Differentiation of Stem Cells into Pancreatic Endocrine Cells

Abstract

An in vitro method for generating a population of pancreatic endocrine (PEC) cells comprises: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine precursors, the differentiating comprising treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor; and ii) differentiating pancreatic endocrine precursor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine precursor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

Description

Technical Field

[0001] The present invention relates to an in vitro method for obtaining pancreatic endocrine (PEC) cells, the method comprising the step of treating the cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor. The method provides islet-like cells, particularly beta-like cells.

Background Art

[0002] Insulin therapy is life-saving, but it can be difficult to obtain stable blood glucose with exogenous insulin, and inadequate control is associated with serious late complications (Non-Patent Document 1). Transplantation of islets isolated from human donors into patients with type 1 diabetes has shown good results in that some patients have become completely independent of insulin (Non-Patent Document 2). Despite such progress, one of the major challenges for islet transplantation is the limited availability of donor islets. This shortage of donor material can be overcome by generating functional insulin-secreting cells in vitro by the differentiation of human embryonic stem cells (hESCs). Protocols for the generation of functional insulin-secreting cells in vitro from stem cells are continuously being developed (Non-Patent Documents 3 and 4, Patent Documents 1, 2, and 3).

[0003] Beta cell (BC) transplantation has the potential to bring about a definitive cure for type I diabetes. However, the use of this treatment as a clinical therapy is restricted due to the limited availability of donor beta cells. Pluripotent stem (PS) cells can proliferate indefinitely and differentiate into many cell types. Therefore, PS cells are a promising source for beta cells, but they need to differentiate efficiently and reproducibly into pancreatic cells before they can be used to treat diabetes.

[0004] During vertebrate embryogenesis, pluripotent stem cells give rise to the three germ layers: ectoderm, mesoderm, and endoderm. Induction of definitive endoderm (DE) is the first step towards the formation of endoderm-derived tissues. Generation of pancreatic endoderm (PE) from DE cells is required for the production of pancreatic endocrine precursors (EP) and ultimately insulin-producing beta cells. PE cells that have the potential to become beta cells are characterized by the co-expression of two important transcription factors, PDX1 and NKX6.1.

[0005] Stepwise in vitro differentiation protocols have been established to generate pancreatic cells from PS cells. These protocols generally mimic the major events of pancreatic development, including the formation of DE, primitive streak, posterior foregut, PE, EP, and PEC (pancreatic endocrine cells) that co-express SOX17 and FOXA2. To date, efficient DE differentiation of hESCs has been achieved by treatment with activin A and treatment with a Wnt pathway agonist. The next major step in the generation of pancreatic beta cells is to generate PE that co-expresses PDX1 and NKX6.1. Several groups have developed in vitro protocols that can differentiate PS cells into DE and PE, and almost all published protocols include the step of adding a retinoic acid receptor (RAR) agonist in the induction of PE.

[0006] In summary, patients with type 1 diabetes can be treated using transplantation of islets from human donors, and some patients achieve insulin independence. However, donor islets are in short supply and have variable quality, and PS cell-derived insulin-producing cells offer an attractive alternative to islets. The major differentiation step is the differentiation into EP cells and PEC cells to obtain functional beta cells (BC).

[0007] Current protocols for differentiating human pluripotent stem cells (hPSCs) into EP cells and specific PEC cells are not efficient processes. Further, with current protocols, non-pancreatic endocrine cells, such as unwanted enterochromaffin cells, as well as non-endocrine cells, are highly formed during differentiation. Thus, it is necessary to increase the efficiency of current differentiation protocols.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0010] The present invention is an in vitro method for generating pancreatic endocrine progenitor cells and / or pancreatic endocrine (PEC) cells, comprising: i) a step of differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells, wherein the differentiating comprises treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, and / or ii) a step of differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0011] In particular, the present invention is an in vitro method for generating pancreatic endocrine (PEC) cells, comprising: i) a step of differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, and ii) a step of differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0012] In some embodiments, the present invention is an in vitro method for generating pancreatic endocrine (PEC) cells, comprising: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor; or ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0013] In some embodiments, the present invention is an in vitro method for generating pancreatic endocrine (PEC) cells, comprising: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitor (EP) cells by treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and a platelet-derived growth factor (PDGF) receptor; and ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and a platelet-derived growth factor (PDGF) receptor.

[0014] In multiple embodiments, an in vitro method for generating a population of pancreatic endocrine progenitor (EP) cells comprises: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors by treating the PE cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0015] In multiple embodiments, an in vitro method for generating a population of pancreatic endocrine (PEC) cells comprises: ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0016] In multiple embodiments, an in vitro method for generating a population of pancreatic endocrine (PEC) cells comprises i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors by treating the PE cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, and ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells.

[0017] In multiple embodiments, an in vitro method for generating a population of pancreatic endocrine (PEC) cells comprises i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine progenitors, and ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

[0018] An inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor suitable for use in the methods of the present invention is linifanib or 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea, also designated as ABT869.

[0019] The inventors have found that the use of an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor in the above step i) and / or step ii) has the advantage of increasing the proportion of beta-like cells obtained compared to a method in which a transforming growth factor beta (TGFb) receptor kinase inhibitor or a TGFβR1 kinase inhibitor or a TGFβ1R kinase inhibitor or a TGFbR1 inhibitor is used instead of an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor. Furthermore, the proportion of pancreatic endocrine (PEC) cells also increases.

[0020] The concentration of an inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor used in step ii) is in the range of 1 nM to 12 μM, for example 5 nM to about 15 μM, 5 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 μM, 2.5 μM to 5.5 μM, 2.5 μM to 5 μM, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 2 μM to about 3 μM, about 3 μM to about 12 μM, about 3 μM to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM, about 6 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM or in the range of about 11 μM to about 12 μM, or 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.It is at a concentration of 5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM or 15 μM.

[0021] The concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) used in step i) is in the range of 1 nM to 12 μM, for example 5 nM to about 15 μM, 5 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 μM, 2.5 μM to 5.5 μM, 2.5 μM to 5 μM, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 2 μM to about 3 μM, about 3 μM to about 12 μM, about 3 μM to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM, about 6 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM or in the range of about 11 μM to about 12 μM, or 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.It is at a concentration of 5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM or 15 μM.

[0022] In some embodiments, the pancreatic endocrine (PEC) cells obtained from step ii) contain islet-like cells. The islet-like cells include alpha-like cells, beta-like cells, epsilon-like cells, delta-like cells, and gamma-like cells. Steps i) and ii) of the method of the present invention do not require a TGFβ receptor kinase inhibitor.

[0023] In some embodiments, step i) does not require a TGFβ receptor kinase inhibitor.

[0024] In some embodiments, step ii) does not require a TGFβ receptor kinase inhibitor.

[0025] As shown in Figure 1B, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 20%, such as at least 25%, such as at least 28%, or at least 30% beta-like cells, based on the total number of cells obtained after step ii), and the beta-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+).

[0026] As described above, the number of beta-like cells obtained in the population of pancreatic endocrine (PEC) cells obtained after step ii) is increased compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), and the beta-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+). Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1%, for example, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, or at least 20% more beta-like cells that are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+) compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). Further, when using standard methods, only about 40% of the cells are ISL1+ whereas more than 60% of the cells are ISL1+ (see, for example, FIG. 3B).

[0027] The number of enterochromaffin (EC) cells produced by the method of the present invention is decreased compared to in vitro methods for producing pancreatic endocrine (PEC) cells, and the pancreatic endocrine (PEC) cells are obtained by using a TGFb receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains up to 35%, for example, up to 30%, or up to 27% enterochromaffin cells, and the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+) (see, for example, FIG. 3B).

[0028] The population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1%, for example, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35% fewer enterochromaffin cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). Enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+) (see, for example, FIG. 3B).

