Method for inducing differentiation into pancreatic alpha cells

Abstract

A method for inducing differentiation into pancreatic α cells, said method comprising: (a) a step for culturing endodermal cells, which are obtained by induced differentiation from pluripotent stem cells, in the presence of a bone morphogenetic protein (BMP) signal inhibitor and retinoic acid or a retinoic acid analog to thereby induce the differentiation thereof into primitive gut tube (PGT) cells; (b) a step for culturing the PGT cells and thus inducing the differentiation thereof into pancreatic endocrine precursor (EP) cells; and (c) a step for culturing the EP cells and thus inducing the differentiation thereof into pancreatic α cells, wherein the aforesaid steps (b) and (c) are carried out in the absence of ascorbic acid.

Description

[Technical Field]

[0001] The present invention relates to a method for inducing differentiation into pancreatic α cells. This application claims priority based on Japanese Patent Application No. 2020-52420, filed on March 24, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Regenerative medicine is expected to play a major role in the development of alternatives to organ transplants, which are currently facing a shortage of donors, and new treatments for intractable diseases. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are pluripotent and have the ability to proliferate indefinitely, making them promising cell sources for the preparation of cells needed in regenerative medicine. To commercialize regenerative medicine using these pluripotent stem cells, it is necessary to establish a technology that can efficiently induce differentiation of pluripotent stem cells into the desired somatic cells, and a variety of differentiation induction methods have been reported.

[0003] Pancreatic islets (islets of Langerhans) are endocrine tissues present inside the pancreas that secrete insulin and glucagon, primarily regulating blood glucose. Pancreatic islets are composed of various cells, including α cells, β cells, and δ cells. Of these, pancreatic β cells are useful in cell therapy for diabetes, and therefore methods for efficiently producing pancreatic β cells from pluripotent stem cells have been investigated. Patent Document 1 describes a method for producing pancreatic β cells from endodermal cells induced to differentiate from pluripotent stem cells.

[0004] In recent years, it has been reported that abnormalities occur not only in pancreatic beta cells but also in pancreatic alpha cells, which promote gluconeogenesis in response to hypoglycemia, in the pancreatic islets of diabetic patients. In such patients, pancreatic alpha cells remain activated even in hyperglycemic conditions and continue to secrete glucagon, which increases blood glucose levels. Thus, glucagon secreted from pancreatic alpha cells is known to play an important role in regulating blood glucose levels in the body, and there is great hope for elucidating the mechanism of glucagon secretion in order to develop new diabetes treatments.

[0005] However, there is a chronic shortage of pancreatic alpha cells for research, and securing new sources of pancreatic alpha cells is necessary to accelerate this research. Pluripotent stem cells, such as iPS cells and ES cells, are capable of differentiating into almost all cell types that make up the body, and are therefore expected to serve as new cell sources for pancreatic alpha cells. For practical application of pluripotent stem cell-derived pancreatic alpha cells, a technique for efficiently inducing the differentiation of pluripotent stem cells into functional pancreatic alpha cells is required. Non-Patent Document 1 reports a method for inducing the differentiation of human embryonic stem cells into pancreatic alpha cells with glucagon secretion function, but few techniques have been reported to date for efficiently inducing the differentiation of pluripotent stem cells into functional pancreatic alpha cells, and no pancreatic alpha cell line with sufficient functionality has been established. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 208788 [Non-patent literature]

[0007] [Non-Patent Document 1] Alireza Rezania et al.,Diabetes,2011 Jan;60(1):239-247 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, from the perspective of elucidating the mechanism of glucagon secretion from pancreatic alpha cells and constructing development tools for new diabetes treatments, it is necessary to provide a technology that can efficiently induce differentiation of pluripotent stem cells into functional pancreatic alpha cells.

[0009] Therefore, an object of the present invention is to provide a method for efficiently inducing differentiation of pluripotent stem cells into functional pancreatic α cells. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that endoderm cells induced to differentiate from pluripotent stem cells can be induced to differentiate into pancreatic alpha cells by culturing them under specific conditions, and that the resulting pancreatic alpha cells have the function of secreting glucagon. The present invention was completed based on these findings.

[0011] That is, according to the present specification, the following inventions are provided. [1] (a) A step of inducing differentiation of endodermal cells, which have been induced to differentiate from pluripotent stem cells, into primitive gut cells (PGT) by culturing them in the presence of a bone morphogenetic protein (BMP) signal inhibitor and retinoic acid or a retinoic acid analogue: (b) culturing the primitive intestinal cells (PGT) to induce differentiation into pancreatic endocrine precursor cells (EP); and (c) culturing the pancreatic endocrine precursor cells (EPs) to induce differentiation into pancreatic α cells: Including, A method for inducing differentiation into pancreatic α cells, wherein the steps (b) and (c) are carried out in the absence of ascorbic acid. [2] The method for inducing differentiation into pancreatic alpha cells according to [1], wherein the step (c) is carried out under oxygen supply conditions. [3] The method for inducing differentiation into pancreatic alpha cells described in [2], wherein the oxygen supply conditions are controlled so that the dissolved oxygen concentration in the culture medium is 20 to 50%, when the saturated dissolved oxygen concentration in the culture medium at 37°C under 1 atm is taken as 100%. [4] The method for inducing differentiation into pancreatic α cells according to any one of [1] to [3], wherein the step (a) is carried out in the presence of a ROCK signal inhibitor. [5] The method for inducing differentiation into pancreatic alpha cells according to any one of [1] to [4], wherein the endodermal cells induced to differentiate from the pluripotent stem cells are endodermal cells induced to differentiate by culturing the pluripotent stem cells in a medium containing a TGFβ superfamily signal activator, followed by culturing the pluripotent stem cells in a medium to which FGF2 and BMP4 are not added. [6] The step (b) (b1) inducing differentiation of the primitive gut cells (PGT) into posterior foregut cells (PFG) by culturing the PGT in the presence of a protein kinase C (PKC) activator: (b2) inducing differentiation of the posterior foregut cells (PFG) into pancreatic progenitor cells (PP) by culturing the PFG in the presence of retinoic acid or an analog thereof; and (b3) inducing differentiation of the pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP) by culturing the pancreatic progenitor cells (PP) in the presence of a Notch signal inhibitor and a ROCK signal inhibitor: The method for inducing differentiation into pancreatic α cells according to any one of [1] to [5], comprising: [7] The method for inducing differentiation into pancreatic alpha cells according to any one of [1] to [6], wherein the step (c) comprises a step of inducing differentiation into pancreatic alpha cells by culturing the pancreatic endocrine precursor cells (EP) in the presence of an insulin receptor signal activator, transferrin and selenious acid. [8] The method for inducing differentiation into pancreatic α cells according to any one of [1] to [7], wherein the culture is performed in suspension culture. [9] The method for inducing differentiation into pancreatic α cells according to any one of [2] to [8], wherein the oxygen supply is carried out under agitated culture. [Effects of the Invention]

[0012] The method for inducing differentiation of pancreatic alpha cells according to the present invention makes it possible to efficiently induce differentiation of pluripotent stem cells into functional pancreatic alpha cells. Furthermore, the pancreatic alpha cells produced according to the present invention are useful as a tool for elucidating the mechanism of glucagon secretion and for developing novel therapeutic drugs for diabetes. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows the results of analyzing the expression of the INS gene, the GCG gene, the NKX6.1 gene, and the PDX1 gene in cells obtained by the methods of Example 1 and Comparative Example 1. [Figure 2]1 shows the results of measuring the amount of glucagon secreted from primitive gut cells (PGT) that were induced to differentiate from human iPS cells in the presence or absence of ascorbic acid. [Figure 3] 1 shows the results of measuring the amount of glucagon secreted from cells obtained by the method of Example 2 and cells obtained by the method of Comparative Example 1. [Figure 4] 1 shows the results of analyzing the expression of the GCG gene, the ARX gene, the GC gene, the INS gene, and the NKX6.1 gene in cells obtained by the method of Example 2 and cells obtained by the method of Comparative Example 1. [Figure 5] This shows the oxygen concentration in the culture medium when cultured under oxygen concentration control #3-1 in the process of inducing differentiation of pancreatic endocrine precursor cells (EP) into pancreatic α cells by culturing them. [Figure 6] 1 shows the results of analyzing the expression of the GCG gene in cells induced to differentiate by culturing pancreatic endocrine precursor cells (EP) under controlled oxygen concentration (#3-1 to #3-3) or uncontrolled oxygen concentration (Comparative Example 1). [Figure 7] The figures show the results of analyzing the expression of the INS gene in cells induced to differentiate by culturing pancreatic endocrine precursor cells (EP) under controlled oxygen concentration (#3-1 to #3-3) or uncontrolled oxygen concentration (Comparative Example 1). DETAILED DESCRIPTION OF THE INVENTION

[0014] The following describes in detail the embodiments of the present invention. However, the following description is intended to facilitate understanding of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiments described below are also included in the scope of the present invention.

[0015] [Terminology] In the present invention, "in the absence of an inhibitor" means "in a medium to which the inhibitor is not added."

[0016] With respect to the medium of the present invention, the term "unsupplemented" refers to the absence of exogenously added factors such as proteins, peptides, and compounds identified as not being added in the culture or conditioned medium. If factors such as proteins, peptides, and compounds identified as not being added in the culture or conditioned medium are introduced through continuous culturing operations, they are adjusted to less than 1% (volume / volume), less than 0.5% (volume / volume), less than 0.1% (volume / volume), less than 0.05% (volume / volume), less than 0.01% (volume / volume), or less than 0.001% (volume / volume).

[0017] With respect to gene expression level, the term "improved" refers to an increase in gene expression compared to the expression level of a particular gene in a comparison cell population, and includes an increase in gene expression compared to the comparison cell population by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 2.1-fold or more, 2.2-fold or more, 2.3-fold or more, 2.4-fold or more, 2.5-fold or more, 2.6-fold or more, 2.7-fold or more. Above, 2.8x or more, 2.9x or more, 3.0x or more, 3.1x or more, 3.2x or more, 3.3x or more, 3.4x or more, 3.5x or more, 3.6x or more, 3.7x or more, 3.8x or more, 3.9x or more, 4.0x or more, 4.1x or more, 4.2 4.3x or more, 4.4x or more, 4.5x or more, 4.6x or more, 4.7x or more, 4.8x or more, 4.9x or more, 5.0x or more, 5.1x or more, 5.2x or more, 5.3x or more, 5.4x or more, 5.5x or more, 5.6x or more, 5 .7x or more, 5.8x or more, 5.9x or more, 6.0x or more, 6.1x or more, 6.2x or more, 6.3x or more, 6.4x or more, 6.5x or more, 6.6x or more, 6.7x or more, 6.8x or more, 6.9x or more, 7.0x or more, 7.1x or more , 7.2x or more, 7.3x or more, 7.4x or more, 7.5x or more, 7.6x or more, 7.7x or more, 7.8x or more, 7.9x or more, 8.0x or more, 8.1x or more, 8.2x or more, 8.3x or more, 8.4x or more, 8.5x or more, 8.6x or more, 8.7 times or more, 8.8 times or more, 8.9 times or more, 9.0 times or more, 9.1 times or more, 9.2 times or more, 9.3 times or more, 9.4 times or more, 9.5 times or more, 9.6 times or more, 9.7 times or more, 9.8 times or more, 9.9 times or more, 10.0 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 250 times or more, 500 times or more, 750 times or more, 1000 times or more, 5000 times or more, 10000 times or more.

[0018] With respect to gene expression levels, the term "decreased" refers to a decrease in gene expression compared to the expression level of a particular gene in a comparative cell population, and includes a decrease in gene expression compared to the comparative cell population of 1.1-fold or less, 1.2-fold or less, 1.3-fold or less, 1.4-fold or less, 1.5-fold or less, 1.6-fold or less, 1.7-fold or less, 1.8-fold or less, 1.9-fold or less, 2.0-fold or less, 2.1-fold or less, 2.2-fold or less, 2.3-fold or less, 2.4-fold or less, 2.5-fold or less, 2.6-fold or less, 2.7-fold or more. Lower, 2.8x or less, 2.9x or less, 3.0x or less, 3.1x or less, 3.2x or less, 3.3x or less, 3.4x or less, 3.5x or less, 3.6x or less, 3.7x or less, 3.8x or less, 3.9x or less, 4.0x or less, 4.1x or less, 4.2 less than 4.3 times, less than 4.4 times, less than 4.5 times, less than 4.6 times, less than 4.7 times, less than 4.8 times, less than 4.9 times, less than 5.0 times, less than 5.1 times, less than 5.2 times, less than 5.3 times, less than 5.4 times, less than 5.5 times, less than 5.6 times, 5 .7x or less, 5.8x or less, 5.9x or less, 6.0x or less, 6.1x or less, 6.2x or less, 6.3x or less, 6.4x or less, 6.5x or less, 6.6x or less, 6.7x or less, 6.8x or less, 6.9x or less, 7.0x or less, 7.1x or less , 7.2x or less, 7.3x or less, 7.4x or less, 7.5x or less, 7.6x or less, 7.7x or less, 7.8x or less, 7.9x or less, 8.0x or less, 8.1x or less, 8.2x or less, 8.3x or less, 8.4x or less, 8.5x or less, 8.6x The following are the rates: 8.7 times or less, 8.8 times or less, 8.9 times or less, 9.0 times or less, 9.1 times or less, 9.2 times or less, 9.3 times or less, 9.4 times or less, 9.5 times or less, 9.6 times or less, 9.7 times or less, 9.8 times or less, 9.9 times or less, 10.0 times or less, 20 times or less, 30 times or less, 40 times or less, 50 times or less, 60 times or less, 70 times or less, 80 times or less, 90 times or less, 100 times or less, 250 times or less, 500 times or less, 750 times or less, 1000 times or less, 5000 times or less, and 10000 times or less.

[0019] <Aggregation> With respect to the aggregates of the present invention, the terms "clump," "cluster," or "spheroid" may be used interchangeably and generally refer to a collection of cells that have not been dissociated into single cells.

[0020] <Pluripotent stem cells> In the present invention, pluripotent stem cells refer to cells with the multipotency (pluripotency) to differentiate into all or multiple types of cells that constitute a living organism, and can continue to proliferate indefinitely while maintaining pluripotency when cultured in vitro under appropriate conditions. Specific examples include embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (EG cells: Proc Natl Acad Sci U.S.A. 1998, 95:13726-31), pluripotent stem cells derived from testes (GS cells: Nature. 2008, 456:344-9), induced pluripotent stem cells (iPS cells), and somatic stem cells (tissue stem cells). Pluripotent stem cells are preferably iPS cells or ES cells, more preferably iPS cells. The term "embryonic" refers to embryos derived by somatic cell nuclear transfer as well as embryos derived by gamete fusion.

[0021] ES cells can be derived from any warm-blooded animal, preferably a mammal. Mammals include, for example, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. Preferably, cells derived from humans can be used.

