density for a representative 6-well tablet and oxygen level.

Table 1: Beta Cell Differentiation

2-6 days 1-3 days 1-3 days 3-6 days Stage 1 Stage 2 Stage 3 Stage 4 Source cells iPSCs Endoderm Pancreatic progenitor cells Pancreatic endocrine cells Culture vessels Ultra-low adhesion plate/flask Cultivation plate/flask Cultivation plate/flask a Cultivation plate/flask Coating No Matrigel Matrigel/Collagen I Matrigel/Collagen I Dissociation EDTA TypeLE TypeLE TypeLE Environment names and main components MEMa, DMEM/F12, DMEM B27
10-50 µM insulin
5% KSR
Transfection of SOX17 mRNA DMEM/F12, MCDB131+8 mM D-glucose
Transfection of PDX1 mRNA DMEM/F12, MCDB131+20 mM D-glucose
Transfection of PDX1 and NKX6.1 mRNA CMRL
Transfection of NKX6.1 and MAFA mRNA Seeding density
(for 6-well plate) 1×10 ⁵ - 4×10 ⁵ cells per well 1×10 ⁵ - 3×10 ⁵ cells per well 3×10 ⁵ cells per well 3×10 ⁵ cells per well l Oxygen Normal oxygen Normal oxygen Normal oxygen Normal oxygen

[00117] Cell populations ranging from 1 x 10 ⁵ to 4 x 10 ⁵ per well have been successfully used in a 6-well plate. iPSCs were considered ready to differentiate if a sufficient number of typical iPSC colonies had well-defined edges, the cells were compact, and colony growth was not excessive. The quality of the iPSCs according to the invention, obtained in accordance with these criteria, plays a major role for differentiation, as was confirmed when comparing the iPSC lines according to the invention with Ipsc lines that were obtained by other methods.

[00118] At this stage, iPSCs are induced to differentiate into mesoendoderm lineage cells. Surprisingly, suspension cell systems have been found to be most suitable for scale-up at this stage, even though the use of adherent cell monolayers is generally preferred by most current differentiation protocols. More specifically, iPSCs grown in suspension for induction were more resistant to chemical toxicity and were easier to reculture at later stages. Ultra-low adhesion plates (Sigma-Aldrich) or other low adhesion plates were used to stimulate the growth of the iPSC cell suspension.

[00119] If iPS cells are to be passaged, it is important to dissociate iPSC colonies according to a protocol that provides low cytotoxicity and produces even smaller iPSC clusters that can rapidly form spheres if a suspension culture is desired. iPSCs were dissociated using TripLE ^™ (ThermoFisher), Accutase (Life Technology) or EDTA by dissociation with 0.1 mM EDTA and sometimes 0.5 mM or 1 mM in DPBS (Fisher Scientific), at 37°C in within 5 minutes. This step has been successfully carried out at various dissociation times, including, in some embodiments, 1 to 2 minutes, and sometimes 10 to 20 minutes. In some aspects of the invention, the dissociation time may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes.

[00120] To obtain the media, MEMA, DMEM/F12 and DMEM B27 were tested using 10-50 μM insulin and 5% KSR, and these media were suitable for obtaining the results desired at this stage of differentiation. iPSCs were then induced to terminate the pluripotent stage of cells and differentiate into mesoendoderm in the presence of GSK3 inhibitors, such as CHIR99021, CHIR98014, BIO or GSK inhibitor IX and SB-216763, which activate the functions of the Wnt pathway genes, BMP4 and activin A. In some embodiments In carrying out the invention, insulin is present at a concentration of, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 μM, or at intervals between any the values listed above. GSK3 inhibitors were administered at time windows of 1, 2, 3 days, and FoxA2-, CXCR4-positive cells used here were counted in a quality control assay over a period of time and at inhibitor concentrations, e.g., 5 mM, 8 mM, 10 mM, and etc. In some embodiments, the inhibitors are present at concentrations of 5, 6, 7, 8, 9, or 10 mM, or in the range of any of the above values.