[0029] As shown in FIG. 3E, the population of pancreatic endocrine (PEC) cells obtained after step ii) is also increased compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). Thus, the population or cells of pancreatic endocrine (PEC) cells obtained after step ii) contain at least 70%, for example, at least 75%, at least 78%, or at least 80% pancreatic endocrine cells, and the endocrine cells are CHGA+.

[0030] The population or cells of pancreatic endocrine (PEC) cells obtained after step ii) contain at least 1%, for example, at least 2%, at least 3%, at least 4%, or at least 5% more pancreatic endocrine (PEC) cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii). The pancreatic endocrine (PEC) cells are CHGA+ (see, for example, FIG. 3E).

[0031] In a further aspect, a cell population or composition is disclosed according to the present invention for use, for example, as an agent in the treatment of type I diabetes. Transplantation of a homogeneous cell population is thought to improve the safety and efficacy profile of cell therapy and reduce the risk of unwanted side effects from unwanted cell types. Treatment with a cell population or composition according to the present invention results in a high percentage of beta-like cells, thereby rendering it suitable for the prevention, amelioration and / or treatment of conditions that require administration of such cells.

Brief Description of the Drawings

[0032]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0033] A method for obtaining pancreatic endocrine cells from pluripotent stem cells is provided.

[0034] The pancreatic endocrine cells obtained by the method described in this specification can be used in and / or are intended for use in a method of providing pancreatic endocrine function to a mammal lacking the production of at least one pancreatic hormone.

[0035] In some embodiments, the invention is a method of providing pancreatic endocrine function to a mammal lacking the production of at least one pancreatic hormone, the method comprising transplanting endocrine cells obtained by any of the methods described in the invention in an amount sufficient to obtain a measurable amount of the at least one pancreatic hormone in the mammal.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology known to those of ordinary skill in the art.

[0037] It should be noted that all headings and subheadings are used herein for convenience only and should in no way be construed as limiting the invention.

[0038] The use of any and all examples or exemplary language (e.g., "such as") presented herein is for illustrative purposes only and, unless otherwise claimed, does not limit the scope of the invention.

[0039] As used herein, "a", "an", or "the" may mean one or more. Unless otherwise indicated herein, terms presented in the singular include the plural. As used herein, "and / or" refers to any and all possible combinations and subcombinations of the one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or"), and encompasses them. Further, the present invention also contemplates that, in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0040] General Definitions hESC: human embryonic stem cell hiPSC: human induced pluripotent stem cell hPSC: human pluripotent stem cell DE: definitive endoderm PE: pancreatic endoderm EP: endocrine progenitor cell PEC: pancreatic endocrine cell BC: beta cell, insulin-producing beta cell

[0041] Stem Cells A "stem cell" should be understood as an undifferentiated cell having the ability to proliferate (especially self-renewal ability) while maintaining the ability to differentiate. The term "stem cell" includes classifications such as pluripotent stem cells, multipotent stem cells, and the like according to the differentiation ability.

[0042] As used herein, the term "pluripotent stem cell" (PSC) refers to a stem cell that can be cultured in vitro and has the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm) and / or extraembryonic tissues (pluripotency).

[0043] As used herein, the term "multipotent stem cell" means a stem cell that has the ability to differentiate into a plurality of types of tissues or cells, although not all types, and is typically limited to one germ layer.

[0044] As used herein, the term "unipotent stem cell" means a stem cell that has the ability to differentiate into only one specific tissue or cell type.

[0045] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, stem cells in tissues, somatic cells, and the like. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs), EG cells (embryonic germ cells), induced pluripotent stem cells (iPSCs), and the like.

[0046] As used herein, the term "induced pluripotent stem cell" (also known as iPS cell or iPSC) means a type of pluripotent stem cell that can be generated directly from adult cells. By introduction of the products of a specific set of pluripotency-related genes, non-pluripotent cells can be converted into pluripotent stem cells. Pluripotent embryonic stem cells may also be derived from parthenogenetic organisms, as described, for example, in WO 2003 / 046141, the contents of which are incorporated herein by reference in their entirety. In addition, embryonic stem cells can be produced from a single blastomere or by culturing the inner cell mass obtained without destroying the embryo. Embryonic stem cells are available from a given tissue and are also commercially available. Preferably, in some embodiments, the methods and products described herein are based on stem cells derived from either hPSCs, i.e., induced pluripotent stem cells or embryonic stem cells including parthenogenetic organisms.

[0047] Definitive endoderm (DE) As used herein, the term "definitive endoderm", "definitive endoderm cell", or "DE" refers to cells characterized by the expression of the marker SOX17. Optionally, additional markers of DE are one or more of the following: FOXA2 and CXCR4. Definitive endoderm cells are important, for example, for the development of pancreatic cells.

[0048] 「SOX17」(SRY-box 17), as used herein, is a member of the SOX (SRY-related HMG box) family of transcription factors involved in the control of embryonic development and cell fate determination.

[0049] 「FOXA2」(forkhead box A2), as used herein, is a member of the forkhead class of DNA-binding proteins

[0050] 「CXCR4」(C-X-C motif chemokine receptor 4), as used herein, is a CXC chemokine receptor specific for stromal cell-derived factor-1.

[0051] Non-limiting examples of DE induction protocols are the conventional D’Amour protocol (Nature Biotechnology 2006, 2008) and the protocol described in International Publication No. WO 2012 / 175633, which is incorporated herein by reference in its entirety.

[0052] Pancreatic endoderm (PE) As used herein, the terms “pancreatic endoderm,” “pancreatic endoderm cells,” “pancreatic precursor,” or “PE” refer to cells characterized by expressing the markers PDX1 and NKX6.1. In some embodiments, at least 5% of the cells are NKX6.1+ / PDX1+ double positive. Optionally, additional markers of PE are one or more of SOX9 and PTF1A.

[0053] 「PDX1」, as used herein, refers to a homeodomain transcription factor involved in pancreatic development.

[0054] 「NKX6.1」, as used herein, is a member of the NKX transcription factor family.

[0055] 「SOX9」(SRY-box transcription factor 9), as used herein, is a transcription factor that plays an important role during embryonic development and cell lineage assignment.

[0056] As used herein, "PTF1A" is a component of the pancreatic transcription factor 1 complex (PTF1) and is a protein known to play a role in mammalian pancreatic development.

[0057] As used herein, "CPA1" is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas.

[0058] Non-limiting examples of PE induction protocols are described in International Publication No. WO 2014 / 033322, which is hereby incorporated by reference in its entirety.

[0059] Pancreatic endocrine progenitor (EP) cells As used herein, the terms "pancreatic endocrine precursor", "endocrine progenitor cell", or "EP" refer to cells characterized by the expression of NEUROG3 and, optionally, one or more of NeuroD and NKX2.2, which are characteristics of EP cells involved in the endocrine cell fate.

[0060] As used herein, "NEUROG3" is a member of the neurogenin family of basic helix-loop-helix transcription factors.

[0061] As used herein, "NKX2.2" and "NKX6.1" are members of the NKX transcription factor family.

[0062] As used herein, "NeuroD" is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors.

[0063] Protocols for generating pancreatic endocrine progenitor cells are described in International Publication No. WO 2015 / 028614, which is hereby incorporated by reference in its entirety.

[0064] Pancreatic endocrine cells (PEC) As used herein, the term "pancreatic endocrine cell" or "PEC" refers to cells that express CHGA and ISL1.

[0065] Pancreatic endocrine (PEC) cells obtained using the methods of the present invention include islet-like cells. Islet-like cells include alpha-like cells, beta-like cells, epsilon-like cells, delta-like cells, and gamma-like cells.

[0066] As used herein, the term "islet-like cell" refers to islet cells obtained in vitro after culturing of stem cells. Islet-like cells include beta cells, alpha cells, delta cells, and gamma cells.

[0067] As used herein, the term "alpha cell" refers to cells that express GCG, and optionally one or more of ISL1 and ARX. In the pancreas, alpha cells produce the hormone glucagon.