[0022] Specific examples of ES cells include mammalian ES cells established by culturing preimplantation early embryos, ES cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei, and ES cells obtained by modifying the genes on the chromosomes of these ES cells using genetic engineering techniques. Each type of ES cell can be prepared according to methods commonly used in the field or known literature. Mouse ES cells were established in 1981 by Evans et al. (Evans et al., 1981, Nature 292:154-6) and Martin et al. (Martin GR. et al., 1981, Proc Natl Acad Sci 78:7634-8). Human ES cells were established by Thomson et al. (Science, 1998, 282:1145-7) in 1998 and are available from the WiCell Research Institute (WiCell Research Institute, website: http: / / www.wicell.org / , Madison, Wisconsin, USA), the National Institutes of Health, Kyoto University, the National Center for Child Health and Development, and can also be purchased from, for example, Cellartis (Website: http: / / www.cellartis.com / , Sweden).

[0023] Induced pluripotent stem cells (iPS cells) are cells with pluripotency that can be obtained by reprogramming somatic cells. Several groups have successfully generated iPS cells, including the group of Professor Shinya Yamanaka et al. at Kyoto University, the group of Rudolf Jaenisch et al. at Massachusetts Institute of Technology, the group of James Thomson et al. at the University of Wisconsin, and the group of Konrad Hochedlinger et al. at Harvard University. For example, International Publication WO 2007 / 069666 describes somatic nuclear reprogramming factors, including gene products of the Oct family genes, Klf family genes, and Myc family genes, as well as somatic nuclear reprogramming factors, including gene products of the Oct family genes, Klf family genes, Sox family genes, and Myc family genes. It also describes a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, including the step of contacting somatic cells with the nuclear reprogramming factors.

[0024] The type of somatic cells used to produce iPS cells is not particularly limited, and any somatic cells can be used. In other words, somatic cells encompass all cells other than germ cells that make up a living organism, and may be differentiated somatic cells or undifferentiated stem cells. The somatic cells may be derived from any of mammals, birds, fish, reptiles, and amphibians, but are not particularly limited thereto. They are preferably mammals (e.g., rodents such as mice, or primates such as humans), and are particularly preferably mice or humans. Furthermore, when human somatic cells are used, they may be from a fetus, a newborn, or an adult. Specific examples of somatic cells include fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, white blood cells (B cells, T cells, etc.), bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiac myocytes), hepatic parenchymal cells, non-hepatic parenchymal cells, adipocytes, osteoblasts, cells that constitute periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells that constitute the kidney, eye, and ear.

[0025] iPS cells are stem cells that have the ability to self-renew over a long period of time under specific culture conditions (e.g., conditions for culturing ES cells) and have the pluripotency to differentiate into any of ectodermal, mesodermal, or endodermal cells under specific differentiation-inducing conditions. iPS cells may also be stem cells that have the ability to form teratomas when transplanted into test animals such as mice.

[0026] To produce iPS cells from somatic cells, at least one reprogramming gene is first introduced into the somatic cells. Reprogramming genes encode reprogramming factors that reprogram somatic cells to iPS cells. Specific examples of combinations of reprogramming genes include, but are not limited to, the following: (i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

[0027] In addition to the above, methods that further reduce the number of introduced genes (Nature. 2008 Jul 31; 454(7204): 646-50), methods that use low molecular weight compounds (Cell Stem Cell. 2009 Jan 9; 4(1): 16-9, Cell Stem Cell. 2009 Nov 6; 5(5): 491-503), and methods that use transcription factor proteins instead of genes (Cell Stem Cell. 2009 May 8; 4(5): 381-4) have also been reported, and iPS cells produced by any of these methods may be used.

[0028] The manner in which the reprogramming factors are introduced into cells is not particularly limited, and examples include gene transfer using a plasmid, transfer of synthetic RNA, and direct transfer as a protein. iPS cells produced by a method using microRNA, RNA, low-molecular-weight compounds, etc. may also be used. Pluripotent stem cells, including ES cells and iPS cells, may be commercially available or provided, or may be newly produced.

[0029] Examples of iPS cells include the 253G1 strain, 253G4 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1231A3 strain, 1383D2 strain, 1383D6 strain, iPS-TIG120-3f7 strain, iPS-TIG120-4f1 strain, and iPS-TI Examples of strains that can be used include the G114-4f1 strain, RPChiPS771-2 strain, 15M63 strain, 15M66 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC 38-2 strain, MSC-iPSC1 strain, and BJ-iPSC1 strain.

[0030] Examples of ES cells that can be used include KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SEES1, SEES2, SEES3, HUES8, CyT49, H1, H9, and HS-181 strains. Newly generated clinical-grade iPS cells or ES cells may also be used.

[0031] <Endodermal cells> Endodermal cells have the ability to differentiate into tissues of organs such as the digestive tract, lung, thyroid gland, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most of the urethra, and part of the ureter), and are sometimes referred to as definitive endoderm (DE). Differentiation of pluripotent stem cells into endodermal cells can be confirmed by measuring the expression levels of genes specific to endodermal cells. Examples of genes specific to endodermal cells include SOX17, FOXA2, CXCR4, AFP, GATA4, and EOMES. Note that, throughout this specification, endodermal cells may be referred to as definitive endoderm.

[0032] <Primitive intestinal cells> Primitive gut cells form the foregut, midgut, and hindgut. The midgut is connected to the yolk sac, and the extraembryonic allantois branches from the hindgut. The foregut also forms the pharynx of the respiratory system. Some organs differentiate directly from the gut, such as the stomach and intestines, while others bud from the gut, such as the liver, gallbladder, pancreas, and spleen (lymphatic organ). Differentiation of endodermal cells into primitive gut cells can be confirmed by measuring the expression levels of genes specific to primitive gut cells. Examples of genes specific to primitive gut cells include HNF-1β and HNF-4α.

[0033] <Posterior foregut cells (PFG)> The differentiation of primitive gut cells into posterior foregut cells can be confirmed by measuring the expression levels of genes specific to posterior foregut cells, such as PDX1 and HNF6.

[0034] <Pancreatic progenitor cells (PP)> Pancreatic progenitor cells are cells differentiated from posterior foregut cells and can differentiate into exocrine and endocrine cells of the pancreas. Differentiation of posterior foregut cells into pancreatic progenitor cells can be confirmed by measuring the expression levels of genes specific to pancreatic progenitor cells. Examples of genes specific to pancreatic progenitor cells include PDX1 and NKX6.1.

[0035] <Pancreatic endocrine progenitor cells (EP)> Pancreatic endocrine precursor cells are cells differentiated from pancreatic precursor cells and can differentiate into pancreatic endocrine cells (α cells, β cells, δ cells, ε cells, PP cells, etc.). Differentiation into pancreatic endocrine precursor cells can be confirmed by measuring the expression levels of genes specific to pancreatic endocrine precursor cells. Examples of genes specific to pancreatic endocrine precursor cells include PDX1, NKX6.1, NeuroG3, and NeuroD1.

[0036] <Pancreatic alpha cells> Pancreatic alpha cells are cells differentiated from pancreatic endocrine precursor cells and secrete glucagon. Differentiation of pancreatic endocrine precursor cells into pancreatic alpha cells can be confirmed by measuring the expression levels of genes specific to pancreatic alpha cells. Examples of genes specific to pancreatic alpha cells include glucagon (GCG), ARK, IRX2, GC, TM4SF4, TTR, and CRYBA2.

[0037] <Pancreatic beta cells> Pancreatic beta cells are cells differentiated from pancreatic endocrine precursor cells and secrete insulin. Differentiation of pancreatic endocrine precursor cells into pancreatic beta cells can be confirmed by measuring the expression levels of genes specific to pancreatic beta cells. Examples of genes specific to pancreatic beta cells include insulin, NKX6.1, MAFA, and PDX1.

[0038] <Signals and Factors> (Bone morphogenetic protein (BMP) signal inhibitor) Bone morphogenetic protein (BMP) signaling, mediated by bone morphogenetic protein (BMP) ligands, plays diverse roles in vertebrates. During embryogenesis, the dorsal-ventral axis is established by a BMP signaling gradient formed by the coordinated expression of ligands, receptors, co-receptors, and soluble antagonists. BMPs are key regulators of gastrulation, mesoderm induction, organogenesis, and endochondral bone formation, and control the fate of pluripotent stem cell populations.

[0039] BMP receptors consist of a complex of type I receptors (activin receptor-like kinases; ALK-1, ALK-2, ALK-3, or ALK-6) and type II receptors (ActRII, ActRIIB, or BMPRII). Activated type I receptor kinases phosphorylate two serine residues at the C-terminus of R-Smad (receptor-regulated Smad) proteins. R-Smads (Smad1, Smad5, and Smad8) are phosphorylated upon binding of the ligand (BMP) to the receptor and are called BR-Smads (BMP R-Smads). Two phosphorylated R-Smad molecules form a heterotrimer with Smad4, translocate into the nucleus, and regulate the transcription of target genes.

[0040] The bone morphogenetic protein (BMP) signal inhibitor is not particularly limited as long as it is a substance that inhibits BMP signaling initiated by binding of a ligand (such as BMP-4) to a receptor, but is preferably a substance that inhibits at least one of ALK-1, ALK-2, ALK-3, and ALK-6. In addition, a substance that prevents the binding of a ligand to a receptor (such as an antagonist antibody) can be used as a BMP signal inhibitor.

[0041] Bone morphogenetic protein (BMP) signal inhibitors are not particularly limited, but inhibitors that act on type I receptors (ALK-1, ALK-2, ALK-3, or ALK-6) can effectively inhibit BMP signals, and examples include dorsomorphin, LDN193189, LDN-214117, LDN-212854, K02288, and ML347.

[0042] (retinoic acid) Retinoic acid is a carboxylic acid derivative of vitamin A and exists in several stereoisomers, including all-trans retinoic acid (tretinoin), 9-cis retinoic acid (alitretinoin), and 13-cis retinoic acid (isotretinoin). Retinoic acid is a natural ligand for the retinoic acid receptor (RAR), a nuclear receptor, and plays a key role in mediating the physiological activities of retinoids and carotenoids in vivo. RAR forms a heterodimer with the retinoid X receptor (RXR; its ligand is 9-cis retinoic acid) and functions as a ligand-inducible transcription factor, positively or negatively regulating the expression of specific target genes by binding to their promoters. Even compounds with chemical structures completely dissimilar to vitamin A, including synthetic compounds that exhibit very high binding affinity to these specific receptors, are called retinoids.

[0043] (Retinoic acid analogue) Retinoic acid (RA) is known to promote cell differentiation, cell division cycle arrest, and apoptosis, and is also used to induce differentiation of pluripotent stem cells. Retinoic acid analogs include, but are not limited to, substances that activate nuclear receptors (retinoic acid receptors (PAR) and retinoid X receptors (RXR)). Examples include EC23, EC19, AC 261066, AC 55649, Adapalene, AM 580, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch 55, isotretinoin, tazarotene, and TTNPB. EC23 is a stable compound that is less susceptible to photodegradation than retinoic acid, making it suitable for use.

[0044] (Ascorbic Acid) Ascorbic acid, also known as vitamin C, is a water-soluble vitamin and is an organic compound with a lactone structure. Ascorbic acid is an optically active compound and exists in both L and D forms, with the L form being preferred. The L form is the form commonly known as vitamin C. Ascorbic acid has strong reducing power and participates in various redox reactions in the body. It is absorbed through the upper small intestine and transported via the portal vein to the liver, where it is partially metabolized. Most of it is absorbed into tissues such as the adrenal glands and pituitary gland. Ascorbic acid is essential for inhibiting peroxide production, detoxifying xenobiotics, metabolizing tyrosine, and promoting the absorption of nonheme iron. It also participates in hydroxylation in reactions such as collagen, catecholamine, and carnitine biosynthesis and cholesterol metabolism.

[0045] (ROCK signal inhibitor) ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase) converts myosin light chains into their active conformation through phosphorylation of myosin light chains and myosin light chain phosphatases. Furthermore, ROCK inactivates the actin depolymerizing factor cofilin through phosphorylation of LIM kinase, thereby suppressing actin depolymerization. ROCK also phosphorylates numerous substrates and is involved in various biological functions, such as cell motility, cell polarity, cell adhesion, cell division, apoptosis, and transcriptional regulation. Examples of ROCK signal inhibitors include Y27632, Thiazovivin, Fasudil (HA-1077) HCl, GSK429286A, RKI-1447, GSK180736A (GSK180736), Hydroxyfasudil (HA-1100) HCl, Y-39983 HCl, Netarsudil (AR-13324) 2HCl, GSK269962A HCl, Ripasudil (K-115) hydrochloride dihydrate, KD025 (SLx-2119), and AT13148.

[0046] (TGFβ superfamily signal activator) The TGF-β superfamily of signaling pathways plays crucial roles in the regulation of cell proliferation, differentiation, and development in a wide range of biological systems. Generally, signaling is initiated by ligand-induced oligomerization of serine / threonine receptor kinases and phosphorylation of intracellular signaling molecules, such as Smad1 / 5 / 8 in the bone morphogenetic protein (BMP) pathway, and Smad2 / 3 in the TGF-β / activin and NODAL / activin pathways. Phosphorylation of the carboxyl terminus of Smads by activated receptors results in their partnering with a common signal transducer, Smad4, and their nuclear translocation. Activated Smads are known to regulate various biological effects by partnering with transcription factors, thereby regulating transcription in a cell-state-specific manner.

[0047] Genes involved in the TGFβ superfamily signaling pathway include the Activin A gene, BMP2 gene, BMP3 gene, BMP4 gene, BMP5 gene, BMP6 gene, BMP7 gene, BMP8 gene, BMP13 gene, GDF2 (Growth differentiation factor 2) gene, GDF3 gene, GDF5 gene, GDF6 gene, GDF7 gene, GDF8 gene, GDF11 gene, TGF-β1 gene, TGF-β2 gene, TGF-β3 gene, AMH (anti-mullerian hormone) gene, paired like homeodomain 2 (PITX2) gene, and NODAL gene.

[0048] The TGFβ superfamily signal activator is not particularly limited as long as it activates signals in the bone morphogenetic protein (BMP) pathway, the TGFβ / activin pathway, and / or the NODAL / activin pathway, and examples thereof include activin A, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP13, GDF2, GDF5, GDF6, GDF7, GDF8, GDF11, TGF-β1, TGF-β2, TGF-β3, AMH, PITX2, and / or NODAL. In particular, substances that activate signals in the TGFβ / activin pathway are preferably used, and specifically, it is preferable to use at least one selected from the group consisting of activin A and BMP4, and it is particularly preferable to use activin A and BMP4.