[00121] In one experiment, cells were then transfected with FoxA2 and/or Sox17 mRNA at a dose of 20 ng per well using Stemgent transfection reagent (Stemgent). This transfection was also performed using GATA4 mRNA and/or GATA6 mRNA and was repeated 3, 4, 5, and 6 times, sometimes at a 10-fold higher dose of mRNA using Stemgent transfection reagent or other commercially available transfection reagents. Cells at this stage had a morphology closer to that of epithelial cells than iPSC-derived mesenchymal cells at different times of mRNA transfection and at different initial densities (Fig. 1).

[00122] In another experiment, as an alternative to the method described in Table 1, iPSC cells cultured as spheres in suspension were directly transfected by electroporation (eg, using a MaxCyte STX electroporator) without coating the plate surface. In one embodiment of the invention, 2 million original iPSCs in the form of spheres were transfected into suspension with other mRNAs, for example, Sox17 or Pax6, or were mock transfected. The amount of mRNA tested, as shown in Figure 8, was 2500 ng. Cells were then cultured in NBM in the case of Soxl7 transfection, or MEMalpha with KSR in the case of Pax6 transfection. Transfection can be repeated 1, 2, 3, 4, 5 or even more times if the transition takes a longer period of time. As a result, after the first transfection of Sox17 mRNA, cell clusters became significantly smaller and less compact spheres, losing a defined “edge” or outer boundary. In contrast, mock-transfected spheres retained well-defined and clearly visible outer “edges” in 2D photographs. Smaller spheres of untransfected or mock-transfected iPSCs had a transparent appearance, whereas larger spheres appeared less transparent and had thick cell layers. In comparison, iPSC spheres transfected with Pax6 mRNA (neural differentiation TF) moved toward the ectoderm, i.e., neural progenitor cells, causing the spheres to become darker and have a less sharp “edge” than mock-transfected spheres, but in size were larger and had sharper boundaries than Sox17-transfected spheres.

[00123] By the same principle and by the same methods, germ layer-specific intermediate cells, such as endodermal cells, and subsequent intermediate cells, such as hepatocyte progenitor cells, pancreatic progenitor cells, and the like, can also be transfected with additional TF mRNAs in spheres. Cells transfected in this manner are more resistant to toxicity caused by small molecules, growth factors, or other elements in cell cultures, and generally should differentiate more efficiently than cells 2D transfected using chemical reagents. This observation, which went unnoticed in scientific publication, was made accidentally during testing of electroporation equipment, and served as a stimulating method during the disclosure of the present invention.

Example 2: Production of pancreatic progenitor cells from endodermal cells

[00124] Endoderm cells were seeded into commercially available cell culture vessels. 6-well plates were used in the experiments illustrated in FIG. 2, but other standard commercially available plates with suitable well sizes may also be used. The plates were pre-coated with Matrigel (BD Biosciences), and then 1 x 10 ^{5 -} 1 x 10 ⁶ cells were seeded in DMEM/F12 or MCDB131 medium supplemented with 8 mM D-glucose.

[00125] In one experiment, cells were then transfected with PDX1 mRNA. In addition, cells were transfected or cotransfected with Hlxb9, Ptf1a, ixl1, HNF1a and b, and Sox9 mRNA at approximately 50 ng per well to achieve stronger effects using Stemgent transfection reagent (Stemgent) in culture medium, and this transfection was repeated 2 , 3 or more times in doses ranging from at least 10 ng and up to a maximum of 200 ng per well using Stemgent transfection reagent or other commercially available transfection reagents. In some embodiments, the initial or repeat dose of Stemgent may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1120, 130, 140, 150, 160, 170, 180, 190 or 200 ng per well or any value between any of the above values. Cells at this stage are darker than endodermal cells and tend to form clusters (Fig. 2).