[0068] As used herein, the term "β cell" or "β-like cell" refers to cells that express INS, and optionally one or more of PDX1, ISL1, and NKX6.1. In the pancreas, beta cells produce the hormones insulin and amylin.

[0069] As used herein, the term "delta cell" refers to cells that express SST, and optionally one or more of ISL1 and HHEX. In the pancreas, delta cells secrete the peptide hormone somatostatin.

[0070] As used herein, the term "epsilon cell" refers to cells that express GHRL, and optionally one or more of ISL1, ARX, and ETV1. In the pancreas, epsilon cells produce the hormone ghrelin.

[0071] As used herein, the term "gamma cell" is used interchangeably in the present context with "pancreatic polypeptide cell", "PP cell", "γ-cell", or "F cell", and refers to an endocrine cell that expresses PPY, and optionally one or more of ISL1 and PAX6. In the pancreas, this serves to regulate the synthesis and release of pancreatic polypeptide (PP).

[0072] As used herein, the term "enterochromaffin cell" is used interchangeably with "EC cell" and "Kultschitzky cell", and refers to an endocrine cell that expresses TPH1, and optionally one or more of LMX1A and FEV. Enterochromaffin cells are a type of enteroendocrine and neuroendocrine cell. In humans, they are located in the epithelial layer throughout the gastrointestinal tract. EC cells regulate neuronal signaling in the enteric nervous system (ENS) via the secretion of the neurotransmitter serotonin and other peptides.

[0073] Expression of markers As used herein, the term "expression level" refers to the degree of gene expression and / or gene product activity in a cell. The expression level can be determined in any absolute or normalized units (relative to a known expression level of a control reference).

[0074] As used herein, the term "marker" refers to a naturally occurring identifiable expression produced by a cell, which may correlate with a particular property of the cell and serves to identify, predict, or characterize the cell or cell population. A marker may be referred to as a gene. A marker may be in the form of mRNA or a protein, such as a protein on the cell surface.

[0075] As used herein, the term "expression" when referring to a marker refers to the presence or absence of a molecule that can be detected in a cell. In certain embodiments, the molecule that is expressed is mRNA or protein. Expression of a marker can be detected at any suitable level, such as the mRNA or protein level. One of ordinary skill in the art will readily understand that cells can be defined by positive or negative expression of a marker, i.e., the characteristics and state of a cell may be equally and mutually associated based on the expression and absence of a particular marker. When referring to a specific marker, the presence or absence of expression may be indicated by + (plus) or - (minus), respectively.

[0076] Steps of the method As used herein, the term "step" with respect to the methods described herein should be understood as a stage at which something is being worked on and / or an operation is being performed. It will be understood by one of ordinary skill in the art that the steps being performed and / or worked on may be simultaneous and / or sequential and / or continuous.

[0077] Throughout this application, the terms "method" and "protocol" when referring to a process for differentiating cells may be used interchangeably.

[0078] As used herein, the term "day" with respect to a protocol and similarly the number of days in vitro (DIV) refers to a specific time for performing a particular step during a differentiation procedure.

[0079] Generally, and unless otherwise specified, "day 0" refers to the start of the protocol, which may include, for example, but is not limited to, plating cells, transferring cells to an incubator, or contacting cells in the current cell culture medium with a compound prior to cell transplantation. Typically, the start of the protocol is effected by transferring cells, such as undifferentiated stem cells, definitive endoderm cells, pancreatic endoderm cells, pancreatic endocrine progenitor (EP) cells or pancreatic endocrine (PEC) cells, to another cell culture medium and / or container, for example, but not limited to, by plating or incubating, and / or by first contacting the cells with one or more compounds that affect the undifferentiated stem cells in such a way that the differentiation process is initiated.

[0080] When referring to "day X", such as day 1, day 2, etc., this is relative to the start of the protocol on day 0. One of ordinary skill in the art will recognize that the exact date and time for performing the steps may vary unless otherwise specified. Thus, "day X" is meant to include a time range such as ±10 hours, ±8 hours, ±6 hours, ±4 hours, ±2 hours, or ±1 hour.

[0081] As used herein, the phrase "from about day X to about day Y" refers to the day on which the event begins. This phrase provides the interval of days during which the event can begin. For example, when "the cells are contacted with a differentiation factor from about day 3 to about day 5", this is to be interpreted as encompassing all options: "the cells are contacted with the differentiation factor starting on about day 3", "the cells are contacted with the differentiation factor starting on about day 4", and "the cells are contacted with the differentiation factor starting on about day 5". Thus, this phrase should not be interpreted as an event that occurs only between day 3 and day 5. This applies mutatis mutandis to the phrase "from about day X to about day Y".

[0082] Differentiation As used herein, "differentiating" or "differentiation" refers to the process by which a cell progresses from an undifferentiated state to a differentiated state, from an immature state to a less immature state, or from an immature state to a mature state. For example, early undifferentiated pancreatic embryonic cells can proliferate and express characteristic markers such as PDX1, NKX6.1, and PTF1a. Mature or differentiated pancreatic cells do not proliferate and secrete high levels of pancreatic endocrine hormones or digestive enzymes. For example, fully differentiated beta cells secrete high levels of insulin in response to glucose. Changes in cell interactions and maturation occur when a cell loses markers of undifferentiated cells or acquires markers of differentiated cells. Loss or acquisition of a single marker can indicate that a cell has "matured or fully differentiated." The term "differentiation factor" refers to a compound that is added to pancreatic cells to enhance their differentiation into mature endocrine cells that also contain insulin-producing beta cells. Exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor, platelet-derived growth factor, and glucagon-like peptide 1. In some embodiments, differentiation of a cell comprises culturing the cell in a medium containing one or more differentiation factors.

[0083] Exemplary differentiation factors include hepatocyte growth factor, keratinocyte growth factor, exendin-4, basic fibroblast growth factor, insulin-like growth factor-1, nerve growth factor, epidermal growth factor, platelet-derived growth factor, glucagon-like peptide 1, indractam V, and retinoic acid.

[0084] In an embodiment of the invention, differentiation of a cell comprises culturing the cell in a medium containing one or more differentiation factors.

[0085] In a preferred embodiment, the method is performed in vitro. The term "in vitro" means that the cells are provided and maintained outside of a human or animal body. In one embodiment, the cells are non-natural. The term "non-natural" means that cells that are derived from pluripotent stem cells but may be of human origin are artificial constructs that do not exist in nature. As used herein, the term "artificial" may include materials that are naturally occurring in nature but have been modified into constructs that do not occur naturally. This includes human stem cells that differentiate into non-natural cells that mimic human body cells.

[0086] Known protocols for the individual differentiation steps from hPSCs to pancreatic endocrine cells References for known protocols for the individual differentiation steps from hPSCs to pancreatic endocrine cells are described below. The protocols, as well as the references presented in the "Background Art" paragraph of the present invention, can be used to provide definitive endoderm cells, pancreatic endoderm (PE) cells, or pancreatic endocrine precursors.

[0087] hPSCs are differentiated stepwise through distinct stages towards pancreatic endocrine (PEC) cells. These stages include definitive endoderm (DE), pancreatic endoderm (PE), endocrine progenitor (EP) cells (EP) and finally pancreatic islet cells (also referred to as PEC) (Madsen et al. - Nat Biotechnol. - 2006 Dec, 24(12):1481-3).