[0049] (Protein kinase C (PKC) activator) PKC is a protein involved in the control of many cellular functions, such as cell proliferation, cell death, gene transcription and translation, cell morphology, and cell-cell contact. PKC is a type of protein kinase that phosphorylates the hydroxyl groups of serine and threonine residues in substrate proteins, and there are at least 10 isozymes. Isozymes are classified into three subfamilies: classical, novel, and atypical, depending on their structure, activation mechanism, and physiological activity. Classical PKC isozymes (α, βI, βII, γ) require diacylglycerol (DAG) and Ca for activation. 2+ DAG is produced together with inositol 1,4,5-triphosphate (IP3) by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). IP3 diffuses within the cell and binds to IP3-sensitive Ca2+ receptors on the endoplasmic reticulum. 2+ Upon binding to the channel, Ca 2+ ions are released into the cytoplasm. 2+ When ions bind to PKC, PKC moves to the cell membrane. There, PKC interacts with DAG via the C1 domain. PKC then undergoes a conformational change that separates the regulatory region from its catalytic domain, resulting in its activated form. Novel PKC isozymes (δ, ε, θ, η) are activated only by DAG. This is because the affinity of the C1 domain of novel PKC isozymes for DAG is much higher than that of conventional PKC isozymes. Atypical PKC isozymes (ζ, Mζ, ι / λ) require both DAG and Ca for activation. 2+ It does not require a specific ATP-dependent pathway and is activated by various lipid metabolite second messengers (Khalil, 2010; Wu-Zhang and Newton, 2013; Mochly-Rosen et al., 2012).

[0050] The PKC activator may be any substance that activates at least one of the above-mentioned PKC isozymes. Examples of PKC activators that can be used include, but are not limited to, indolactam V (ILV), Okadaic Acid, Phorbol-12-Myristate-13-Acetate (PMA), Bryostatin 1, 1alpha,25-Dihydroxyvitamin D3, Prostratin, 1,2-Dioctanoyl-sn-glycerol, 1-Oleoyl-2-acetyl-sn-glycerol (OAG), Oleic Acid, Ingenol 3-angelate, DCP-LA, PIP2, Phorbol-12,13-dibutyrate, 8(S)-HETE, and derivatives thereof.

[0051] (Notch signal inhibitor) Notch signaling is deeply involved in the survival, proliferation, and differentiation of many cells, and is also involved in ontogeny, along with cytokines / tyrosine kinases, Wnt, TGF-β family / Smad, hedgehog, and integrins. Abnormal Notch signaling is also involved in carcinogenesis, cancer cell proliferation, and survival of cancer stem cells. Notch receptors are single-pass transmembrane proteins consisting of a functional extracellular domain (NECD), a transmembrane domain (TM), and an intracellular domain (NICD). Processing of Notch receptors in the endoplasmic reticulum (ER) and Golgi apparatus within the signal-receiving cells results in cleavage (S1 cleavage), followed by the binding of sugar chains and the release of calcium ions (Ca 2+) stabilizes the Notch signaling receptor, forming a heterodimer that consists of a membrane-inserted TM-NICD and a non-covalently attached NECD. This processed receptor then translocates to the plasma membrane and becomes capable of ligand binding. In mammals, members of the Delta-like (DLL1, DLL3, DLL4) and Jagged (JAG1, JAG2) families present in signaling cells act as ligands for Notch signaling receptors. Upon ligand binding, the NECD is cleaved from the TM-NICD domain by TACE (ADAM metalloprotease TNF-α converting enzyme) (S2 cleavage). The NECD remains bound to the ligand, and the complex undergoes endocytosis and recycling / degradation within the signaling cell. Within the signal-receiving cell, γ-secretase (also implicated in Alzheimer's disease) releases NICD from the TM (S3 cleavage), allowing it to translocate into the nucleus, where it associates with the CSL (CBF1 / Su(H) / Lag-1) family transcription factor complex, resulting in the subsequent activation of the canonical Notch target genes Myc, p21, and HES family members.

[0052] Examples of Notch signal inhibitors include IMR-1, FLI-06, and crenigacestat (LY3039478). Also useful as Notch signal inhibitors are gamma-secretase inhibitors such as DBZ (dibenzazepine), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester), LY411575, dibenzazepine (YO-01027), RO4929097, nirogacestat (PF-03084014, PF-3084014), L-685,458, semagacestat (LY450139), avalagacestat (BMS-708163), and MK-0752.

[0053] (Hedgehog (HH) signal inhibitor) Hedgehog (HH) signaling is known as a cell growth factor and morphogenetic factor during embryonic development. It has also been shown to function in adult homeostasis, tissue regeneration, and regulation of tissue stem cells. Abnormal HH signaling during embryonic development can cause congenital disorders such as holoprosencephaly, while persistent HH signaling activity in adults is associated with various cancers, including basal cell carcinoma and medulloblastoma. Three HH ligands (SHH; Sonic hedgehog, IHH; Indian hedgehog, and DHH; Desert hedgehog) are known to act as HH signaling ligands in mammals. In the absence of HH ligands (off state), Patched, the receptor for Hedgehog family ligands, binds normally to the G protein-coupled transmembrane protein Smoothened (Smo) and inhibits its association with the membrane. In this off state, SuFu and COS2 (Kif7 in vertebrates) sequester a population of microtubule-bound transcription factors, Gli, in the first cilium, which are phosphorylated by PKA, CKI, and GSK-3, leading to either β-TrCP-mediated degradation of Gli activators (Gli1 and Gli2 in mammals) or, in a conserved pathway, to generate Gli repressors (Gli3 or truncated Ci in Drosophila), which lead to repression of Hedgehog target genes. In the activated (on) state, Hedgehog ligand binding to Patched allows β-arrestin-mediated translocation of Smoothened to the first cilium, where its associated G protein activity inhibits inhibitory kinase activity on Gli, freeing Gli to translocate to the nucleus and activate Hedgehog target genes, including cyclin D, cyclin E, Myc, and Patched.

[0054] The hedgehog (HH) signal inhibitor is not particularly limited as long as it inhibits the hedgehog signal, and examples thereof include substances that inhibit the signal by acting on Smo. Antagonist antibodies that inhibit the binding of hedgehog ligands to receptors such as Patched can also be used as hedgehog signal inhibitors.

[0055] Examples of hedgehog (HH) signal inhibitors include, but are not limited to, SANT1, cyclopamine, sonidegib, PF-5274857, glasdegib, taladegib, BMS-833923, MK-4101, vismodegib, GANT61, Jervine, HPI-4, etc. For example, SANT1 is a cell-permeable, potent HH signal antagonist that inhibits by directly binding to the Smo receptor, and can therefore be suitably used.

[0056] (TGF-β receptor signal inhibitor) TGF-β receptor (TGFβ) signaling is a signal transduction pathway involving the ligand of transforming growth factor β (TGFβ), which plays a central role in cellular processes such as cell growth, proliferation, differentiation, and apoptosis. TGFβ signaling involves the binding of a TGFβ ligand to a type II receptor (serine / threonine kinase), which recruits and phosphorylates a type I receptor (ALK5). This type I receptor then phosphorylates a receptor-regulated SMAD (R-SMAD; e.g., SMAD1, SMAD2, SMAD3, SMAD5, SMAD8, or SMAD9) that binds to SMAD4, and then this SMAD complex enters the nucleus where it plays a role in transcriptional regulation.

[0057] The TGFβ signal inhibitor is not particularly limited as long as it inhibits the TGFβ signal, but is, for example, a substance that acts on ALK5 to inhibit its phosphorylation. In addition, antagonist antibodies that inhibit the binding of TGFβ to its receptor can also be used as TGFβ signal inhibitors.

[0058] The TGFβ signal inhibitor is not particularly limited, but inhibitors that act on ALK5 can be suitably used, such as SB431542, Galunisertib, LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, SD-208, Vactosertib, and LDN-212854.

[0059] (Insulin receptor signal activator) Insulin receptors are expressed in liver, skeletal muscle, adipose tissue, and neurons, and insulin receptor signaling is known to be involved in the formation, maintenance, and repair of neural circuits. Insulin is an important hormone that regulates important energy functions such as glucose and lipid metabolism. It activates the insulin receptor tyrosine kinase (IR), which recruits and phosphorylates different substrate adaptors, such as the insulin receptor substrate (IRS) family. Tyrosine-phosphorylated IRSs provide binding sites for numerous signaling partners. Among these, phosphoinositide 3-kinase (PI3K) plays an important role in insulin function, primarily through activation of Akt (protein kinase B) and PKC (protein kinase C). Activated Akt induces glycogen synthesis via inhibition of glycogen synthase kinase-3 (GSK-3), protein synthesis via mammalian target of rapa (mTOR) and downstream factors, and cell survival through inhibition of pro-apoptotic factors (e.g., Bad, Forkhead family transcription factors, and GSK-3). Insulin receptor signaling also has cell growth and mitogenic effects that primarily involve the Akt cascade as well as activation of the Ras / MAPK pathway.

[0060] The insulin receptor signal activator is not particularly limited as long as it activates the insulin receptor signal, and examples thereof include ligands that bind to insulin receptors and IGF receptors. It may also be a substance that directly or indirectly activates PI3K, PKC, or Akt.

[0061] Preferred examples of insulin receptor signal activators include insulin, insulin-like growth factor-1 (IGF-1), IGF-2, etc. PI3K activators such as PI3-kinase activator (SantaCruz, product number: sc-3036) and 740 YP can also be used as insulin receptor signal activators.

[0062] (FGF receptor signal activator) FGF (fibroblast growth factor) receptor signaling is a signal transduction pathway mediated by FGF receptors, which flows through the RAS-MAPK pathway and the PI3K-AKT pathway and is involved in various cellular functions such as cell proliferation, cell death, angiogenesis, and epithelial-mesenchymal transition (EMT). It also plays an important role in regulating embryonic and postnatal skeletal development. FGF receptor signal activators can be any substance that activates the signal transduction described above, and representative examples include ligands (FGF family) that bind to FGF receptors. Activators of the RAS-MAPK pathway and the PI3K-AKT pathway can also be used as FGF receptor signal activators.

[0063] Examples of FGF receptor signal activators include the FGF family, preferably FGF7, FGF3, FGF10, FGF22, FGF1, FGF2, FGF4, FGF5, FGF6, FGF8, FGF17, FGF18, FGF9, FGF16, FGF20, FGF19, FGF21, FGF23, etc., with FGF7 being particularly preferred.

[0064] (EGF receptor signal activator) Epidermal growth factor (EGF) is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds. It binds to the epidermal growth factor receptor (EGFR) on the cell surface as a ligand and plays an important role in regulating cell growth and proliferation. EGF is an example of an EGF receptor signal activator.

[0065] (GLP-1 (Glucagon-like peptide-1) receptor signal activator) Glucagon-like peptide-1 (GLP-1), an incretin hormone secreted by the body, increases cAMP by acting through the GLP-1 receptor, promoting insulin secretion in a glucose-dependent manner. Examples of GLP-1 receptor signal activators include GLP-1 and Exendin 4.

[0066] (growth factor stabilizer) A growth factor stabilizer refers to a substance that has the effect of stabilizing a growth factor. The stabilization of a growth factor is not particularly limited as long as it is an effect of suppressing the reduction of the amount of extracellular growth factor, such as an effect of suppressing the degradation of the growth factor or an effect of promoting the secretion of the growth factor into the extracellular space. Examples of growth factor stabilizers include heparin and heparan sulfate.

[0067] (hepatocyte growth factor) Hepatocyte growth factor (HGF) is a cytokine involved in tissue regeneration and repair in vivo. HGF acts on various cells in addition to hepatocytes, promoting the proliferation of epithelial and endothelial cells, and acting as a neurotrophic factor for nerve cells.

[0068] (insulin-like growth factor) Insulin-like growth factors (IGFs) are polypeptides with a structure similar to proinsulin and are known to be involved in cell proliferation, survival, migration, and the production of extracellular matrix, including collagen. They exhibit mitogenic activity by binding to a common receptor, insulin-like growth factor receptor-1 (IGFR1), on various cell types, including tumor cells. Insulin-like growth factors include insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II).

[0069] (Adenylate cyclase activator (a substance that increases intracellular cAMP concentration)) Adenylate cyclase is an enzyme that catalyzes the conversion of ATP to 3',5'-cyclic AMP (cAMP) and pyrophosphate. cAMP is a second messenger molecule that is important for signal transduction in eukaryotes. Adenylate cyclase activators include forskolin and NKH477.

[0070] (WNT signal activator) WNT signaling refers to a series of actions that promote the nuclear translocation of β-catenin and exert its function as a transcription factor. WNT signaling is caused by cell-cell interactions, and includes a series of events in which, for example, a protein called WNT3A secreted from one cell acts on another cell, causing β-catenin in the cell to translocate to the nucleus and act as a transcription factor. This series of events triggers the first phenomenon in organ construction, such as epithelial-mesenchymal interactions. WNT signaling is mediated by the β-catenin pathway, PCP pathway, and Ca 2+ It is known that by activating these three pathways, it controls various cellular functions such as cell proliferation and differentiation, organ formation, and cell movement during early development. Genes involved in the WNT signaling pathway include the WNT3A gene.

[0071] The WNT signal activator is not particularly limited, but may be any compound that exhibits inhibitory activity against glycogen synthase kinase-3 (GSK-3), such as bis-indolo(indirubin) compounds (BIO) ((2'Z,3'E)-6-bromoindirubin-3'-oxime), its acetoxime analogue BIO-acetoxime (2'Z,3'E)-6-bromoindirubin-3'-acetoxime), thiadiazolidine (TDZD) analogues (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), oxothiadiazolidine-3-thione analogues (2,4-di benzyl-5-oxothiadiazolidine-3-thione), thienyl α-chloromethyl ketone compound (2-chloro-1-(4,4-dibromo-thiophen-2-yl)-ethanone), phenyl α-bromomethyl ketone compound (α-4-dibromoacetophenone), thiazole-containing urea compound (N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea), and GSK-3β peptide inhibitors such as H-KEAPPAPPQSpP-NH2 can be used, and CHIR99021 (CAS: 252917-06-9) is particularly preferred. WNT3A can also be used.

[0072] [1] Method for inducing differentiation into pancreatic alpha cells The method for inducing differentiation into pancreatic α cells according to the present invention comprises the steps of inducing differentiation of endodermal cells, which have been induced to differentiate from pluripotent stem cells, into primitive gut tubule cells (PGT), inducing differentiation of the primitive gut tubule cells (PGT) into pancreatic endocrine precursor cells (Endocrine Precursor Cells), and inducing differentiation of the pancreatic endocrine precursor cells into pancreatic α cells. Pancreatic α cells can be produced from pluripotent stem cells by the method for inducing differentiation into pancreatic α cells according to the present invention.

[0073] A method for inducing differentiation into pancreatic α-cells from endodermal cells that have been induced to differentiate from pluripotent stem cells will be described. In step (a) of the present invention, endodermal cells induced to differentiate from pluripotent stem cells are cultured in the presence of a bone morphogenetic protein (BMP) signal inhibitor and retinoic acid or a retinoic acid analog to induce differentiation into primitive gut cells (PGT). In step (b) of the present invention, the primitive gut cells (PGT) are cultured to induce differentiation into pancreatic endocrine precursor cells (EP). In step (c) of the present invention, the pancreatic endocrine precursor cells (EP) are cultured to induce differentiation into pancreatic α cells. The steps (b) and (c) are carried out in the absence of ascorbic acid. Each step will be explained below. The differentiation of pluripotent stem cells into endodermal cells will be described later.