Example 3: Production of Pancreatic Endocrine Cells from Pancreatic Progenitor Cells

[00126] Pancreatic progenitor cells were cultured in 6-well plates or other plates pre-coated with Matrigel (BD Biosciences) or collagen I (Sigma) in DMEM/F12 or MCDB131 supplemented with 8 mM D-glucose. Other similar cell adhesion culture media are also suitable for this purpose.

[00127] Pancreatic progenitor cells were transfected with PDX1, NKX6.1 and/or NKX2.2, Pax6, Pax4, Hlxb9 Ngn3 mRNA at a dose of 10-200 ng per well using Stemgent transfection reagent (Stemgent) in culture medium supplemented with 200 ng/ml B18R. In some embodiments, the dose of Stemgent may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1120, 130, 140, 150, 160, 170, 180, 190, or 200 ng per well or any value between any of the above values. The initial or repeat dose of Stemgent can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, 180, 190, or 200 ng per well, or in intervals between any of the above values. Most commercially available transfection reagents can also be used.

[00128] At this stage, pancreatic progenitor cells are optionally treated with a number of growth factors for subsequent differentiation. Cells were cultured in S5 medium containing MCDB131 + 20 mM D-glucose + 1.754 g/l NaHCO ₃ + 2% FAF-BSA + ITS-X 1:200 + 2 mM Glutamax + 0.25 mM vitamin C + 1% penicillin/ streptomycin + 10 μg/ml heparin (Sigma; H3149). Cells were treated with S5 medium containing 0.25 µM Sant 1 + 100 nM RA + 1 µM XXI (EMD Millipore) + 10 µM Alk5i II (Axxora) + 1 µM T3 (EMD Millipore) + 20 ng/ml beta-cellulin (Thermo Fisher Scientific) every day for four days. On the fifth day, cells were treated with S5 medium containing 25 nM RA + 1 µM XXI + 10 µM Alk5i II + 1 µM T3 + 20 ng/ml beta-cellulin every day for four days.

[00129] At this stage, the cells further clustered and initiated flotation from the monolayer, which is an early sign of differentiation and formation of pancreatic endocrine cells (Fig. 3).

Example 4: Maturation of Pancreatic Endocrine Cells

[00130] Pancreatic endocrine cells were cultured in commercially available cell culture vessels. 6-well plates were used in these experiments, but all other types of plates can also be used. Plates were precoated with Matrigel (BD Biosciences) or collagen I (Sigma) in CMRL (Mediatech). After cluster collection, ultra-low adhesion plates or flasks were used.

[00131] These cells were then transfected with 10-200 ng of KX6.1/MAFA or MAFA mRNA per well using Stemgent transfection reagent (Stemgent) in culture medium supplemented with 200 ng/ml B18R. Repeat transfections were performed 3 times, and optional additional transfections could be performed if necessary. Other commercially available transfection reagents may also be used.

[00132] Pancreatic endocrine cells are also optionally treated with a number of growth factors for subsequent maturation. Cells were cultured in S6 medium containing medium supplemented with CMRL 1066 (Mediatech; 99-603-CV)+10% FBS (HyClone, VWR; 16777)+1% penicillin/streptomycin. Cells were treated with S6 medium containing 10 μM Alk5i II + 1 μM T3 every day for 14 days.

[00133] At this stage, clusters of pancreatic endocrine cells, such as beta cells, form clusters ranging in size from several tens to several thousand cells that have independently separated from the monolayer of mature pancreatic endocrine cells. At this stage, cell cultures have been exemplified in FIG. 4. These cells were stained with antibodies against protein markers specific for this cell stage, such as KX6.1 and insulin, and the results of such staining confirmed that some of these cells were indeed pancreatic beta cells (Fig. 5). These cells also showed specific markers of cell status throughout this artificially induced differentiation pathway into beta cell-like cells (Fig. 6). In addition, glucose-responsive beta cell clusters were added to the culture medium, and the beta cells secreted insulin in response to glucose (Figure 7).