[0088] DE is generally induced by treating hPSCs with transforming growth factor β and WNT / β-catenin agonists (D’Amour et al., Nat Biotechnol., 2005 Dec;23(12):1534-41, Rezania et al., Diabetes, 2011 Jan;60(1):239-47, Kubo et al., Development, 2004 Apr;131(7):1651-62, Rezania et al., Nat Biotechnol., 2014 Nov;32(11):1121-33, Funa et al., Cell Stem Cell., 2015 Jun 4;16(6):639-52). DE is further specified in vitro into a PDX1+NKX6.1+PE population. Fibroblast growth factor, retinoic acid, sonic hedgehog, epidermal growth factor, and bone morphogenetic protein signaling pathways have all been involved in pancreatic development, and manipulation of these pathways at distinct stages of differentiation promotes a highly enriched PE population (D’Amour et al., Nat Biotechnol., 2006 Nov;24(11):1392-401, Kroon et al., Nat Biotechnol., 2008 Apr;26(4):443-52, Nostro et al., Development, 2011 Mar;138(5):861-71, Rezania et al., Diabetes, 2012 Aug;61(8):2016-29, Mfopou et al., Gastroenterology, 2010 Jun;138(7):2233-45, Ameri et al., Stam cells, 2010 Jan;28(1):45-56, Russ et al., EMBO J., 2015 Jul 2;34(13):1759-72).Additional pathways, including protein kinase C, nicotinamide, WNT, Rho-associated kinase, and TGFβ, have also been identified as being involved in the specification of hPSCs towards the pancreatic lineage (Chen et al.-Nat Chem Biol.-2009 Apr;5(4):258-65, Rezania et al.-Diabetes-2012 Aug;61(8):2016-29, Rezania etal.-Stem Cell-2013 Nov;31(11):2432-42,Nostro et al.-Stem Cell Reports-2015 Apr 14;4(4):591-604,Sharon et al.-Cell Rep.-2019 May 21;27(8):2281-2291.e5,Toyoda et al.-Stem Cell Rep.-2017 Aug 8;9(2):419-428).

[0089] The specification of pancreatic endocrine from PE depends on the expression of the transcription factor NEUROG3 (McGrath et al. - Diabetes - 2015 Jul;64(7):2497 - 505, Zhang et al. - Dev. Cell - 2019 Aug 5;50(3):367 - 380.e7.). Several approaches for inducing EP and PEC from PE have been investigated. Culturing PE in a gas - liquid interface medium resulted in upregulation of NEUROG3 transcripts as well as the pancreatic hormones insulin (INS) and glucagon (GCG) compared to cells cultured in planar media (Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121 - 33). Expression of the NEUROG3 transgene in PE has been shown to induce endocrine differentiation (Zhu et al. - Cell Stem Cell. - 2016 Jun 2;18(6):755 - 768). Modulation of the actin cytoskeleton, as well as dispersion of PE into single cells followed by re - aggregation into cell clusters, can induce NEUROG3 expression and differentiation into EP cells and hPSC - endocrine cells (Mamidi - Nature. - 2018 Dec;564(7734):114 - 118, Hogrebe et al. - Nat Biotechnol. - 2020 Apr;38(4):460 - 470). Inhibition of TGFβ signaling and Notch signaling progressed PE to a pancreatic endocrine phenotype (Rezania et al. - Diabetes. - 2011 Jan;60(1):239 - 47, Nostro et al. - Development. - 2011 Mar;138(5):861 - 71, Pagliuca et al. - Cell. - 2014 Oct 9;159(2):428 - 39, Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121 - 33, Rezania etal. - Differentiation of human embryonic stem cells - 2015 Jun 23).However, enabling TGFβ signaling appears to be necessary for differentiation into more mature beta-like cells (Velazco-Cruz et al.-Stem Cell Reports.-2019 Feb 12;12(2):351-365).

[0090] Several other signaling pathways have been shown to promote EP induction and further differentiation into hPSC-endocrine cells. Bromodomain and extra-terminal (BET) protein inhibition using I-BET151 or JQ1 enhanced the number of NEUROG3 endocrine progenitor (EP) cells (Huijbregts et al.-Diabetes.-2019 Apr;68(4):761-773). Sodium cromoglycate (SCG) was identified by small molecule screening and induced pancreatic endocrine differentiation in PE via inhibition of the bone morphogenetic protein 4 signaling pathway (Kondo et al.-Diabetologia.-2017 Aug;60(8):1454-1466.). Bone morphogenetic proteins are involved in endocrine induction (Nostro et al.-Development.-2011 Mar;138(5):861-71 Sharon et al.-Cell Rep.-2019 May 21;27(8):2281-2291.e5.), and inhibitors of this pathway are commonly used for differentiation of hPSCs into EPs and PECs. Treatment of PE with the WNT-tankyrase inhibitor IWR1-endo increased the expression of endocrine markers and downregulated progenitor markers, demonstrating that small molecule WNT inhibitors increase endocrine induction (Sharon et al.-Cell Rep.-2019 May 21;27(8):2281-2291.e5.).

[0091] Finally, specific pancreatic endocrine cell types similar to their in vivo counterparts are derived and characterized in detail. Glucagon-expressing alpha-like cells derived from hPSCs exhibit the molecular and functional characteristics of bona fide pancreatic alpha cells (Rezania et al. - Diabetes - 2011 Jan;60(1):239-47, Peterson et al. - Nat Commun. - 2020 May 7;11(1):2241). Differentiation protocols for maturing hPSC-derived beta-like cells capable of secreting insulin in response to elevated glucose concentrations have recently been reported (Rezania et al. - Nat Biotechnol. - 2014 Nov;32(11):1121-33, Pagliuca et al. - Cell. - 2014 Oct 9;159(2):428-39, Velazco-Cruz et al. - Stem Cell Reports. - 2019 Feb 12;12(2):351-365, Hogrebe et al. - Nat Biotechnol. - 2020 Apr;38(4):460-470, Liu et al. - Nat Commun. - 2021 Jun 7;12(1):3330, Nair et al. - Nat Cell Biol. - 2019 Feb;21(2):263-274).

[0092] Single-cell gene expression analysis has visually shown the differentiation pathway of hPSCs towards pancreatic endocrine (PEC) cells, including beta-like cells (Petersen et al.-Stem Cell Reports.-2017 Oct 10;9(4):1246-1261, Ramond et al.-Development.-2018 Aug 15;145(16):dev165480, Docherty et al.-Diabetes.-2021 Nov;70(11):2554-2567, Veres et al.-Nature.-2019 May;569(7756):368-373). Detailed characterization of hPSC-derived beta-like cells in both in vitro and in vivo has revealed many similarities to native pancreatic beta cells (Velazco-Cruz et al.-Stem Cell Reports.-2019 Feb 12;12(2):351-365, Augsornworawat et al.-Cell Rep.-2020 Aug 25;32(8):108067, Balboa et al.-Functional, metabolic and transcriptional maturation of stem cell derived beta cells-2021.03.31.437748v1). Interestingly, the formation of non-endocrine cells as well as non-pancreatic enterochromaffin cells has been recently reported for protocols aimed at differentiating hPSCs towards the pancreatic endocrine lineage (Petersen et al.-Stem Cell Reports.-2017 Oct 10;9(4):1246-1261, Veres et al.-Nature.-2019 May;569(7756):368-373).

[0093] The method according to the present invention is described in more detail by non-limiting embodiments and examples below.

[0094] In vitro method for obtaining pancreatic endocrine cells Described herein is an in vitro method for obtaining pancreatic endocrine cells from pluripotent stem cells that is useful and / or intended for use in a method of providing pancreatic endocrine function to a mammal lacking the production of at least one pancreatic hormone.

[0095] The present invention identifies small molecule inhibitors (lenvatinib) of vascular endothelial growth factor receptor (VEGFR) and / or platelet-derived growth factor receptor (PDGFR) that promote the differentiation of pancreatic endoderm into pancreatic endocrine progenitor (EP) cells and pancreatic endocrine cells (including beta-like cells).

[0096] The present invention is an in vitro method for generating a population of pancreatic endocrine (PEC) cells, i) a step of differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine precursors, the differentiating comprising treating the pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, and / or ii) a step of differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

[0097] In some embodiments, step i) is performed by treating pancreatic endoderm (PE) cells with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor.