[0074] <Culture conditions> The cells cultured during the differentiation induction from endodermal cells to pancreatic α cells may be either adherent culture or suspension culture, but suspension culture is preferred. The cells may be cultured in suspension by adhering them to microcarriers or the like, or may be cultured in suspension in the form of cell aggregates composed only of cells, or may contain polymers such as collagen mixed within the cell aggregates; the morphology is not particularly limited.

[0075] The culture temperature for inducing differentiation of endodermal cells into pancreatic alpha cells is not particularly limited as long as it is a culture temperature suitable for culturing the cells used, but is generally 30°C to 40°C, and preferably approximately 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0076] <Culture period> The culture period for inducing differentiation of endodermal cells into primitive gut cells (PGT) in step (a) is generally 24 to 120 hours, preferably about 48 to 96 hours, for example, 72 hours.

[0077] The differentiation induction of primitive gut cells (PGT) into pancreatic endocrine precursor cells (EP) in step (b) includes the differentiation induction of the primitive gut cells (PGT) into posterior foregut cells (PFG), the differentiation induction of the posterior foregut cells (PFG) into pancreatic precursor cells (PP), and the differentiation induction of the pancreatic precursor cells (PP) into pancreatic endocrine precursor cells (EP).

[0078] The culture period for differentiation culture of primitive gut cells (PGT) to posterior foregut cells (PFG) is not particularly limited as long as the cell type exhibits the cell characteristics of posterior foregut cells (PFG), but may be, for example, within 2 weeks, more specifically, 1 to 10 days, more preferably 2 to 7 days, and even more preferably 3 to 5 days, an example of which is 4 days.

[0079] The culture period for differentiation culture of posterior foregut cells (PFG) into pancreatic progenitor cells (PP) is not particularly limited as long as the cell type exhibits the cell characteristics of pancreatic progenitor cells (PP), but may be, for example, within 2 weeks, more specifically, 1 to 10 days, more preferably 2 to 7 days, and even more preferably 2 to 4 days, an example of which is 3 days.

[0080] The culture period for differentiation culture of pancreatic progenitor cells (PP) to pancreatic endocrine precursor cells (EP) is not particularly limited as long as the cell type exhibits the cell characteristics of pancreatic endocrine precursor cells (EP), but may be, for example, within 2 weeks, more specifically, 1 to 10 days, more preferably 3 to 10 days, and even more preferably 5 to 9 days, an example of which is 7 days.

[0081] The culture period for the differentiation culture into pancreatic alpha cells in step (c) is not particularly limited as long as the cells have become of a cell type exhibiting the cell characteristics of pancreatic alpha cells, but may be, for example, within 3 weeks, more specifically, between 3 and 20 days, more preferably between 5 and 14 days, and even more preferably between 7 and 12 days, and an example is 10 days.

[0082] <Culture medium> The media used in steps (a) to (c) include MEM medium, BME medium, DMEM medium, DMEM / F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, and Essential 6 medium, depending on the type of cells. TM The medium may be a mixture of two or more media selected from the above media (Thermo Fisher Scientific). The medium may contain glucose, and the glucose concentration is preferably 1 mM to 100 mM, more preferably 2 mM to 50 mM, and even more preferably 5 mM to 30 mM.

[0083] The medium may further contain bovine serum albumin (BSA) or human serum albumin (HSA). Preferably, the BSA or HSA contains 2 mg / g or less of lipids and 0.2 mg / g or less of free fatty acids. The lower limit of the amount of BSA added to the medium is preferably 0.01%, more preferably 0.05%, more preferably 0.10%, more preferably 0.15%, more preferably 0.20%, and more preferably 0.25%. The upper limit of the amount of BSA added to the medium is preferably 1.00%, more preferably 0.90%, more preferably 0.80%, more preferably 0.70%, more preferably 0.60%, more preferably 0.50%, more preferably 0.40%, more preferably 0.30%, and more preferably 0.25%.

[0084] The medium may further contain antibiotics such as penicillin and streptomycin. For example, the medium may contain 0.1 to 2% (volume / volume) penicillin and 0.1 to 2% (volume / volume) streptomycin.

[0085] The medium may also contain B27® supplement. The lower limit of the amount of B27® supplement added to the culture medium is preferably 0.01%, more preferably 0.1%, more preferably 0.2%, more preferably 0.3%, more preferably 0.4%, more preferably 0.5%, more preferably 0.6%, more preferably 0.7%, more preferably 0.8%, more preferably 0.9%. The upper limit of the amount of B27® supplement added to the culture medium is preferably 10%, more preferably 9%, more preferably 8%, more preferably 7%, more preferably 6%, more preferably 5%, more preferably 4%, more preferably 3%, more preferably 2%, more preferably 1%.

[0086] The medium may contain insulin, transferrin, and selenite, which may be included in the medium in the form of a commercially available mixture, such as B27 supplement.

[0087] The lower limit of the amount of transferrin added to the medium is preferably 0.001 mg / L, more preferably 0.01 mg / L, more preferably 0.1 mg / L, more preferably 1 mg / L, more preferably 1.1 mg / L, more preferably 1.2 mg / L, more preferably 1.3 mg / L, and more preferably 1.4 mg / L. The upper limit of the amount of transferrin added to the medium is preferably 1000 mg / L, more preferably 500 mg / L, more preferably 100 mg / L, more preferably 90 mg / L, more preferably 80 mg / L, more preferably 70 mg / L, more preferably 60 mg / L, more preferably 50 mg / L, more preferably 40 mg / L, more preferably 30 mg / L, more preferably 20 mg / L, more preferably 10 mg / L, more preferably 9 mg / L, more preferably 8 mg / L, more preferably 7 mg / L, more preferably 6 mg / L, more preferably 5 mg / L, more preferably 4 mg / L, and more preferably 3 mg / L.

[0088] The lower limit of the amount of selenious acid to be added to the medium is preferably 0.001 μg / L, more preferably 0.01 μg / L, more preferably 0.1 μg / L, more preferably 1 μg / L, more preferably 1.1 μg / L, more preferably 1.2 μg / L, more preferably 1.3 μg / L, more preferably 1.4 μg / L, more preferably 1.5 μg / L, more preferably 1.6 μg / L, and more preferably 1.7 μg / L. The upper limit of the amount of selenious acid to be added to the medium is preferably 1000 μg / L, more preferably 500 μg / L, more preferably 100 μg / L, more preferably 90 μg / L, more preferably 80 μg / L, more preferably 70 μg / L, more preferably 60 μg / L, more preferably 50 μg / L, more preferably 40 μg / L, more preferably 30 μg / L, more preferably 20 μg / L, more preferably 10 μg / L, more preferably 9 μg / L, more preferably 8 μg / L, more preferably 7 μg / L, more preferably 6 μg / L, more preferably 5 μg / L, and more preferably 4 μg / L.

[0089] [2] Differentiation-inducing factors and other additives used to induce differentiation of endodermal cells into primitive intestinal cells In step (a) of the present invention, endodermal cells induced to differentiate from pluripotent stem cells are cultured in the presence of a bone morphogenetic protein (BMP) signaling inhibitor and retinoic acid or a retinoic acid analog under culture conditions suitable for inducing differentiation into primitive gut cells (PGT), thereby inducing differentiation into primitive gut cells (PGT). In step (a), endodermal cells induced to differentiate from pluripotent stem cells are cultured in the presence of a bone morphogenetic protein (BMP) signaling inhibitor and retinoic acid or a retinoic acid analog, thereby inducing differentiation into primitive gut cells (PGT), thereby enabling efficient induction of differentiation from primitive gut cells (PGT) into pancreatic α cells. Culture conditions suitable for inducing differentiation into primitive gut cells (PGT) are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of endodermal cells induced to differentiate from pluripotent stem cells into primitive gut cells (PGT). The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of endodermal cells into primitive gut cells (PGT), but one embodiment is culturing in the differentiation-inducing medium described below.

[0090] When the bone morphogenetic protein (BMP) signal inhibitor is dorsomorphin, the lower limit of the amount of the added bone morphogenetic protein (BMP) signal inhibitor in the medium used in step (a) is preferably 0.01 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.3 μmol / L, more preferably 0.5 μmol / L, more preferably 0.6 μmol / L, more preferably 0.7 μmol / L, more preferably 0.8 μmol / L, more preferably 0.9 μmol / L, more preferably 1 μmol / L. The upper limit of the dorsomorphin content in the medium is preferably 20 μmol / L, more preferably 15 μmol / L, more preferably 10 μmol / L, more preferably 7 μmol / L, more preferably 5 μmol / L, more preferably 4 μmol / L, more preferably 3 μmol / L, more preferably 2 μmol / L, more preferably 1 μmol / L.

[0091] When the bone morphogenetic protein (BMP) signal inhibitor is LDN193189, the lower limit of the content of LDN193189 in the medium is preferably 0.01 μ mol / L, more preferably 0.02 μ mol / L, more preferably 0.05 μ mol / L, more preferably 0.1 μ mol / L, more preferably 0.2 μ mol / L, more preferably 0.3 μ mol / L, more preferably 0.4 μ mol / L, more preferably 0.5 μ mol / L, more preferably 0.6 μ mol / L. The upper limit of the content of LDN193189 in the medium is preferably 10 μ mol / L, more preferably 5 μ mol / L, more preferably 1 μ mol / L, more preferably 0.9 μ mol / L, more preferably 0.8 μ mol / L, more preferably 0.7 μ mol / L.

[0092] The lower limit of the amount of retinoic acid or retinoic acid analogs (EC23, etc.) added to the medium used in step (a) is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.03 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.2 μmol / L, more preferably 0.3 μmol / L, more preferably 0.4 μmol / L, more preferably 0.5 μmol / L, more preferably 0.6 μmol / L, more preferably 0.7 μmol / L, more preferably 0.8 μmol / L, more preferably 0.9 μmol / L, and more preferably 1.0 μmol / L. The upper limit of the amount of retinoic acid analogs (EC23, etc.) added to the medium is preferably 5.0 μmol / L, more preferably 4.0 μmol / L, more preferably 3.0 μmol / L, more preferably 2.0 μmol / L, and more preferably 1.0 μmol / L.

[0093] The step (a) may be carried out in the presence of a ROCK signal inhibitor (Y27632, etc.). When step (a) is performed in the presence of a ROCK signal inhibitor (e.g., Y27632), the lower limit of the content of the ROCK signal inhibitor (e.g., Y27632) in the medium is preferably 0.1 μmol / L, more preferably 0.5 μmol / L, more preferably 1 μmol / L, more preferably 1.5 μmol / L, more preferably 2.0 μmol / L, and more preferably 2.5 μmol / L. The upper limit of the content of the ROCK signal inhibitor (e.g., Y27632) in the medium is preferably 100 μmol / L, more preferably 50 μmol / L, more preferably 40 μmol / L, more preferably 30 μmol / L, more preferably 20 μmol / L, more preferably 10 μmol / L, more preferably 5 μmol / L, more preferably 4 μmol / L, more preferably 3 μmol / L, and more preferably 2.5 μmol / L.

[0094] The medium used in the step (a) is preferably a medium containing an insulin receptor signal activator.

[0095] The lower limit of the amount of insulin receptor signal activator to be added to the culture medium is preferably 0.001 mg / L, more preferably 0.01 mg / L, more preferably 0.1 mg / L, more preferably 1 mg / L, more preferably 2 mg / L, more preferably 3 mg / L, more preferably 4 mg / L, and more preferably 5 mg / L. The upper limit of the amount of insulin receptor signal activator to be added to the culture medium is preferably 1000 mg / L, more preferably 500 mg / L, more preferably 100 mg / L, more preferably 90 mg / L, more preferably 80 mg / L, more preferably 70 mg / L, more preferably 60 mg / L, more preferably 50 mg / L, more preferably 40 mg / L, more preferably 30 mg / L, more preferably 20 mg / L, and more preferably 10 mg / L.

[0096] The medium used in the step (a) is preferably a medium containing insulin, transferrin and selenious acid. Insulin, transferrin, and selenite may be included in the medium in the form of a commercially available mixture, such as a B27 supplement. In addition to insulin, transferrin, and selenite, ethanolamine may also be included.

[0097] The lower limit of the amount of transferrin to be added to the culture medium is preferably 0.001 mg / L, more preferably 0.01 mg / L, more preferably 0.1 mg / L, more preferably 1 mg / L, more preferably 2 mg / L, more preferably 2.1 mg / L, more preferably 2.2 mg / L, more preferably 2.3 mg / L, more preferably 2.4 mg / L, more preferably 2.5 mg / L, more preferably 2.6 mg / L, and more preferably 2.7 mg / L. The upper limit of the amount of transferrin to be added to the culture medium is preferably 1000 mg / L, more preferably 500 mg / L, more preferably 100 mg / L, more preferably 90 mg / L, more preferably 80 mg / L, more preferably 70 mg / L, more preferably 60 mg / L, more preferably 50 mg / L, more preferably 40 mg / L, more preferably 30 mg / L, more preferably 20 mg / L, more preferably 10 mg / L, more preferably 9 mg / L, more preferably 8 mg / L, more preferably 7 mg / L, more preferably 6 mg / L, more preferably 5 mg / L, more preferably 4 mg / L, more preferably 3 mg / L, more preferably 2.9 mg / L, and more preferably 2.8 mg / L.

[0098] The lower limit of the amount of selenious acid to be added to the medium is preferably 0.001 μg / L, more preferably 0.01 μg / L, more preferably 0.1 μg / L, more preferably 1 μg / L, more preferably 2 μg / L, more preferably 2.1 μg / L, more preferably 2.2 μg / L, more preferably 2.3 μg / L, more preferably 2.4 μg / L, more preferably 2.5 μg / L, more preferably 2.6 μg / L, more preferably 2.7 μg / L, more preferably 2.8 μg / L, more preferably 2.9 μg / L, more preferably 3 μg / L, more preferably 3.1 μg / L, more preferably 3.2 μg / L, and more preferably 3.3 μg / L. The upper limit of the amount of selenious acid to be added to the medium is preferably 1000 μg / L, more preferably 500 μg / L, more preferably 100 μg / L, more preferably 90 μg / L, more preferably 80 μg / L, more preferably 70 μg / L, more preferably 60 μg / L, more preferably 50 μg / L, more preferably 40 μg / L, more preferably 30 μg / L, more preferably 20 μg / L, more preferably 10 μg / L, more preferably 9 μg / L, more preferably 8 μg / L, more preferably 7 μg / L, more preferably 6 μg / L, more preferably 5 μg / L, and more preferably 4 μg / L.

[0099] The medium used in step (a) is preferably a medium containing an FGF receptor signal activator, although from the viewpoint of achieving more efficient differentiation induction, the culture in step (a) is preferably carried out in the absence of FGF2.

[0100] The lower limit of the amount of FGF receptor signal activator to be added to the culture medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 20 ng / mL, more preferably 30 ng / mL, more preferably 40 ng / mL, and more preferably 50 ng / mL. The upper limit of the amount of FGF receptor signal activator to be added to the culture medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 90 ng / mL, more preferably 80 ng / mL, more preferably 70 ng / mL, more preferably 60 ng / mL, and more preferably 50 ng / mL.