Example 5: iPSC-Derived Beta Cells Respond to Glucose in vitro

[00134] Clusters of cells formed as 3-dimensional structures from monolayer pancreatic endocrine cells, as shown in FIG. 4, were collected and transferred into 15 ml tubes, 2 x 10 ^{5 -} 1 x 10 ⁶ cells per tube, and the culture medium was removed by centrifugation (300 x g for 2 minutes) and washed twice with 1 ml of Krebs buffer per sample ( published by Milton 2014). 3 ml of Krebs medium with a low concentration of glucose (2 mM) was added to the cells and incubated for 2 hours. The medium was removed by centrifugation, and the cells were washed 2 times with 1 ml of Krebs buffer per sample. 1 ml of Krebs buffer with a low concentration of glucose was added to each tube and incubated for 30 minutes at 37°C in an incubator with the lid loosely closed to allow air exchange. Thereafter, the supernatant was collected for analysis by centrifugation, and then the cells were washed once with 1 ml of Krebs buffer, incubated in Krebs buffer with high glucose (20 mM) for 30 minutes, and then centrifuged to collect the supernatant for analysis. The cells were washed twice and then the low-high glucose cycle was repeated for a total of 3 times. Supernatants were collected at each stage, pooled after 4 to 6 independent replicates, and analyzed for insulin levels using a commercially available ELISA kit (ALPCO) and a plate reader (Tecan or SpectroMax). For comparison, the analysis was also carried out with beta cells obtained essentially according to the protocol of Milton (2014), and the results showed that beta cells obtained by the method according to the invention had a lower baseline level of insulin expression and a higher level insulin in response to an increase in glucose concentration in the medium compared to the Milton method (Fig. 7), which indicates unexpected and surprising advantageous properties of the variants of the present invention.

Example 6: iPSC-Derived Beta Cells Respond to Glucose in Animal Models

[00135] To further confirm the functions of representative mature pancreatic beta cells obtained in accordance with the present invention, the function of glucose-sensing and insulin-secreting iPSC-derived pancreatic beta cells was tested in a mouse model of diabetes, such as: 1) NOD mouse with spontaneous type I diabetes model; 2) model of STZ-induced diabetes type I; 3) ob/ob or db/db model with type II diabetes; or in non-human primates such as: macaques/green monkeys or baboons with a model of STZ-induced type I diabetes; or macaques/green monkeys or baboons with a model of type II diabetes induced spontaneously or after consumption of a high-fat diet.

[00136] Method of delivery: Representative spheres of pancreatic beta cells were administered by infusion into the hepatic or renal capsules or into the omentum. Testing: Serum insulin levels were measured without glucose stimulation (weeks 2, 4, 8, 12, 16, 24); serum insulin levels were measured without glucose stimulation (weeks 2, 4, 8, 12, 16, 24); We assessed blood glucose levels in fasting animals (weeks 2, 4, 8, 12, 16, 24); IHC analysis of the transplant site was performed for insulin, and staining for C-peptide and glucagon was performed. The results demonstrated elimination or reduction of diabetes symptoms such as high blood glucose and associated organ effects.

Example 7: iPSC-Derived Beta Cells Respond to Glucose in the Treatment of Diabetic Patients

[00137] Clinical trials using human pancreatic beta cells derived from iPSCs, conducted in accordance with the described protocols adapted for cGMP procedures, were carried out by dosing in animal studies in comparison with other types of cell therapy, and even if in While there are currently no therapies using iPSC-derived pancreatic beta cells, human ESC-derived pancreatic progenitor cells are currently being used in clinical trials to treat type I diabetes. Autologous, preferred type, or allogeneic iPS cell-derived beta cells are administered via the portal vein to treat, for example, type I induced diabetes, type II “skinny” diabetes, advanced diabetes where a person's own beta cells are dysfunctional or depleted, or another's diabetes type associated with impaired beta cell function. Representative engineered beta cells produced by the inventive methods described herein are injected into other organs of the human body, such as muscles, connective tissues, parts of the pancreas, or certain areas of other organs.