[0098] Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitor (EP) cells - step i) In some embodiments, the pancreatic endoderm (PE) cells for use in step i) of the methods described herein are cells having the markers PDX1 and NKX6. [Table 1]

[0099] In some embodiments, in step i) of culturing pancreatic endoderm (PE) cells into pancreatic endocrine precursors, the pancreatic endoderm cells are treated with one or more compounds selected from the group consisting of thyroid hormone, an epidermal growth factor (EGF) agonist, staurosporine, a NOTCH pathway inhibitor, a BMP pathway inhibitor, an EZH2 histone methyltransferase inhibitor, a JNK pathway inhibitor, and a TGFbR1 inhibitor.

[0100] In some embodiments, suitable examples of thyroid hormone are T3 (CAS number 6893-02-3) or GC1 (CAS number 211110-63-3). In multiple embodiments, the thyroid hormone is T3.

[0101] For use in the methods of the present invention, suitable examples of EGF agonists (EGF pathway activators) are betacellulin (gene name BTC), epidermal growth factor (gene name EGF), amphiregulin (gene name AREG), transforming growth factor alpha (gene name TGFA), and neuregulin 1 (gene name (NRG1)). In multiple embodiments, the EGF pathway activator is betacellulin.

[0102] In some embodiments, suitable examples of NOTCH pathway inhibitors are DBZ (XX) (CAS number 209984-56-5), DAPT (CAS number 208255-80-5), compound E (CAS number 209986-17-4), and L-685,485 (CAS number 292632-98-5). In multiple embodiments, the NOTCH pathway inhibitor is XX.

[0103] In some embodiments, suitable examples of BMP pathway inhibitors include LDN 193189 dihydrochloride (CAS number 1435934-00-1), DMH-1 (CAS number 1206711-16-1), dorsomorphin dihydrochloride (CAS number 1219168-18-9), and Noggin. In multiple embodiments, the BMP pathway inhibitor is LDN.

[0104] In some embodiments, suitable examples of EZH2 histone methyltransferase inhibitors are 3-deazaneplanocin A hydrochloride (DZNep) (CAS number 120964-45-6), GSK 126 (CAS number 1346574-57-9), and EPZ005687 (CAS number 1396772-26-1). In multiple embodiments, the EZH2 histone methyltransferase inhibitor is DZNep.

[0105] In some embodiments, suitable examples of JNK pathway inhibitors are TCS JNK 6o / JNK inhibitor VIII (CAS number 894804-07-0), SP600125 (CAS number 129-56-6), TCS JNK 5a (CAS number 312917-14-9), and JNK-IN-8 (CAS number 1410880-22-6). In multiple embodiments, the JNK pathway inhibitor is JNK inhibitor VIII.

[0106] In some embodiments, staurosporine is a broad-spectrum protein kinase inhibitor (CAS number 62996-74-1). Other broad-spectrum protein kinase inhibitors for use in the methods of the present invention include apigenin, H-7 dihydrochloride, 5-iodotubercidin, K 252a, PKC 412, and Ro 31-8220 mesylate. In multiple embodiments, the broad-spectrum protein kinase inhibitor is staurosporine.

[0107] In some embodiments, suitable examples of TGFβ receptor kinase inhibitors include RepSox (ALK5i II) (CAS number 446859-33-2), SB431542 (CAS number 301836-41-9), LY 364947 (CAS number 396129-53-6), and A 83-01 (CAS number 909910-43-6). In some embodiments, the TGFβ receptor kinase inhibitors are RepSox and SB431542.

[0108] The differentiation in step i) may also include inhibitors of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, such as, for example, Rho kinase (ROCK inhibitor), such as Tiger, Chroman-1 (CAS number 1273579-40-0) and thiazovivin (CAS number 1226056-71-8), heparin, as well as other differentiation factors such as forskolin or NKH 477.

[0109] In step i), the pancreatic endoderm (PE) cells may also be treated with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor. Lenvatinib is an example of such a substance. Lenvatinib is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. In one embodiment, the cells are treated with such an inhibitor.

[0110] The concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) used in step i) is in the range of 5 nM to about 15 μM, for example, 5 nM to about 12 μM, 1 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 μM, 2.5 μM to 5.5 μM, 2.5 μM to 5 μM, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 2 μM to about 3 μM, about 3 μM to about 12 μM, about 3 μM to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM, about 6 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM or in the range of about 11 μM to about 12 μM, or 5 nM, 50 nM, 500 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.It is at a concentration of 5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM or 15 μM.

[0111] When the pancreatic endoderm (PE) cells of step i) are treated with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, step i) does not require a TGFβ receptor kinase inhibitor. No other changes to the protocol are made, allowing for a direct comparison with the protocol using a TGFβ receptor kinase inhibitor. Examples of TGFβR inhibitors include RepSox (ALK5i II) (CAS number 446859-33-2), SB431542 (CAS number 301836-41-9), LY 364947 (CAS number 396129-53-6), and A 83-01 (CAS number 909910-43-6).

[0112] Such a population of pancreatic endoderm (PE) cells can be used as a starting material in step i) of the method of the present invention. In another aspect of the present invention, pancreatic endoderm (PE) cells can also be used as a starting material in step i) when obtained using a previously published method.

[0113] The pancreatic endocrine precursors obtained in step i) typically have the markers NEUROG3, NKX2.2, and NEUROD1.

[0114] In some embodiments, differentiation is carried out in a suitable culture medium such as MCDB131 (basal medium), RPMI, DMEM, DMEM / F12, CMRL, MEM, and the like.

[0115] The medium may be supplemented with, for example, human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium bicarbonate, ITSX, GlutaMAX, ascorbic acid, and zinc sulfate.

[0116] In some embodiments, the differentiation of pancreatic endoderm into pancreatic endocrine progenitor (EP) cells is carried out over a period of about 1 to about 15 days or about 1 to about 12 days, about 1 to 8 days, for example, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days. Generally, it is about 4 days.

[0117] The obtained pancreatic endocrine progenitor (EP) cells contain at least one of the markers NEUROG3, NKX2.2, and NEUROD1.

[0118] Differentiation of pancreatic endocrine progenitors into pancreatic endocrine (PEC) cells - Step ii) In some embodiments, the population obtained from step i) contains pancreatic endocrine progenitor (EP) cells that express at least one of the markers NEUROG3, NKX2.2, and NEUROD1. Such a population obtained from step i) can be used as the starting material for step ii) of the method of the present invention. However, in another aspect of the present invention, pancreatic endocrine progenitor (EP) cells can also be used as the starting material for step ii) when obtained using previously published methods.

[0119] In some embodiments, in step ii), the pancreatic endocrine progenitor (EP) cells are treated with one or more compounds selected from the group consisting of thyroid hormones, staurosporine, BMP pathway inhibitors, and EZH2 histone methyltransferase inhibitors.

[0120] Examples of thyroid hormones, staurosporine, BMP pathway inhibitors, EZH histone methyltransferase inhibitors, and TGFb receptor kinase inhibitors are described in the above paragraph "Differentiation of pancreatic endoderm (PE) cells to pancreatic endocrine progenitor (EP) cells - Step i)", and they are also suitable for use in step ii) of the method of the present invention.

[0121] Differentiation in step ii) may also include, for example, inhibitors of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor, such as Tiger, Chroman-1 (CAS number 1273579-40-0) and thiazobibine (CAS number 1226056-71-8), heparin, and other differentiation factors such as forskolin or NKH 477.

[0122] In some embodiments, the pancreatic endocrine precursors of step ii) are treated with an inhibitor of vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor. Lenvatinib is an example of such a substance. Lenvatinib is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. In one embodiment, the cells are treated with such an inhibitor.