[0101] The medium used in the step (a) is preferably a medium containing B27 (registered trademark) supplement and / or FGF7.

[0102] The lower limit of the amount of B27® supplement added to the culture medium is preferably 0.01%, more preferably 0.1%, more preferably 0.2%, more preferably 0.3%, more preferably 0.4%, more preferably 0.5%, more preferably 0.6%, more preferably 0.7%, more preferably 0.8%, more preferably 0.9%. The upper limit of the amount of B27® supplement added to the culture medium is preferably 10%, more preferably 9%, more preferably 8%, more preferably 7%, more preferably 6%, more preferably 5%, more preferably 4%, more preferably 3%, more preferably 2%, more preferably 1%.

[0103] The lower limit of the amount of FGF7 added to the medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 20 ng / mL, more preferably 30 ng / mL, more preferably 40 ng / mL, and more preferably 50 ng / mL. The upper limit of the amount of FGF7 added to the medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 90 ng / mL, more preferably 80 ng / mL, more preferably 70 ng / mL, more preferably 60 ng / mL, and more preferably 50 ng / mL.

[0104] The culture in the step (a) is culture in the absence of a hedgehog (HH) signal inhibitor, and more preferably culture in the absence of a TGFβ signal inhibitor.

[0105] Depending on the cell type, various media are available: MEM medium, BME medium, DMEM medium, DMEM / F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, and Essential 6 medium. TM Culture medium (Thermo Fisher Scientific), or a mixture thereof, etc. can be used.

[0106] The medium may further contain bovine serum albumin (BSA) or human serum albumin (HSA). Preferably, the BSA or HSA contains 2 mg / g or less of lipids and 0.2 mg / g or less of free fatty acids. The lower limit of the amount of BSA added to the medium is preferably 0.01% (wt %), more preferably 0.05%, more preferably 0.10%, more preferably 0.15%, more preferably 0.20%, and more preferably 0.25%. The upper limit of the amount of BSA added to the medium is preferably 1.00%, more preferably 0.90%, more preferably 0.80%, more preferably 0.70%, more preferably 0.60%, more preferably 0.50%, more preferably 0.40%, more preferably 0.30%, and more preferably 0.25%.

[0107] The medium may further contain sodium pyruvate. The lower limit of the amount of sodium pyruvate to be added to the medium is preferably 0.01 mmol / L, more preferably 0.05 mmol / L, more preferably 0.1 mmol / L, more preferably 0.2 mmol / L, more preferably 0.5 mmol / L, more preferably 0.6 mmol / L, more preferably 0.7 mmol / L, more preferably 0.8 mmol / L, more preferably 0.9 mmol / L, and more preferably 1 mmol / L. The upper limit of the amount of sodium pyruvate to be added to the medium is preferably 20 mmol / L, more preferably 15 mmol / L, more preferably 10 mmol / L, more preferably 5 mmol / L, more preferably 4 mmol / L, more preferably 3 mmol / L, more preferably 2 mmol / L, and more preferably 1 mmol / L.

[0108] The medium may further contain NEAA (for example, 1× non-essential amino acids (NEAA; Wako)). The lower limit of the NEAA content in the medium is preferably 0.05xNEAA, more preferably 0.1xNEAA, more preferably 0.5xNEAA, more preferably 0.6xNEAA, more preferably 0.7xNEAA, more preferably 0.8xNEAA, more preferably 0.9xNEAA, and more preferably 1xNEAA. The upper limit of the NEAA content in the medium is preferably 20xNEAA, more preferably 15xNEAA, more preferably 10xNEAA, more preferably 5xNEAA, more preferably 4xNEAA, more preferably 3xNEAA, more preferably 2xNEAA, and more preferably 1xNEAA.

[0109] The medium may further contain a growth factor stabilizer (such as heparin). The lower limit of the amount of growth factor stabilizer (e.g., heparin) added to the culture medium is preferably 0.2 μg / mL, more preferably 0.5 μg / mL, more preferably 1 μg / mL, more preferably 2 μg / mL, more preferably 3 μg / mL, more preferably 4 μg / mL, and more preferably 5 μg / mL. The upper limit of the amount of growth factor stabilizer (e.g., heparin) added to the culture medium is preferably 100 μg / mL, more preferably 90 μg / mL, more preferably 80 μg / mL, more preferably 70 μg / mL, more preferably 60 μg / mL, more preferably 50 μg / mL, more preferably 40 μg / mL, more preferably 30 μg / mL, more preferably 20 μg / mL, more preferably 10 μg / mL, more preferably 9 μg / mL, more preferably 8 μg / mL, more preferably 7 μg / mL, more preferably 6 μg / mL, and more preferably 5 μg / mL.

[0110] The medium may further contain antibiotics such as penicillin and streptomycin.

[0111] The culture temperature is not particularly limited as long as it is suitable for culturing the pluripotent stem cells used, but is generally 30°C to 40°C, and preferably about 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0112] The culture may be performed with stirring. The stirring speed is not particularly limited, but the upper limit is preferably 200 rpm, more preferably 150 rpm, even more preferably 120 rpm, more preferably 110 rpm, more preferably 100 rpm, more preferably 90 rpm, more preferably 80 rpm, more preferably 70 rpm, particularly preferably 60 rpm, and most preferably 55 rpm. The lower limit is preferably 1 rpm, more preferably 10 rpm, even more preferably 20 rpm, more preferably 30 rpm, more preferably 40 rpm, particularly preferably 50 rpm, and most preferably 55 rpm.

[0113] [3] Differentiation-inducing factors, other additives, and culture conditions used to induce differentiation of primitive intestinal cells into pancreatic endocrine precursor cells In step (b) of the present invention, primitive gut cells (PGTs) that have been induced to differentiate from endodermal cells are induced to differentiate into pancreatic endocrine precursor cells (EPs) under culture conditions suitable for inducing differentiation into EPs. Culture conditions suitable for inducing differentiation into pancreatic endocrine precursor cells (EP) are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of primitive gut cells (PGT), which have been induced to differentiate from endodermal cells, into pancreatic endocrine precursor cells (EP). The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of primitive gut cells (PGT) into pancreatic endocrine precursor cells (EP), but one embodiment is culturing in the differentiation-inducing medium described below. Step (b) in the present invention is carried out in the absence of ascorbic acid, from the viewpoint of efficiently inducing differentiation of pancreatic α-cells from primitive gut cells (PGT).

[0114] Step (b) in the present invention comprises the steps of inducing differentiation of posterior foregut cells (PFG) from primitive gut cells (PGT) that have been induced to differentiate from endodermal cells, inducing differentiation of the posterior foregut cells (PFG) into pancreatic progenitor cells (PP), and inducing differentiation of the pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP).

[0115] Step (b) in the present invention is (b1) inducing differentiation of the primitive gut cells (PGT) into posterior foregut cells (PFG) by culturing the PGT in the presence of a protein kinase C (PKC) activator: (b2) inducing differentiation of the posterior foregut cells (PFG) into pancreatic progenitor cells (PP) by culturing the PFG in the presence of retinoic acid or an analog thereof; and (b3) inducing differentiation of the pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP) by culturing the pancreatic progenitor cells (PP) in the presence of a Notch signal inhibitor and a ROCK signal inhibitor: may also include:

[0116] [3-1] Differentiation-inducing factors and other additives used to induce differentiation from primitive intestinal cells to posterior foregut cells In the step (b1), primitive gut cells (PGT) induced to differentiate from endodermal cells are cultured in the presence of a protein kinase C (PKC) activator under culture conditions suitable for inducing differentiation into posterior foregut cells (PFG), thereby inducing differentiation into posterior foregut cells (PFG). Culture conditions suitable for inducing differentiation into posterior foregut cells (PFG) are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of primitive gut cells (PGT) induced to differentiate from endodermal cells into posterior foregut cells (PFG). The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of primitive gut cells (PGT) into posterior foregut cells (PFG), but one embodiment is culturing in the differentiation-inducing medium described below.

[0117] The lower limit of the content of the PKC activator in the medium used in step (b1) is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.2 μmol / L, and more preferably 0.3 μmol / L. The upper limit of the content of the PKC activator in the medium is preferably 10 μmol / L, more preferably 5 μmol / L, more preferably 1 μmol / L, more preferably 0.8 μmol / L, more preferably 0.5 μmol / L, more preferably 0.4 μmol / L, and more preferably 0.3 μmol / L.

[0118] The medium used in the step (b1) may contain a hedgehog (HH) signal inhibitor (SANT1, etc.). The lower limit of the amount of HH signal inhibitor (SANT1, etc.) added to the medium is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.03 μmol / L, more preferably 0.05 μmol / L, more preferably 0.10 μmol / L, more preferably 0.15 μmol / L, more preferably 0.20 μmol / L, more preferably 0.25 μmol / L. The upper limit of the amount of HH signal inhibitor (SANT1, etc.) added to the medium is preferably 5.0 μmol / L, more preferably 4.0 μmol / L, more preferably 3.0 μmol / L, more preferably 2.0 μmol / L, more preferably 1.0 μmol / L, more preferably 0.80 μmol / L, more preferably 0.70 μmol / L, more preferably 0.60 μmol / L, more preferably 0.50 μmol / L, more preferably 0.40 μmol / L, more preferably 0.30 μmol / L, more preferably 0.25 μmol / L.

[0119] The medium used in the step (b1) may contain a bone morphogenetic protein (BMP) signal inhibitor. When the bone morphogenetic protein (BMP) signal inhibitor is LDN193189, the lower limit of the LDN193189 content in the medium is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.2 μmol / L. The upper limit of the LDN193189 content in the medium is preferably 10 μmol / L, more preferably 5 μmol / L, more preferably 1 μmol / L, more preferably 0.8 μmol / L, more preferably 0.5 μmol / L, more preferably 0.4 μmol / L, more preferably 0.3 μmol / L, more preferably 0.2 μmol / L.

[0120] The medium used in the step (b1) may contain an EGF receptor signal activator. The lower limit of the amount of EGF receptor signal activator to be added to the culture medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 15 ng / mL, more preferably 20 ng / mL, and more preferably 25 ng / mL. The upper limit of the amount of EGF receptor signal activator to be added to the culture medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 50 ng / mL, more preferably 40 ng / mL, more preferably 30 ng / mL, and more preferably 25 ng / mL.

[0121] The medium used in step (b1) may contain a retinoic acid analogue (EC23, etc.). The amount of the retinoic acid analogue added to the medium is as described in [2] above.

[0122] The medium used in step (b1) may contain NEAA (e.g., 1× non-essential amino acids (NEAA; Wako)). The amount of NEAA added to the medium is as described in [2] above.

[0123] The medium in step (b1) may contain an FGF receptor signal activator, particularly preferably FGF7. The amount of FGF receptor signal activator added to the medium is as described in [2] above.

[0124] [3-2] Differentiation-inducing factors and other additives used to induce differentiation of posterior foregut cells (PFG) into pancreatic progenitor cells (PP) In the step (b2), the posterior foregut cells (PFG) are induced to differentiate into pancreatic progenitor cells (PP) by culturing them in the presence of retinoic acid or an analog thereof under culture conditions suitable for inducing differentiation into pancreatic progenitor cells (PP). The culture conditions suitable for inducing differentiation into pancreatic progenitor cells (PP) are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of posterior foregut cells (PFG) into pancreatic progenitor cells (PP). The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of posterior foregut cells (PFG) into pancreatic progenitor cells (PP), but one embodiment is culturing in the differentiation-inducing medium described below.

[0125] The amount of retinoic acid or a retinoic acid analog (EC23, etc.) added to the medium used in the step (b2) is as described in [2] above.

[0126] The medium used in step (b2) may contain a hedgehog (HH) signaling inhibitor (e.g., SANT1). The amount of the hedgehog (HH) signaling inhibitor (e.g., SANT1) added to the medium is as described in [3-1] above.

[0127] The medium used in step (b2) may also contain NEAA (e.g., 1× non-essential amino acids (NEAA; Wako)). The amount of NEAA added to the medium is as described in [2] above.

[0128] The medium in step (b2) may contain a bone morphogenetic protein (BMP) signaling inhibitor. The amount of bone morphogenetic protein (BMP) signaling inhibitor added to the medium is as described in [3-1] above.

[0129] The medium in step (b2) may contain an FGF receptor signal activator, particularly preferably FGF10. The amount of FGF receptor signal activator added to the medium is as described in [2] above.

[0130] The medium in step (b2) may contain a protein kinase C (PKC) activator, the amount of which is as described in [3-1] above.

[0131] The medium in step (b2) may contain an EGF receptor signal activator, the amount of which is as described in [3-1] above.

[0132] The medium in step (b2) may contain a TGF-β receptor signal inhibitor (such as RepSOX). The lower limit of the content of the TGF-β receptor signal inhibitor (RepSOX, etc.) in the medium is preferably 0.01 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.3 μmol / L, more preferably 0.5 μmol / L, more preferably 0.6 μmol / L, more preferably 0.7 μmol / L, more preferably 0.8 μmol / L, more preferably 0.9 μmol / L, more preferably 1.0 μmol / L. The upper limit of the content of the TGF-β receptor signal inhibitor (RepSOX, etc.) in the medium is preferably 10 μmol / L, more preferably 8 μmol / L, more preferably 5 μmol / L, more preferably 4 μmol / L, more preferably 3 μmol / L, more preferably 2 μmol / L, more preferably 1.5 μmol / L, more preferably 1.2 μmol / L, more preferably 1.0 μmol / L.

[0133] The medium in step (b2) may contain ZnSO4. The lower limit of the ZnSO content in the medium is preferably 0.1 μmol / L, more preferably 0.5 μmol / L, more preferably 1 μmol / L, more preferably 3 μmol / L, and more preferably 5 μmol / L. The upper limit of the ZnSO content in the medium is preferably 100 μmol / L, more preferably 80 μmol / L, more preferably 50 μmol / L, more preferably 40 μmol / L, more preferably 30 μmol / L, more preferably 20 μmol / L, more preferably 15 μmol / L, more preferably 12 μmol / L, more preferably 10 μmol / L, and more preferably 5 μmol / L.

[0134] [3-3] Differentiation-inducing factors and other additives used to induce differentiation of pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP) In step (b3) of the present invention, pancreatic progenitor cells (PP) are induced to differentiate into pancreatic endocrine precursor cells (EP) by culturing them in the presence of a Notch signal inhibitor and a ROCK signal inhibitor under culture conditions suitable for inducing differentiation into pancreatic endocrine precursor cells (EP). The culture conditions suitable for inducing differentiation into endocrine precursor cells (EP) are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of posterior foregut cells (PP) into pancreatic endocrine precursor cells (EP). The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of posterior foregut cells (PP) into pancreatic endocrine precursor cells (EP), but one embodiment is culturing in the differentiation-inducing medium described below.