Claims (34)

1. A method of inducing differentiation of stem cells into glucose-sensitive insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing induced pluripotent stem cells (iPSCs) as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and period of time, wherein the first combination of mRNAs comprises at least one of FoxA2 mRNA and Sox17 mRNA; (d) converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs, wherein the second combination of mRNAs comprises at least one of PDX1, Hlxb9, Ptf1a, ixl1, HNF1a and b, and Sox9 mRNAs; (e) maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs, wherein the third combination of mRNAs comprises at least one of PDX1, NKX6.1, NKX2.2, Pax6, Pax4, Hlxb9 and Ngn3 mRNAs; And (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, where induced pluripotent stem cells are not isolated from a human embryo. 2. The method of claim 1, wherein said first mRNA combination comprises FoxA2 mRNA. 3. The method of claim 1, wherein said first mRNA combination comprises Soxl7 mRNA. 4. The method of claim 1, wherein said first mRNA combination comprises FoxA2 and Soxl7 mRNA. 5. The method of claim 1, wherein said first mRNA combination comprises FoxA2, Sox17, GATA4 and GATA6 mRNA. 6. The method of claim 1, wherein said second mRNA combination comprises at least PDX1 mRNA. 7. The method of claim 1, wherein said third mRNA combination comprises at least PDX1 and NKX6.1 mRNA. 8. The method of claim 1, comprising maturing pancreatic endocrine cells from step (e) into pancreatic beta cells using a fourth mRNA combination, wherein the fourth mRNA combination comprises NKX6.1 and MAFA mRNA or MAFA mRNA. 9. The method of claim 1, wherein said iPSCs are isolated from the physiological fluid or tissue of an individual. 10. A method of eliminating or reducing symptoms of diabetes in an individual, which includes (a) culturing induced pluripotent stem cells (iPSCs) as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and time period, wherein the first combination of mRNAs comprises at least one of FoxA2 mRNA and Sox17 mRNA; (d) converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs, wherein the second combination of mRNAs comprises at least one of PDX1, Hlxb9, Ptf1a, ixl1, HNF1a and b, and Sox9 mRNAs; (e) maturation of said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs, wherein the third combination of mRNAs comprises at least one of PDX1, NKX6.1, NKX2.2, Pax6, Pax4, Hlxb9 and Ngn3 mRNAs; (f) collecting clusters enriched in pancreatic beta cells that are responsive to environmental glucose and capable of secreting insulin in response to glucose; And (g) administering glucose-sensitive insulin-secreting pancreatic beta cells to a subject; where iPSCs are not isolated from human embryos. 11. The method of claim 10, wherein said iPSCs are isolated from a recipient individual. 12. A method for producing induced glucose-sensitive insulin-secreting pancreatic beta cells, where the method includes the steps of: (a) culturing induced pluripotent stem cells as parent cells under differentiation conditions; (b) inducing said parent cells to transition from a pluripotent state to a mesoendoderm lineage state; (c) directing the cells to differentiate into endodermal cells by transfecting the cell culture with a first combination of mRNAs at an effective dose and period of time, wherein the first combination of mRNAs comprises at least one of FoxA2 mRNA and Sox17 mRNA; (d) converting said endodermal cells into pancreatic progenitor cells by transfection with a second combination of mRNAs, wherein the second combination of mRNAs comprises at least one of PDX1, Hlxb9, Ptf1a, ixl1, HNF1a and b, and Sox9 mRNAs; (e) maturing said pancreatic progenitor cells into pancreatic endocrine cells using a third combination of mRNAs, wherein the third combination of mRNAs comprises at least one of PDX1, NKX6.1, NKX2.2, Pax6, Pax4, Hlxb9 and Ngn3 mRNAs; And (f) collecting clusters enriched in pancreatic beta cells that are receptive to environmental glucose and capable of secreting insulin in response to glucose, where induced pluripotent stem cells are not isolated from a human embryo.

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