[0123] In some embodiments, the concentration of the inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor used in step ii) is in the range of 5 nM to about 15 μM, for example, 5 nM to about 12 μM, 1 nM to about 12 μM, 10 nM to about 11 μM, 25 nM to 10 μM, 50 nM to 10 μM, 100 nM to 10 μM, 250 nM to 10 μM, 500 nM to 10 μM, 750 nM to 10 nM, 1 μM to 10 μM, 2 μM to 10 μM, 2.5 μM to 10 μM, 2.5 μM to 8 μM, 2.5 μM to 7 μM, 2.5 μM to 6 μM, 2.5 μM to 5 μM, 2.5 μM to 5.5 μM, 2.5 μM to 5 μM, or in the range of about 3 μM to about 4 μM, or in the range of about 1 μM to about 12 μM, about 1 μM to about 11 μM, about 1 μM to about 10 μM, about 1 μM to about 9 μM, about 1 μM to about 8 μM, about 1 μM to about 7 μM, about 1 μM to about 6 μM, about 1 μM to about 5 μM, about 1 μM to about 4 μM, about 1 μM to about 3 μM, about 1 μM to about 2 μM, about 2 μM to about 12 μM, about 2 μM to about 11 μM, about 2 μM to about 10 μM, about 2 μM to about 9 μM, about 2 μM to about 8 μM, about 2 μM to about 7 μM, about 2 μM to about 6 μM, about 2 μM to about 5 μM, about 2 μM to about 4 μM, about 2 μM to about 3 μM, about 3 μM to about 12 μM, about 3 μM to about 11 μM, about 3 μM to about 10 μM, about 3 μM to about 9 μM, about 3 μM to about 8 μM, about 3 μM to about 7 μM, about 3 μM to about 6 μM, about 3 μM to about 5 μM, about 3 μM to about 4 μM, about 4 μM to about 12 μM, about 4 μM to about 11 μM, about 4 μM to about 10 μM, about 4 μM to about 9 μM, about 4 μM to about 8 μM, about 4 μM to about 7 μM, about 4 μM to about 6 μM, about 4 μM to about 5 μM, about 5 μM to about 12 μM, about 5 μM to about 11 μM, about 5 μM to about 10 μM, about 5 μM to about 9 μM, about 5 μM to about 8 μM, about 5 μM to about 7 μM, about 5 μM to about 6 μM, about 6 μM to about 12 μM, about 6 μM to about 11 μM, about 6 μM to about 10 μM, about 6 μM to about 9 μM, about 6 μM to about 8 μM, about 6 μM to about 7 μM, about 7 μM to about 12 μM, about 8 μM to about 11 μM, about 9 μM to about 10 μM, about 10 μM to about 12 μM, about 10 μM to about 11 μM or about 11 μM to about 12 μM, or 5 nM, 50 nM, 500 nM, 1 μM, 1 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.It is at a concentration of 5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM or 15 μM.

[0124] When the pancreatic endocrine (EP) cells of step ii) are treated with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PGDF) receptor, step ii) does not require a TGFβ receptor kinase inhibitor. No other changes to the protocol are made, allowing for a direct comparison with the protocol using a TGFβ receptor kinase inhibitor. Examples of TGFβR inhibitors include RepSox (ALK5i II) (CAS number 446859-33-2), SB431542 (CAS number 301836-41-9), LY 364947 (CAS number 396129-53-6), and A 83-01 (CAS number 909910-43-6).

[0125] Differentiation is typically carried out in a suitable culture medium such as MCDB131 (basal medium), or in one of the above-mentioned culture media or their equivalents.

[0126] The medium may be supplemented with, for example, human serum albumin (HSA), antibiotics such as penicillin and / or streptomycin, glucose, sodium bicarbonate, ITSX, GlutaMAX, ascorbic acid, and zinc sulfate.

[0127] Generally, the differentiation of pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells is carried out over a period of about 2 to about 30 days or about 2 to 25 days, about 2 to 20 days, about 3 to about 12 days, for example about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 15 days, about 20 days, about 25 days, or about 30 days. Lenifanib is usually added to the culture medium when the culture medium is changed. The culture medium is typically changed 1 or 2 days after the start of the culture.

[0128] In some embodiments, an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor is administered about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days after the start of step i), as described herein.

[0129] If the pancreatic endocrine (PEC) cell population contains beta cells, the culture can be continued. The culture medium may be the same as above, but generally zinc sulfate is excluded. Furthermore, the above-described differentiation factors are not included, but an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor may be included for further culture for at least 2 days, such as 2 days, 3 days, or 4 days.

[0130] If the resulting population of endocrine cells contains pancreatic endocrine cell aggregates, the aggregates can be dissociated into single cells.

[0131] Pancreatic endocrine (PEC) cells can be further processed using a cryopreservation medium and the temperature can be lowered to obtain cryopreserved single cells. A method suitable for cryopreserving pancreatic endocrine (PEC) cells is described in International Publication No. WO 2019 / 048690, which is hereby incorporated by reference in its entirety.

[0132] Cryopreservation can be performed after culturing the cells for 1 day or more, for example, after culturing for about 1 to about 30 days.

[0133] In some embodiments, the obtained pancreatic endocrine (PEC) cells include islet-like cells such as beta cells (having markers INS, PDX1, and / or NKX6.1), alpha cells (having markers GCG and / or ARX), and delta cells (having markers SST and / or HHEX).

[0134] In some embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) comprises at least 20%, such as at least 25%, such as at least 28% or at least 30% beta-like cells, based on the total number of cells obtained after step ii), and the beta-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+), see, for example, FIG. 1B.

[0135] Accordingly, the population of pancreatic endocrine (PEC) cells obtained after step ii) can contain at least 1%, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30% more beta-like cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in step i) and / or step ii), and the beta-like cells are double positive for ISL1 and NKX6.1 (ISL1+NKX6.1+), see, for example, FIG. 3B.

[0136] In a plurality of embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains up to 35%, such as up to 30% or up to 27% enterochromaffin cells, and the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+), see, for example, FIG. 3B

[0137] Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1%, such as at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35% fewer enterochromaffin cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in steps i) and ii). Enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1-NKX6.1+); see, for example, FIG. 3B.

[0138] In a plurality of embodiments, the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 78%, such as at least 80% pancreatic endocrine cells, and the endocrine cells are CHGA+; see, for example, FIG. 3E.

[0139] Thus, the population of pancreatic endocrine (PEC) cells obtained after step ii) can contain at least 1%, such as at least 2%, at least 3%, at least 4%, or at least 5% more pancreatic endocrine (PEC) cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of an inhibitor of VEGF and / or PDGF in steps i) and ii). The pancreatic endocrine (PEC) cells are CHGA+; see, for example, FIG. 3E.

[0140] Differentiation of stem cells into pancreatic endocrine cells In some embodiments, the hPSCs are obtained from any suitable source mentioned above. In some embodiments, the methods described herein include culturing hPSCs. The term "culturing" means that the hPSCs are cultured in a cell culture medium and that they are suitable for survival in their current developmental state. In some embodiments, culturing the stem cells requires transferring them to a different environment, such as by seeding the stem cells onto a new substrate or suspending them in an incubator. One of ordinary skill in the art will recognize that stem cells are vulnerable to such transfers, that the procedures require care, and that maintaining the stem cells in the original cell culture medium can facilitate more sustainable transfer of the cells before replacing the cell culture medium with another cell culture medium more suitable for the differentiation process.

[0141] In some embodiments, the methods described herein are in vitro methods for producing pancreatic endocrine (PEC) cells from human pluripotent stem cells, comprising: i) differentiating hPSC cells into definitive endoderm cells; ii) differentiating definitive endoderm cells into pancreatic endoderm cells; iii) differentiating pancreatic endoderm (PE) cells into pancreatic endocrine progenitor cells; iv) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells (islet-like cells including beta cells), wherein step iii) and / or step iv) are performed using an inhibitor of vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor.