[0135] The lower limit of the amount of Notch signal inhibitor (DBZ, etc.) added to the medium in step (b3) is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.03 μmol / L, more preferably 0.05 μmol / L, more preferably 0.1 μmol / L, more preferably 0.2 μmol / L, more preferably 0.3 μmol / L, more preferably 0.4 μmol / L, more preferably 0.5 μmol / L. The upper limit of the amount of Notch signal inhibitor (DBZ, etc.) added to the medium is preferably 5.0 μmol / L, more preferably 4.0 μmol / L, more preferably 3.0 μmol / L, more preferably 2.0 μmol / L, more preferably 1.0 μmol / L, more preferably 0.9 μmol / L, more preferably 0.8 μmol / L, more preferably 0.7 μmol / L, more preferably 0.6 μmol / L, more preferably 0.5 μmol / L.

[0136] The medium used in step (b3) may contain a hedgehog (HH) signaling inhibitor (e.g., SANT1). The amount of the hedgehog (HH) signaling inhibitor (e.g., SANT1) added to the medium is as described in [3-1] above.

[0137] The medium used in step (b3) may contain a retinoic acid analogue (EC23, etc.). The amount of the retinoic acid analogue added to the medium is as described in [2] above.

[0138] The medium used in step (b3) may contain L-glutamine. The lower limit of the L-glutamine content in the medium is preferably 0.01 mmol / L, more preferably 0.05 mmol / L, more preferably 0.1 mmol / L, more preferably 0.5 mmol / L, more preferably 0.7 mmol / L, more preferably 1.0 mmol / L, more preferably 1.2 mmol / L, more preferably 1.5 mmol / L, and more preferably 2.0 mmol / L. The upper limit of the L-glutamine content in the medium is preferably 100 mmol / L, more preferably 50 mmol / L, more preferably 40 mmol / L, more preferably 30 mmol / L, more preferably 20 mmol / L, more preferably 10 mmol / L, more preferably 9 mmol / L, more preferably 8 mmol / L, more preferably 7 mmol / L, more preferably 6 mmol / L, more preferably 5 mmol / L, more preferably 4 mmol / L, more preferably 3 mmol / L, and more preferably 2 mmol / L.

[0139] The medium in step (b3) may contain a bone morphogenetic protein (BMP) signaling inhibitor. The amount of bone morphogenetic protein (BMP) signaling inhibitor added to the medium is as described in [3-1] above.

[0140] The medium in step (b3) may contain a TGF-β receptor signaling inhibitor, the amount of which is as described in [3-2] above.

[0141] The medium in step (b3) may contain ZnSO4. The amount of ZnSO4 added to the medium is as described in [3-2] above.

[0142] The medium in step (b3) may contain a growth factor stabilizer (e.g., heparin). The amount of growth factor stabilizer (e.g., heparin) added to the medium is as described in [2] above.

[0143] The medium in step (b3) may contain nicotinamide. The lower limit of the nicotinamide content in the medium is preferably 0.1 mmol / L, more preferably 0.5 mmol / L, more preferably 1 mmol / L, more preferably 3 mmol / L, more preferably 5 mmol / L. The upper limit of the nicotinamide content in the medium is preferably 100 mmol / L, more preferably 80 mmol / L, more preferably 50 mmol / L, more preferably 40 mmol / L, more preferably 30 mmol / L, more preferably 20 mmol / L, more preferably 15 mmol / L, more preferably 12 mmol / L, more preferably 10 mmol / L, more preferably 5 mmol / L.

[0144] The medium in step (b3) may contain an EGF receptor signal activator (EGF, etc.). The amount of EGF receptor signal activator (EGF, etc.) added to the medium is as described in [3-1] above.

[0145] The medium in step (b3) may contain a ROCK signal inhibitor (Y27632, etc.). When step (b3) is performed in the presence of a ROCK signal inhibitor (such as Y27632), the lower limit of the content of the ROCK signal inhibitor (such as Y27632) in the medium is preferably 0.1 μmol / L, more preferably 0.5 μmol / L, more preferably 1 μmol / L, more preferably 3 μmol / L, more preferably 5 μmol / L, more preferably 6 μmol / L, more preferably 7 μmol / L, more preferably 8 μmol / L, more preferably 9 μmol / L, and more preferably 10 μmol / L. The upper limit of the content of the ROCK signal inhibitor (such as Y27632) in the medium is preferably 100 μmol / L, more preferably 80 μmol / L, more preferably 50 μmol / L, more preferably 40 μmol / L, more preferably 30 μmol / L, more preferably 20 μmol / L, and more preferably 10 μmol / L.

[0146] [4] Differentiation-inducing factors, other additives, and culture conditions used to induce differentiation of pancreatic endocrine progenitor cells (EP) into pancreatic alpha cells In step (c) of the present invention, pancreatic α-cells are produced by culturing pancreatic endocrine precursor cells (EP) under conditions suitable for inducing differentiation into pancreatic α-cells. The culture conditions suitable for inducing differentiation into pancreatic α-cells are not particularly limited, as long as they are culture conditions that can suitably induce differentiation of pancreatic endocrine precursor cells (EP) into pancreatic α-cells. The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of pancreatic endocrine precursor cells (EP) into pancreatic alpha cells, but one embodiment is culturing in the differentiation-inducing medium described below. Step (c) in the present invention is carried out in the absence of ascorbic acid, from the viewpoint of efficiently inducing differentiation of pancreatic endocrine precursor cells (EP) into pancreatic α-cells.

[0147] Step (c) of the present invention is preferably performed under oxygen-supplying conditions. By performing step (c) under oxygen-supplying conditions, pancreatic endocrine precursor cells (EP) can be efficiently induced to differentiate into pancreatic α cells. The oxygen supply conditions are preferably controlled so that the dissolved oxygen concentration in the culture solution is 20 to 50%, when the saturated dissolved oxygen concentration in the culture solution at 37°C under 1 atm is taken as 100%. The dissolved oxygen concentration in the culture medium can be measured, for example, using a dissolved oxygen meter such as a fluorescent oxygen sensor or a galvanic cell oxygen sensor.

[0148] The oxygen supply method is not particularly limited as long as it can supply oxygen to the medium. Examples include surface aeration, in which air or oxygen is passed over the top of the culture medium; sparging, in which air or oxygen is supplied to the culture medium using a sparger; and methods of supplying oxygen to the culture medium using microbubbles or nanobubbles, with surface aeration being preferred. These oxygen supply methods can also be combined with step (c) performed under stirred culture. The stirring conditions for stirred culture are not particularly limited as long as they allow oxygen to be supplied to the culture medium. The upper limit is preferably 200 rpm, more preferably 150 rpm, even more preferably 120 rpm, more preferably 100 rpm, more preferably 90 rpm, more preferably 80 rpm, even more preferably 70 rpm, particularly preferably 60 rpm, and most preferably 55 rpm. The lower limit is preferably 1 rpm, more preferably 10 rpm, even more preferably 20 rpm, more preferably 30 rpm, more preferably 40 rpm, particularly preferably 50 rpm, and most preferably 55 rpm.

[0149] In step (c) of the present invention, pancreatic endocrine precursor cells (EP) may be cultured in the presence of an insulin receptor signal activator, transferrin, and selenite.

[0150] The amounts of insulin receptor signal activator, transferrin, and selenious acid added to the medium are as described above in <Medium>.

[0151] The medium in step (c) may contain a TGF-β receptor signaling inhibitor, the amount of which is as described in [3-2] above.

[0152] The medium in step (c) may contain ZnSO4. The amount of ZnSO4 added to the medium is as described in [3-2] above.

[0153] The medium in step (c) may contain a GLP-1 (Glucagon-like peptide-1) receptor signal activator (such as Exendin4). The lower limit of the amount of GLP-1 (Glucagon-like peptide-1) receptor signal activator (such as Exendin4) to be added to the medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 20 ng / mL, more preferably 30 ng / mL, more preferably 40 ng / mL, and more preferably 50 ng / mL. The upper limit of the amount of GLP-1 receptor signal activator (such as Exendin4) to be added to the medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 90 ng / mL, more preferably 80 ng / mL, more preferably 70 ng / mL, more preferably 60 ng / mL, and more preferably 50 ng / mL.

[0154] The medium in step (c) may contain a growth factor stabilizer (e.g., heparin), the amount of which is as described in [2] above.

[0155] The medium in step (c) may contain nicotinamide. The amount of nicotinamide added to the medium is as described in [3-3] above.

[0156] The medium in step (c) may contain a TGFβ superfamily signal activator (such as BMP4). The lower limit of the amount of TGFβ superfamily signal activator (BMP4, etc.) added to the culture medium is preferably 0.2ng / mL, more preferably 0.5ng / mL, more preferably 1ng / mL, more preferably 3ng / mL, more preferably 5ng / mL, more preferably 6ng / mL, more preferably 7ng / mL, more preferably 8ng / mL, more preferably 9ng / mL, more preferably 10ng / mL. The upper limit of the amount of TGFβ superfamily signal activator (BMP4, etc.) added to the culture medium is preferably 100ng / mL, more preferably 90ng / mL, more preferably 80ng / mL, more preferably 70ng / mL, more preferably 60ng / mL, more preferably 50ng / mL, more preferably 40ng / mL, more preferably 30ng / mL, more preferably 20ng / mL, more preferably 10ng / mL.

[0157] The medium in step (c) may contain hepatocyte growth factor (HGF). The lower limit of the amount of hepatocyte growth factor (HGF) added to the culture medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 20 ng / mL, more preferably 30 ng / mL, more preferably 40 ng / mL, and more preferably 50 ng / mL. The upper limit of the amount of hepatocyte growth factor (HGF) added to the culture medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 90 ng / mL, more preferably 80 ng / mL, more preferably 70 ng / mL, more preferably 60 ng / mL, and more preferably 50 ng / mL.

[0158] The medium in step (c) may contain an insulin-like growth factor (IGF1, etc.). The lower limit of the amount of insulin-like growth factor (IGF1, etc.) added to the culture medium is preferably 1 ng / mL, more preferably 5 ng / mL, more preferably 10 ng / mL, more preferably 20 ng / mL, more preferably 30 ng / mL, more preferably 40 ng / mL, more preferably 50 ng / mL. The upper limit of the amount of insulin-like growth factor (IGF1, etc.) added to the culture medium is preferably 500 ng / mL, more preferably 400 ng / mL, more preferably 300 ng / mL, more preferably 200 ng / mL, more preferably 100 ng / mL, more preferably 90 ng / mL, more preferably 80 ng / mL, more preferably 70 ng / mL, more preferably 60 ng / mL, more preferably 50 ng / mL.

[0159] The medium in step (c) may contain an adenylate cyclase activator (such as forskolin). The lower limit of the content of adenylate cyclase activator (e.g., forskolin) in the medium is preferably 0.1 μmol / L, more preferably 0.3 μmol / L, more preferably 0.5 μmol / L, more preferably 0.7 μmol / L, more preferably 1 μmol / L, more preferably 2 μmol / L, more preferably 3 μmol / L, more preferably 4 μmol / L, and more preferably 5 μmol / L. The upper limit of the content of adenylate cyclase activator (e.g., forskolin) in the medium is preferably 100 μmol / L, more preferably 80 μmol / L, more preferably 50 μmol / L, more preferably 40 μmol / L, more preferably 30 μmol / L, more preferably 20 μmol / L, more preferably 15 μmol / L, more preferably 10 μmol / L, more preferably 7 μmol / L, and more preferably 5 μmol / L.

[0160] [5] Maintenance culture of pluripotent stem cells In the differentiation induction method of the present invention, endodermal cells induced to differentiate from pluripotent stem cells are used. It is preferable that the pluripotent stem cells before induction of differentiation into endodermal cells be maintained in an undifferentiated state using an undifferentiation maintenance medium. Culture that maintains the undifferentiated state of pluripotent stem cells using an undifferentiation maintenance medium is also called maintenance culture of pluripotent stem cells.

[0161] The undifferentiated state maintenance medium is not particularly limited as long as it is a medium that can maintain the undifferentiated state of pluripotent stem cells. Examples include a medium containing leukemia inhibitory factor, which is known to have the property of maintaining the undifferentiated state of mouse embryonic stem cells and mouse induced pluripotent stem cells, and a medium containing basic FGF (fibroblast growth factor), which is known to have the property of maintaining the undifferentiated state of human iPS cells. For example, human iPS cell culture medium (DMEM / Ham's F12 (Wako) containing 20% ​​knockout serum replacement (KSR; Gibco), 1× non-essential amino acids (NEAA; Wako), 55 μmol / L 2-mercaptoethanol (2-ME; Gibco), 7.5 ng / mL recombinant human fibroblast growth factor 2 (FGF2; Peprotech), 0.5× penicillin and streptomycin (PS; Wako), or Essentail 8 medium (Thermo Fisher Scientific), STEMPRO (registered trademark) hESC SFM (Life Technologies Japan, Inc.), mTeSR1 (Veritas), TeSR2 (Veritas), StemFit (registered trademark), etc. can be used, but is not particularly limited.

[0162] Pluripotent stem cells can be maintained in the undifferentiated state maintenance medium on suitable feeder cells (e.g., SL10 feeder cells, SNL feeder cells, etc.) or on cell culture dishes coated with cell adhesion proteins or extracellular matrices such as vitronectin, fibronectin, laminin, collagen, or Matrigel.

[0163] The culture temperature is not particularly limited as long as it is suitable for culturing the pluripotent stem cells used, but is generally 30°C to 40°C, and preferably about 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0164] Pluripotent stem cells can be maintained in culture for a desired period of time while being subcultured. For example, it is preferable to form aggregates and induce differentiation using pluripotent stem cells that have been subcultured for 1 to 100 passages, preferably 10 to 50 passages, and more preferably 25 to 40 passages after maintenance culture.

[0165] [6] Formation of aggregates in suspension culture of pluripotent stem cells In one embodiment for forming pluripotent stem cell aggregates, undifferentiated cells undergoing maintenance culture are detached from feeder cells using accutase (Innovative Cell Technologies) or accumax (Innovative Cell Technologies), and then rinsed three to four times with human iPS cell medium to remove the feeder cells. The cells are then broken down into small cell clumps or single cells by pipetting, and the cells are suspended in medium and cultured in suspension with stirring or rotation until the pluripotent stem cells in the suspension form aggregates.

[0166] Suspension culture may be static culture using a microwell or the like with a viscous medium or unevenness, or culture under conditions in which the liquid medium is flowing using a spinner or the like, but culture under conditions in which the liquid medium is flowing is preferred. Culture under conditions in which the liquid medium is flowing so as to promote cell aggregation is preferred. Examples of culture under conditions in which the liquid medium is flowing so as to promote cell aggregation include culture under conditions in which the liquid medium is flowing so that cells gather at a single point due to stress (centrifugal force, centripetal force) caused by flow such as swirling flow or oscillating flow, or culture under conditions in which the liquid medium is flowing by linear reciprocating motion, with swirling flow and / or oscillating flow being particularly preferred. Furthermore, cells may be cultured in suspension by adhering them to microcarriers or the like in advance, or in the form of cell aggregates composed only of cells, or polymers such as collagen may be mixed within the cell aggregates; the morphology is not particularly limited.

[0167] The culture vessel used for suspension culture is preferably one with low cell adhesion to the vessel inner surface. Examples of such vessels with low cell adhesion to the vessel inner surface include plates with hydrophilic surface treatment using a biocompatible substance. For example, Nunclon TM Sphera (Thermo Fisher Scientific Inc.) can be used, but is not particularly limited. The shape of the culture vessel is also not particularly limited, and examples include culture vessels in the shape of a dish, flask, well, bag, spinner flask, etc.

[0168] The period for forming the aggregates is not particularly limited as long as it is more than 6 hours, but specifically, it is preferable to form the aggregates over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0169] The suspension culture medium is not particularly limited as long as it contains components that allow pluripotent stem cells to grow. Examples include mTeSR1 (Veritas) medium containing 1 to 100 μM Y-27632 (Cayman), and Essential 8 medium containing 1 to 100 μM Y-27632 (Cayman) and 1 to 100 mg / mL BSA. TM etc. can be used.

[0170] The stirring or rotation conditions for suspension culture are not particularly limited as long as they allow pluripotent stem cells to form aggregates in the suspension. The upper limit is preferably 200 rpm, more preferably 150 rpm, even more preferably 120 rpm, more preferably 100 rpm, more preferably 90 rpm, more preferably 80 rpm, more preferably 70 rpm, more preferably 60 rpm, particularly preferably 50 rpm, and most preferably 45 rpm. The lower limit is preferably 1 rpm, more preferably 10 rpm, even more preferably 20 rpm, more preferably 30 rpm, more preferably 40 rpm, and particularly preferably 45 rpm. The rotation width during rotation culture is not particularly limited, but the lower limit can be, for example, 1 mm, preferably 10 mm, more preferably 20 mm, and most preferably 25 mm. The upper limit of the rotation width can be, for example, 200 mm, preferably 100 mm, preferably 50 mm, more preferably 30 mm, and most preferably 25 mm. The rotation radius during rotation culture is also not particularly limited, but is preferably set so that the rotation width is within the above-mentioned range. The lower limit of the rotation radius is, for example, 5 mm, preferably 10 mm, and the upper limit can be, for example, 100 mm, preferably 50 mm. Setting the rotation culture conditions within this range is preferred because it facilitates the production of cell aggregates of appropriate dimensions.

[0171] Furthermore, suspension culture may be rocking culture, which is performed by moving the liquid medium by rocking agitation. Rocking culture is performed by rocking a culture vessel containing the liquid medium and cells in a plane perpendicular to a generally horizontal plane. The rocking speed is not particularly limited, but can be, for example, 2 to 50 times per minute, preferably 4 to 25 times per minute (one round trip counts as one rotation). The rocking angle is not particularly limited, but can be, for example, 0.1° to 20°, more preferably 2° to 10°. Setting the rocking culture conditions within this range is preferred because it enables the production of cell aggregates of appropriate dimensions.

[0172] Furthermore, the culture can be carried out while stirring by a combination of the above-mentioned rotation and rocking movements.

[0173] Suspension culture using a spinner flask-type culture vessel is a culture in which a liquid medium is stirred using a stirring blade inside the culture vessel. The rotation speed and the amount of medium are not particularly limited. If a commercially available spinner flask-type culture vessel is used, the amount of culture medium recommended by the manufacturer can be suitably used. For example, a spinner flask from ABLE can also be suitably used.

[0174] In the present invention, the seeding density of cells in suspension culture is not particularly limited as long as the cells form aggregates. 5 ~1×10 7 The seeding density of cells is preferably 2 x 10 cells / mL. 5 cells / mL or more, 3×10 5 cells / mL or more, 4×10 5 cells / mL or more, or 5 x 10 5 cells / mL or more is preferred, 9×10 6 cells / mL or less, 8×10 6 cells / mL or less, 7×10 6 cells / mL or less, 6×10 6 cells / mL or less, 5×10 6 cells / mL or less, 4×10 6 cells / mL or less, 3×10 6cells / mL or less, 2×10 6 cells / mL or less, 1.9×10 6 cells / mL or less, 1.8×10 6 cells / mL or less, 1.7×10 6 cells / mL or less, 1.6×10 6 cells / mL or less, 1.5×10 6 cells / mL or less is preferable. In particular, 5 x 10 5 cells / mL to 1.5 × 10 6 A cell density in the range of cells / mL is preferred.

[0175] Cell aggregates contain hundreds to thousands of cells per aggregate. In the present invention, the size (diameter) of the cell aggregate is not particularly limited, but examples include 50 μm or more, 55 μm or more, 60 μm or more, 65 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, and 150 μm or more, and 1000 μm or less, 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, and 400 μm or less. Cell aggregates with diameters in the range of 150 μm to 400 μm are suitable in the present invention. Cell aggregates with diameters outside the above range may also be present.

[0176] The amount of culture solution used for suspension culture can be adjusted appropriately depending on the culture vessel used. For example, in a 12-well plate (where the area of ​​the bottom of each well in plan view is 3.5 cm), 2 When a 6-well plate (where the area of ​​the bottom of each well in a plan view is 9.6 cm²) is used, the amount of the solution can be 0.5 ml or more and 1.5 ml or less per well, and more preferably 1 ml per well. 2When a 125 mL Erlenmeyer flask (with a capacity of 125 mL) is used, the volume can be 1.5 mL or more, preferably 2 mL or more, more preferably 3 mL or more, and 6.0 mL or less, preferably 5 mL or less, more preferably 4 mL or less. For example, when a 125 mL Erlenmeyer flask (with a capacity of 125 mL) is used, the volume can be 10 mL or more, preferably 15 mL or more, more preferably 20 mL or more, more preferably 25 mL or more, more preferably 30 mL or more, and 50 mL or less, more preferably 45 mL or less, more preferably 40 mL or less. For example, when using a 500 mL Erlenmeyer flask (a Erlenmeyer flask with a capacity of 500 mL), the capacity can be 100 mL or more per container, preferably 105 mL or more, more preferably 110 mL or more, more preferably 115 mL or more, more preferably 120 mL or more, and 150 mL or less, more preferably 145 mL or less, more preferably 140 mL or less, more preferably 135 mL or less, more preferably 130 mL or less, and more preferably 125 mL or less. For example, when using a 1000 mL Erlenmeyer flask (a Erlenmeyer flask with a capacity of 1000 mL), the capacity can be 250 mL or more per container, preferably 260 mL or more, more preferably 270 mL or more, more preferably 280 mL or more, more preferably 290 mL or more, and 350 mL or less, more preferably 340 mL or less, more preferably 330 mL or less, more preferably 320 mL or less, and more preferably 310 mL or less. For example, in the case of a 2000 mL Erlenmeyer flask (an Erlenmeyer flask with a capacity of 2000 mL), the capacity can be 500 mL or more per container, more preferably 550 mL or more per container, more preferably 600 mL or more per container, and can be 1000 mL or less per container, more preferably 900 mL or less per container, more preferably 800 mL or less per container, more preferably 700 mL or less per container.For example, in the case of a 3000 mL Erlenmeyer flask (an Erlenmeyer flask with a capacity of 3000 mL), the capacity can be 1000 mL or more per container, preferably 1100 mL or more per container, more preferably 1200 mL or more per container, more preferably 1300 mL or more per container, more preferably 1400 mL or more per container, and more preferably 1500 mL or more per container, and can be 2000 mL or less per container, more preferably 1900 mL or less per container, more preferably 1800 mL or less per container, more preferably 1700 mL or less per container, and more preferably 1600 mL or less per container. For example, in the case of a 2L culture bag (a disposable culture bag with a capacity of 2L), the volume can be 100mL / bag or more, more preferably 200mL / bag or more, more preferably 300mL / bag or more, more preferably 400mL / bag or more, more preferably 500mL / bag or more, more preferably 600mL / bag or more, more preferably 700mL / bag or more, more preferably 800mL / bag or more, more preferably 900mL / bag or more, more preferably 1000mL / bag or more, and 2000mL / bag or less, more preferably 1900mL / bag or less, more preferably 1800mL / bag or less, more preferably 1700mL / bag or less, more preferably 1600mL / bag or less, more preferably 1500mL / bag or less, more preferably 1400mL / bag or less, more preferably 1300mL / bag or less, more preferably 1200mL / bag or less, more preferably 1100mL / bag or less. For example, in the case of a 10 L culture bag (a disposable culture bag with a capacity of 10 L), the capacity can be 500 mL / bag or more, more preferably 1 L / bag or more, more preferably 2 L / bag or more, more preferably 3 L / bag or more, more preferably 4 L / bag or more, more preferably 5 L / bag or more, and can be 10 L / bag or less, more preferably 9 L / bag or less, more preferably 8 L / bag or less, more preferably 7 L / bag or less, and more preferably 6 L / bag or less.For example, in the case of a 20L culture bag (a disposable culture bag with a capacity of 20L), the capacity can be 1L / bag or more, more preferably 2L / bag or more, more preferably 3L / bag or more, more preferably 4L / bag or more, more preferably 5L / bag or more, more preferably 6L / bag or more, more preferably 7L / bag or more, more preferably 8L / bag or more, more preferably 9L / bag or more, more preferably 10L / bag or more, and can be 20L / bag or less, more preferably 19L / bag or less, more preferably 18L / bag or less, more preferably 17L / bag or less, more preferably 16L / bag or less, more preferably 15L / bag or less, more preferably 14L / bag or less, more preferably 13L / bag or less, more preferably 12L / bag or less, and more preferably 11L / bag or less. For example, in the case of a 50 L culture bag (a disposable culture bag with a capacity of 50 L), the volume can be 1 L or more, more preferably 2 L or more, more preferably 5 L or more, more preferably 10 L or more, more preferably 15 L or more, more preferably 20 L or more, more preferably 25 L or more, and can be 50 L or less, more preferably 45 L or less, more preferably 40 L or less, more preferably 35 L or less, and more preferably 30 L or less. When the volume of the culture medium is within this range, cell aggregates of an appropriate size are likely to form.

[0177] The capacity of the culture vessel to be used can be selected appropriately and is not particularly limited, but the lower limit of the area of ​​the bottom surface of the portion containing the liquid medium when viewed in plan is, for example, 0.32 cm 2 , preferably 0.65 cm 2 , more preferably 0.95 cm 2 , and more preferably 1.9 cm 2 , more preferably 3.0 cm 2 , 3.5cm 2 , 9.0cm 2 , or 9.6 cm 2 The upper limit of the culture vessel is, for example, 1000 cm 2 , preferably 500 cm2 , more preferably 300 cm 2 , more preferably 150 cm 2 , more preferably 75 cm 2 , more preferably 55cm 2 , more preferably 25 cm 2 , and even more preferably 21 cm 2 , and even more preferably 9.6 cm 2 , or 3.5 cm 2 The above culture vessels can be used.

[0178] The culture temperature is not particularly limited as long as it is suitable for culturing the pluripotent stem cells used, but is generally 30°C to 40°C, and preferably about 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0179] [7] Pre-culturing of pluripotent stem cells Before inducing differentiation of the above-mentioned pluripotent stem cell aggregates or pluripotent stem cells into endodermal cells, a cell population can be prepared by carrying out suspension culture using a medium containing 2-mercaptoethanol and stauprimide. The medium used for pre-culture is MEM medium, BME medium, DMEM medium, DMEM / F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, or Essential 6 medium, depending on the cell type. TM Culture medium (Thermo Fisher Scientific) and the like can be used.

[0180] Pre-culture of pluripotent stem cells is carried out in suspension culture, which can be carried out under the conditions for suspension culture described above, and further, the cells may be pre-adhered to microcarriers or the like and then cultured in suspension, or they may be cultured in the form of cell aggregates composed only of cells, or polymers such as collagen may be mixed in the cell aggregates, and the form is not particularly limited.

[0181] The concentration of 2-mercaptoethanol in the medium used for pre-culture is not particularly limited as long as it is within a range that improves the efficiency of differentiation induction, but for example, the concentration of 2-mercaptoethanol is preferably 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, or 50 μM or more, and is preferably 200 μM or less, 150 μM or less, 120 μM or less, 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, or 60 μM or less. The concentration of stauprimide in the medium used for preculture is not particularly limited as long as it is within a range that improves the efficiency of differentiation induction, but for example, the concentration of stauprimide is preferably 0.01 μmol / L, more preferably 0.02 μmol / L, more preferably 0.05 μmol / L, and more preferably 0.1 μmol / L. The upper limit of the stauprimide content in the medium is preferably 10 μmol / L, more preferably 5 μmol / L, more preferably 1 μmol / L, more preferably 0.8 μmol / L, more preferably 0.5 μmol / L, more preferably 0.4 μmol / L, more preferably 0.3 μmol / L, more preferably 0.2 μmol / L, and more preferably 0.1 μmol / L.

[0182] The medium used for pre-culture is also preferably a medium that does not contain FGF2 (Fibroblast Growth Factor 2).The use of a medium that does not contain FGF2 may further improve the efficiency of differentiation into endodermal cells. The medium used for pre-culture is also preferably one that does not contain TGF-β1 (Transforming growth factor-β1).The use of a medium that does not contain TGF-β1 may further improve the efficiency of differentiation into endodermal cells.

[0183] It is also preferable that the medium used for pre-culture does not contain a WNT signaling activator, as the use of a medium not containing a WNT signaling activator may further improve the efficiency of differentiation into endodermal cells. The medium used for pre-culture is also preferably a medium that does not contain activin A (sometimes referred to as "ACTIVIN A" in this specification). Use of a medium that does not contain activin A may further improve the efficiency of differentiation into endodermal cells.

[0184] The preculture medium may contain amino acids, antibiotics, antioxidants, and other additives, such as 0.1 to 2% (volume / volume) NEAA (non-essential amino acids), 0.1 to 2% (volume / volume) penicillin / streptomycin, 0.1 to 20 mg / mL BSA, or 1 to 25% (volume / volume) (preferably 1 to 20% (volume / volume)) knockout serum replacement (KSR).

[0185] The culture temperature is not particularly limited as long as it is suitable for culturing the pluripotent stem cells used, but is generally 30°C to 40°C, and preferably about 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0186] The culture period for pre-culture of pluripotent stem cells is not particularly limited as long as it is the number of days required for culture until pluripotency is improved, but it may be, for example, a period not exceeding one week. More specifically, it is less than 6 days, less than 5 days, less than 4 days, or less than 3 days, or 6 to 48 hours, approximately 12 to 36 hours, or 18 to 24 hours.

[0187] [8] Induction of differentiation into endodermal cells In the present invention, the cell population obtained by the above pre-culture can be induced to differentiate into endodermal cells by culturing it under conditions that allow induction of differentiation into endodermal cells.

[0188] Endodermal cells have the ability to differentiate into tissues of organs such as the digestive tract, lung, thyroid, pancreas, and liver, cells of secretory glands that open into the digestive tract, peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most of the urethra, and part of the ureter), and are sometimes referred to as definitive endoderm (DE). Differentiation of pluripotent stem cells into endodermal cells can be confirmed by measuring the expression levels of genes specific to endodermal cells. Examples of genes specific to endodermal cells include SOX17, FOXA2, CXCR4, AFP, GATA4, and EOMES.