[0142] Step iii) is described as step i) of the section entitled "Differentiation of pancreatic endoderm cells (PE) into pancreatic endocrine progenitors (EP or PEP)-step i)", and step iv) is described as step ii) of the section entitled "Differentiation of pancreatic endocrine progenitor into pancreatic endocrine (PEC) cells-step ii)".

[0143] The step of differentiating hPSC cells into definitive endoderm cells and the step of differentiating definitive endoderm cells into pancreatic endoderm (PE) cells may follow standard protocols such as the following: · D’Amour et al.-Nat Biotechnol.-2005 Dec;23(12):1534-41. · Funa et al.-Cell Stem Cell.-2015 Jun 4;16(6):639-52 · Teo et al.-Stem Cell Reports.-2014 Jun 12;3(1):5-14 · Kubo et al.-Development.-2004 Apr;131(7):1651-62 · Rezania et al.-Nat Biotechnol.-2014 Nov;32(11):1121-33 · Rezania et al.-Diabetes.-2011 Jan;60(1):239-47 · Kroon et al.-Nat Biotechnol.-2008 Apr;26(4):443-52. · Ameri et al.-Stem Cells.-2010 Jan;28(1):45-56 ·Nostro et al.-Development.-2011 Mar;138(5):861-71. ·Rezania et al.-Diabetes.-2012 Aug;61(8):2016-29 ·Nostro et al.-Stem Cell Reports.-2015 Apr 14;4(4):591-604 ·Russ et al.-EMBO J-2015 Jul 2;34(13):1759-72.

[0144] Other aspects of the present invention A composition comprising pancreatic endocrine cells (PEC) obtained by any of the methods of the present invention In a further aspect, an agent comprising pancreatic endocrine cells (PEC) obtained by any of the methods of the present invention according to this description is described herein. In particular, PEC obtained by the methods described herein have: i) more islet cells (ISL1+), more beta-like cells (ISL1+ / NKX6.1+), and fewer EC cells (ISL1- / NKX6.1+) compared to standard methods using TGFbRi.

[0145] In a preferred embodiment, the agent described herein comprises enriched, or homogeneous, thawed and re-aggregated cryopreserved pancreatic endocrine cells (PEC) obtained by any of the methods of the present invention.

[0146] Medical use of pancreatic endocrine (PEC) cells obtained by any of the methods of the present invention In some embodiments, the present invention is a method of providing pancreatic endocrine function to a mammal lacking production of at least one pancreatic hormone, the method comprising transplanting into the mammal a sufficient amount of pancreatic endocrine cells obtained by any of the methods of the present invention to obtain a measurable amount of the at least one pancreatic hormone.

[0147] In the present context, the term "mammal" includes human and veterinary subjects.

[0148] In the present context, the term "mammal" relates to, for example, humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cows, cats, guinea pigs, or rodents. The term does not indicate a particular age or sex.

[0149] Islet cell transplantation can be used, for example, in diabetic mammals to restore insulin production and blood glucose control.

[0150] Methods for treating diabetes (type 1 or type 2) are also provided herein. For example, methods for treating type 1 diabetes in a mammal are provided herein. In some embodiments, the method includes the steps of selecting a mammal having type 1 diabetes and administering to the mammal pancreatic endocrine cells obtained by any of the methods of the present invention. In other embodiments, the method includes preventing type 1 diabetes in a mammal at risk of developing type 1 diabetes by administering to the mammal endocrine cells obtained by any of the methods of the present invention.

[0151] One of ordinary skill in the art can identify and select mammals having type 1 diabetes and mammals at risk of developing type 1 diabetes using diagnostic and any particular methods. For example, the diagnosis is based on an increase in blood glucose level after fasting or a glucose tolerance test. Additionally, the diagnosis of type 1 diabetes includes various physical symptoms and characteristics.

[0152] Identifying mammals at risk of developing type 1 diabetes is also within the skill of one of ordinary skill in the art. For example, a mammal at risk of type 1 diabetes is an individual having a genetic predisposition or an individual in which the pancreas or a part thereof has been surgically removed. Mammals in which the pancreas has been surgically removed include mammals having chronic pancreatitis or mammals having an injury that requires surgical removal of the pancreas.

[0153] Mammals having insulin-dependent type 2 diabetes, or mammals at risk of developing type 2 diabetes, will similarly benefit from the administration of pancreatic endocrine cells obtained by any of the methods of the present invention. Accordingly, provided herein is a method comprising the steps of selecting a mammal having type 2 diabetes, or a mammal at risk of developing type 2 diabetes, and administering to the mammal in a sufficient amount pancreatic endocrine cells obtained by any of the methods of the present invention. Diagnosis is typically based on fasting blood glucose levels, glucose tolerance tests, or blood insulin levels.

[0154] Pancreatic endocrine cells obtained by any of the methods of the present invention are administered in a sufficient amount herein in many ways. The transplant composition is often administered intrahepatically, for example, by percutaneous direct puncture of the liver. The right or left portal branch may be selected for cannulation, and the puncture site is appropriately selected by an interventional radiologist. In some embodiments, several transplants are performed. However, one skilled in the art can readily determine the concentration of cells contained in the transplant composition and recognize the need for a second or subsequent transplant based on hyperglycemia and such clinical signs.

[0155] The methods taught herein for preparing a population of islet cells for transplantation are also combined with treatment. Accordingly, provided herein, for example, is a method of treating diabetes in a mammal comprising the steps of preparing a population of insulin-secreting cells (e.g., a population of islet cells) for transplantation according to any one of the in vitro methods described above, and transplanting the population of cells into the mammal to be treated (i.e., the transplant recipient).

Example

[0156] [Example 1] Pancreatic endocrine precursors (EPs) generated in vitro according to the present invention are obtained through the following steps.

[0157] Pancreatic endoderm cell aggregates generated from hPSCs are cultured in a suitable suspension culture format. The aggregates are washed by sedimentation of the cell aggregates and removal of excess culture medium. Wash medium (MCDB131 medium, Gibco, catalog number 10372019) is added to the cell aggregates and then removed.

[0158] Differentiation into EP is carried out in MCDB131 medium supplemented with GlutaMAX (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 20 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07) and 10 μM ZnSO4 (Merck, catalog number 1088811000). The following compounds: 2 μM of XX (Tocris, catalog number 4489), 1 μM of T3 (Tocris, catalog number 6666), 5 μM of Tiger (Tocris, catalog number 1254), 100 nM of LDN-193189 (Tocris, catalog number 6053), 20 ng / ml of betacellulin (R&D systems, catalog number 261-CE-250), 10 μg / ml of heparin (Merck, catalog number H3393), 3.3 nM of staurosporine (Tocris, catalog number 1258), 0.1 μM of DZNep (Tocris, catalog number 4703), 10 μM of forskolin (Tocris, catalog number 1099), 5 μM of JNKi VIII (Tocris, catalog number 3222), 10 μM of RepSox (Tocris, catalog number 3742) and 6 μM of SB431542 (Tocris, catalog number 1614) are further supplemented to the medium. This protocol is called the standard (STD) protocol. When evaluating the effect of using VEGFR / PDGFR inhibitors, RepSox and SB431542 are excluded from the above protocol and 4 μM of lenvatinib (ABT-869, SelleckChem, catalog number S1003) is added instead.

[0159] The medium is replenished every 48 hours and the differentiation from pancreatic endoderm to endocrine precursors is carried out over 4 days.

[0160] [Example 2] Pancreatic endocrine cells (PECs) generated in vitro according to the present invention are obtained through the following steps.

[0161] Pancreatic endocrine progenitor cell aggregates generated from pancreatic endoderm are cultured in a suitable suspension culture format. The aggregates are washed by sedimentation of the cell aggregates and removal of excess culture medium. Wash medium (MCDB131 medium, Gibco, catalog number 10372019) is added to the cell aggregates and then removed.

[0162] Differentiation into PECs is carried out in MCDB131 medium supplemented with GlutaMAX (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 20 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07), and 10 μM ZnSO4 (Merck, catalog number 1088811000). The following compounds: 1 μM of XX (Tocris, catalog number 4489), 1 μM of T3 (Tocris, catalog number 6666), 5 μM of Tiger (Tocris, catalog number 1254), 100 nM of LDN-193189 (Tocris, catalog number 6053), 10 μg / ml of heparin (Merck, catalog number H3393), 3.3 nM of staurosporine (Tocris, catalog number 1258), 0.1 μM of DZNep (Tocris, catalog number 4703), and 10 μM of RepSox (Tocris, catalog number 3742) are further supplemented to the medium. This protocol is called the standard (STD) protocol. When evaluating the effect of using a VEGFR / PDGFR inhibitor, RepSox is excluded from the above protocol and 4 μM of lenvatinib (ABT-869, SelleckChem, catalog number S1003) is added instead.

[0163] The medium is replenished every 48 hours, and the differentiation from endocrine precursors to pancreatic endocrine cells is carried out over 3 days. At this stage, the cells can be cryopreserved or further differentiated using the following medium composition.

[0164] Differentiate for 2 days in MCDB131 medium supplemented with GlutaMAX (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07) and 10 μM ZnSO4 (Merck, catalog number 1088811000). Further supplement the medium with the following compounds: 1 μM of XX (Tocris, catalog number 4489), 1 μM of T3 (Tocris, catalog number 6666), 5 μM of Tiger (Tocris, catalog number 1254), 10 μg / ml of heparin (Merck, catalog number H3393), 0.1 μM of DZNep (Tocris, catalog number 4703), and further supplement with 10 μM of RepSox (Tocris, catalog number 3742) (STD) or 4 μM of lenvatinib (ABT-869, SelleckChem, catalog number S1003).

[0165] Differentiate for 2 days in MCDB131 medium supplemented with GlutaMAX (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07), and further supplemented with 10 μM RepSox (Tocris, catalog number 3742) (STD) or 4 μM lenvatinib (ABT-869, SelleckChem, catalog number S1003).

[0166] Subsequently, pancreatic endocrine cells may be maintained for several days to several weeks in MCDB131 medium supplemented with GlutaMAX (Gibco, catalog number 35050038), 0.05% human serum albumin (Origin, catalog number ART-3003), 2.5 mM glucose (Sigma-Aldrich, catalog number G8769), 14.64 mM NaHCO3 (Gibco, catalog number 25080094), ITS-X (Gibco, catalog number 51500056), and 0.25 mM ascorbic acid (Fisher Scientific, catalog number 0937-07), and replenished every 48 hours.

[0167] [Example 3] - Cryopreservation Pancreatic endocrine cell aggregates obtained in vitro according to the present invention are subjected to cryopreservation as described in International Publication No. WO2019 / 048690. Briefly, the obtained cells are resuspended in a cryopreservation medium and stored by sequentially reducing the temperature to less than -80°C.

[0168] Thawing of cryopreserved single cells To return the cells to culture, the cells are quickly brought to 37°C and washed once in pre-warmed RPMI 1640 medium (Gibco#61870-044) supplemented with 12% KOSR (Gibco#10828-0280). After counting, the cells are resuspended in stage-specific medium supplemented with 50 μg / mL DNasel (Sigma#11284932001) and 10 μM Rocki (Sigma#Y27632-Y0503).

[0169] Re-aggregation of cells obtained after thawing Cells obtained after thawing can be re-aggregated in a conical flask with a reduced volume having a density of 0.5 - 2 million viable cells / mL. Re-aggregation is carried out at 37°C with horizontal shaking at 70 rpm for 2 days, followed by a medium change.

[0170] After cryopreservation, cells with a survival rate in the range of 60% - 90% are recovered. The glucose-responsive insulin secretion phenotype is improved upon re-aggregation of the cells.

Claims

**Claim 1** An in vitro method for generating a population of pancreatic endocrine (PEC) cells, comprising: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine precursors, said differentiating step comprising treating said pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor; ii) differentiating said pancreatic endocrine precursor (EP) cells into pancreatic endocrine (PEC) cells by treating said pancreatic endocrine precursor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor. **Claim 2** The method of claim 1, wherein said inhibitor of a VEGF receptor and / or a PDGF receptor is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea. **Claim 3** The method of claim 1 or 2, wherein step i) does not require a TGFb receptor kinase inhibitor. **Claim 4** The method according to any one of claims 1 to 3, wherein step ii) does not require a TGFb receptor kinase inhibitor. **Claim 5** The method according to any one of claims 1 to 4, wherein the concentration of the inhibitor of the vascular endothelial growth factor (VEGF) and / or platelet-derived growth factor (PDGF) receptor used in step ii) is in the range of 5 nM to about 15 μM, such as 5 nM to about 12 μM, 1 μM to 10 μM, about 1 μM to about 5 μM, or about 3 μM to about 4 μM. **Claim 6** The method according to any one of claims 2 to 5, wherein the concentration of the inhibitor of the vascular endothelial growth factor (VEGF) receptor and / or platelet-derived growth factor (PDGF) receptor used in step i) is in the range of 5 nM to about 15 μM, such as 5 nM to about 12 μM, 1 μM to about 10 μM, about 1 μM to about 5 μM, or about 3 μM to about 4 μM (Figure 2C, 2D). **Claim 7** The method according to any one of claims 1 to 6, wherein the population of pancreatic endocrine (PEC) cells obtained after step ii) comprises at least 20%, such as at least 25%, such as at least 28%, or at least 30% beta-like cells, based on the total number of cells obtained after step ii), and said beta-like cells are double positive (ISL1+NKX6.1+) for ISL1 and NKX6.

1. **Claim 8** The method according to any one of claims 1 to 7, wherein the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1%, such as at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, or at least 20% more beta-like cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of the inhibitor of VEGF and / or PDGF in step i) and step ii), and the beta-like cells are double positive (ISL1+NKX6.1+) for ISL1 and NKX6.

1.

9. The method according to any one of claims 1 to 8, wherein the population of pancreatic endocrine (PEC) cells obtained after step ii) contains up to 35%, such as up to 30% or up to 27% enterochromaffin cells, and the enterochromaffin cells are negative for ISL1 and positive for NKX6.1 (ISL1−NKX6.1+).

10. The method according to any one of claims 1 to 9, wherein the cells obtained after step ii) contain at least 78%, such as at least 80% pancreatic endocrine cells, and the endocrine cells are CHGA+.

11. The method according to any one of claims 1 to 10, wherein the population of pancreatic endocrine (PEC) cells obtained after step ii) contains at least 1%, such as at least 2%, at least 3%, at least 4%, or at least 5% more pancreatic endocrine (PEC) cells compared to the population of pancreatic endocrine (PEC) cells obtained by using a TGFβ receptor kinase inhibitor instead of the inhibitor of VEGF and / or PDGF in step i) and step ii), and the pancreatic endocrine (PEC) cells are CHGA+.

12. An in vitro method for generating a population of pancreatic endocrine progenitor (EP) cells, comprising: i) differentiating the population of PE cells into pancreatic endocrine precursors by treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor.

13. An in vitro method for generating a population of pancreatic endocrine (PEC) cells, comprising: ii) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, a method. **Claim 14** An in vitro method for generating a population of pancreatic endocrine (PEC) cells, comprising: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine precursors by treating the pancreatic endoderm (PE) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor; and ii) differentiating pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells, a method. **Claim 15** An in vitro method for generating a population of pancreatic endocrine (PEC) cells, comprising: i) differentiating a population of pancreatic endoderm (PE) cells into pancreatic endocrine precursors; and ii) differentiating the pancreatic endocrine progenitor (EP) cells into pancreatic endocrine (PEC) cells by treating the pancreatic endocrine progenitor (EP) cells with an inhibitor of a vascular endothelial growth factor (VEGF) receptor and / or a platelet-derived growth factor (PDGF) receptor, a method.

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