[0189] When inducing differentiation of pluripotent stem cells into endodermal cells, the pluripotent stem cells are cultured using a differentiation-inducing medium. The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of pluripotent stem cells, and examples include serum-containing medium and serum-free medium containing serum substitute components.

[0190] Depending on the type of cells used, media such as medium for primate ES / iPS cells (ReproCell medium), BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and a mixture of two or more media arbitrarily selected from these media can be used. Note that there is no particular limitation on the media as long as they can be used to culture animal cells.

[0191] The differentiation-inducing medium may contain serum components or serum replacement components, such as albumin, insulin, transferrin, fatty acids, collagen precursors, trace elements (e.g., zinc and selenium), B-27 supplement (Thermo Fisher Scientific), N2 supplement, N21 supplement (R&D Systems), NeuroBrew-21 supplement (Miltenyibiotec), knockout serum replacement (KSR), 2-mercaptoethanol, 3′-thiolglycerol, and equivalents thereof.

[0192] The differentiation-inducing medium may further contain various additives, antibiotics, antioxidants, etc. For example, 0.1 mM to 5 mM sodium pyruvate, 0.1 to 2% (volume / volume) non-essential amino acids, 0.1 to 2% (volume / volume) penicillin, 0.1 to 2% (volume / volume) streptomycin, 0.1 to 2% (volume / volume) amphotericin B, catalase, glutathione, galactose, retinoic acid (vitamin A), superoxide dismutase, ascorbic acid (vitamin C), D-α-tocopherol (vitamin E), etc. may be added.

[0193] The differentiation-inducing medium further contains a differentiation-inducing factor, the details of which will be described later.

[0194] The pluripotent stem cells are preferably cultured in suspension during differentiation induction. The cells may be cultured in suspension by adhering them to microcarriers or the like, or in the form of cell aggregates composed of cells only, or the cell aggregates may contain polymers such as collagen, and the morphology is not particularly limited.

[0195] The culture temperature for differentiation induction is not particularly limited as long as it is suitable for culturing the pluripotent stem cells used, but is generally 30°C to 40°C, and preferably about 37°C. It is preferable to use a CO2 incubator or the like to carry out the culture in an atmosphere with a CO2 concentration of about 1 to 10%, preferably 5%.

[0196] The culture period for differentiation culture of pluripotent stem cells into endodermal cells is not particularly limited as long as the cells have become of a cell type exhibiting endodermal cell characteristics, but may be, for example, within 2 weeks, more specifically, between 2 and 8 days, more preferably between 2 and 7 days, and even more preferably between 3 and 6 days, and an example is 4 or 5 days.

[0197] [9] Differentiation-inducing factors and other additives used to induce differentiation into endodermal cells Preferably, the endodermal cells are endodermal cells induced to differentiate by culturing pluripotent stem cells in a medium containing a transforming growth factor-β (TGFβ) superfamily signal activator, followed by culturing in a medium to which FGF2 and BMP4 (bone morphogenetic protein 4) have not been added.

[0198] When activin A is used in a medium containing a TGFβ superfamily signal activator, the initial concentration of activin A added is preferably 1 ng / mL or more, 2 ng / mL or more, 3 ng / mL or more, 5 ng / mL or more, 10 ng / mL or more, 20 ng / mL or more, 30 ng / mL or more, 40 ng / mL or more, or 50 ng / mL or more, and preferably 1,000 ng / mL or less, 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 200 ng / mL or less, 150 ng / mL or less, or 100 ng / mL or less.

[0199] When FGF2 is used in a medium containing a TGFβ superfamily signal activator, the initial concentration of FGF2 added is preferably 1 ng / mL or more, 2 ng / mL or more, 3 ng / mL or more, 5 ng / mL or more, 10 ng / mL or more, 20 ng / mL or more, 30 ng / mL or more, or 40 ng / mL or more, and preferably 1,000 ng / mL or less, 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 200 ng / mL or less, 150 ng / mL, 100 ng / mL or less, 90 ng / mL or less, 80 ng / mL or less, or 70 ng / mL or less.

[0200] When BMP4 is used in a medium containing a TGFβ superfamily signal activator, the initial concentration of BMP4 added is preferably 1 ng / mL or more, 2 ng / mL or more, 3 ng / mL or more, 5 ng / mL or more, 6 ng / mL or more, 7 ng / mL or more, 8 ng / mL or more, 9 ng / mL or more, 10 ng / mL or more, 11 ng / mL or more, 12 ng / mL or more, 13 ng / mL or more, 14 ng / mL or more, or 15 ng / mL or more, and or less than 1,000ng / mL, less than 900ng / mL, less than 800ng / mL, less than 700ng / mL, less than 600ng / mL, less than 500ng / mL, less than 400ng / mL, less than 300ng / mL, less than 200ng / mL, 150ng / mL, less than 100ng / mL, less than 90ng / mL, less than 80ng / mL, less than 70ng / mL, less than 60ng / mL, less than 50ng / mL, less than 40ng / mL, or less than 30ng / mL.

[0201] The medium to which FGF2 and BMP4 are not added preferably contains activin A. When activin A is contained in a culture medium to which FGF2 and BMP4 have not been added, the initial concentration of activin A added is preferably 1 ng / mL or more, 2 ng / mL or more, 3 ng / mL or more, 5 ng / mL or more, 10 ng / mL or more, 20 ng / mL or more, 30 ng / mL or more, 40 ng / mL or more, or 50 ng / mL or more, and preferably 1,000 ng / mL or less, 900 ng / mL or less, 800 ng / mL or less, 700 ng / mL or less, 600 ng / mL or less, 500 ng / mL or less, 400 ng / mL or less, 300 ng / mL or less, 200 ng / mL or less, 150 ng / mL or less, or 100 ng / mL or less.

[0202] The medium to which FGF2 and BMP4 are not added preferably contains at least one selected from the group consisting of insulin, transferrin, sodium selenite, and ethanolamine. The concentration of insulin added is preferably 0.001 μg / mL or more, 0.01 μg / mL or more, 0.05 μg / mL or more, 0.1 μg / mL or more, 0.2 μg / mL or more, and preferably 10,000 μg / mL or less, 1,000 μg / mL or less, 100 μg / mL or less, 10 μg / mL or less, 9 μg / mL or less, 8 μg / mL or less, 7 μg / mL or less, 6 μg / mL or less, 5 μg / mL or less, 4 μg / mL or less, 3 μg / mL or less, 2 μg / mL or less. The concentration of transferrin added is preferably 0.001 μg / mL or more, 0.01 μg / mL or more, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg / mL or more, 0.1 μg / mL or more, 0.11 μg / mL or more, and preferably 10,000 μg / mL or less, 1,000 μg / mL or less, 100 μg / mL or less, 10 μg / mL or less, The concentration of sodium selenite added is preferably 0.001 ng / mL or more, 0.01 ng / mL or more, 0.1 ng / mL or more, and more preferably 10,000 ng / mL or less, 1 ... The concentration of ethanolamine added is preferably 0.001 μg / mL or more, 0.01 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, or 0.04 μg / mL or more, and preferably 10,000 μg / mL or less, 1,000 μg / mL or less, 100 μg / mL or less, 10 μg / mL or less, 1 μg / mL or less, 0.9 μg / mL or less, 0.8 μg / mL or less, 0.7 μg / mL or less, 0.6 μg / mL or less, 0.5 μg / mL or less, or 0.4 μg / mL or less.

[0203] The medium containing a TGFβ superfamily signal activator and / or the medium without FGF2 or BMP4 preferably further contains 2-mercaptoethanol, which can enhance the efficiency of inducing differentiation into endodermal cells.

[0204] It is preferable that the medium containing a TGFβ superfamily signal activator further contains a WNT signal activator.

[0205] When CHIR99021 is used in a medium containing a TGFβ superfamily signal activator, the initial concentration added is preferably 0.01 μM or more, 0.02 μM or more, 0.03 μM or more, 0.04 μM or more, 0.05 μM or more, 0.1 μM or more, 0.2 μM or more, 0.3 μM or more, 0.4 μM or more, 0.5 μM or more, 0.6 μM or more, 0.7 μM or more, 0.8 μM or more, 0.9 μM or more, 1 μM or more, or 2 μM or more, and preferably 100 μM or less, 90 μM or less, 80 μM or less, 70 μM or less, 60 μM or less, 50 μM or less, 45 μM or less, 40 μM or less, 35 μM or less, 30 μM or less, 25 μM or less, 20 μM or less, 15 μM or less, 10 μM or less, or 5 μM or less. More preferably, it is 3 μM or 4 μM.

[0206] The medium containing a TGFβ superfamily signaling activator and / or the medium to which FGF2 and BMP4 are not added contains at least glucose. The lower limit of the glucose concentration in the medium is not particularly limited as long as it allows cell proliferation, but is preferably 0.01 g / L or higher. The upper limit of the glucose concentration in the medium is not particularly limited as long as it does not kill the cells, but is preferably 10 g / L or lower. In another embodiment, from the viewpoint of efficient differentiation into endodermal lineage somatic cells, a medium containing less than 2.0 g / L of glucose is preferred. The glucose concentration in the medium containing a TGFβ superfamily signaling activator and / or the medium to which FGF2 and BMP4 are not added may be 1.0 g / L or lower, 0.9 g / L or lower, 0.8 g / L or lower, 0.7 g / L or lower, or 0.6 g / L or lower. When a medium containing a TGFβ superfamily signal activator and / or a medium to which FGF2 and BMP4 have not been added contains glucose, the lower limit of the glucose concentration is not particularly limited, but may be 0.01 g / L or more, 0.02 g / L or more, 0.05 g / L or more, 0.1 g / L or more, 0.2 g / L or more, 0.3 g / L or more, 0.4 g / L or more, or 0.5 g / L or more.

[0207]

[10] Utilization of pancreatic alpha cells Pancreatic alpha cells obtained by the method of the present invention have the ability to secrete glucagon and are useful as a tool for elucidating the mechanism of glucagon secretion in pancreatic islets and for developing novel diabetes therapeutics. That is, pancreatic alpha cells produced using the method of the present invention can be used to screen for substances that inhibit glucagon secretion from the cells. Substances that inhibit glucagon secretion from pancreatic alpha cells obtained as a result of this screening can be used as novel diabetes therapeutics.

[0208] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples. [Example]

[0209] [Comparative Example 1] Human iPS cells were induced to differentiate into pancreatic beta cells, based on the work of SG Yabe et al., Induction of functional islet-like cells from human iPS cells by suspension culture, Regen. Ther., 10, 69-76, 2019.

[0210] (1) Maintenance culture of human iPS cells Human iPS cells were maintained in an undifferentiated state on mitomycin-C-treated SNL feeder cells in DMEM / HAM'S F12 medium containing 20% ​​(vol / vol) Knockout Serum Replacement (KSR), 1x Non-Essential Amino Acid (NEAA), 55 μM 2-Mercaptethanol (2-ME), 7.5 ng / mL Recombinant Human Fibroblast Growth Factor 2 (FGF2), and 0.5x Penicillin and Streptomycin (PS). Alternatively, they were maintained in an undifferentiated state on vitronectin-coated plates in StemFit medium containing 0.5x Penicillin and Streptomycin. Y-27632 was added to a final concentration of 10 μM only at the time of seeding. Cultures were performed in a 37°C, 5% CO2 incubator.

[0211] (2) Preparation of human iPS cell aggregates After washing the human iPS cells once with PBS(-), Accumax was added. After incubation at 37°C for 5-15 minutes, the cells were dispersed by pipetting to single cells and collected. 4 x 10 7 The cells were suspended in 30 mL of mTeSR1 medium containing 10 μM Y-27632 and seeded into a 30 mL single-use bioreactor (ABLE). The cells were attached to a 6-channel magnetic stirrer (ABLE) and stirred at 45 rpm in a 5% CO2 incubator at 37°C for 2 days.

[0212] (3) Pre-culturing of human iPS cells The cell populations forming the aggregates obtained by the preparation of aggregates were suspended in DMEM / HAM'S F12 containing 20% ​​(vol / vol) KSR, 1x NEAA, 0.1 μM Stauprimide, and 55 μM 2-ME, and transferred to a 30 mL single-use bioreactor. This was attached to a 6-channel magnetic stirrer and stirred at 45 rpm in a 5% CO2 incubator at 37°C for 1 day.

[0213] (4) Induction of differentiation into pancreatic β cells The cell population obtained from the preculture was first induced to differentiate into endodermal cells. Specifically, for the first two days, the cells were cultured in suspension in RPMI 1640 medium containing 0.25 (v / v)% bovine serum albumin (BSA), 1 mM sodium pyruvate, 1x NEAA, 0.4x PS, 80 ng / mL recombinant human activin A, 50 ng / mL FGF2, 20 ng / mL recombinant human bone morphogenetic protein 4 (BMP4), 3 μM CHIR99021, and 55 μM 2-ME. On day three, the cells were cultured in the same medium minus FGF2, BMP4, and CHIR99021. On day four, the cells were cultured in the same medium as day three, supplemented with 0.5 (v / v)% KSR. Suspension culture was carried out in a 30 mL single-use bioreactor (ABLE) equipped with a 6-channel magnetic stirrer in a 5% CO 2 incubator at 37°C while stirring at 45 rpm.

[0214] The endodermal cells obtained above were induced to differentiate into primitive gut cells (PGT). Specifically, they were cultured in suspension for 3 days in RPMI 1640 medium or a 1:1 mixture of DMEM and HAM'S F12 medium containing 0.25 (v / v)% BSA, 1 mM sodium pyruvate, 1x NEAA, 0.4x PS, 1 (v / v)% B-27 supplement, 0.3 (v / v)% insulin-transferrin-selenium-ethanolamine (ITS-X), and 50 ng / mL recombinant human fibroblast growth factor 7 (FGF-7). Suspension culture was performed in a 30 mL single-use bioreactor equipped with a six-channel magnetic stirrer at 45 rpm in a 5% CO2 incubator at 37°C. The resulting primitive gut cells (PGT) were induced to differentiate into posterior foregut cells (PFG). Specifically, the cells were cultured in suspension for 4 days in RPMI 1640 medium or a 3:1 mixture of DMEM and HAM'S F12 medium containing 0.15 (v / v)% BSA, 1x NEAA, 0.4x PS, 1 (v / v)% B-27 Supplement, 0.3 (v / v)% ITS-X, 50 ng / mL FGF-7, 0.3 μM Indolactam V (ILV), 0.2 μM LDN193189, 0.25 μM SANT1, and 0.25 mM ascorbic acid. The suspension culture was performed in a 30 mL single-use bioreactor equipped with a six-channel magnetic stirrer at 45 rpm in a 5% CO2 incubator at 37°C.

[0215] Next, the obtained posterior foregut cells (PGT) were induced to differentiate into pancreatic progenitor cells (PP) in suspension culture for 3 days in DMEM medium containing 0.15 (v / v)% BSA, 1x NEAA, 0.4x PS, 1 (v / v)% B-27 Supplement, 0.25 (v / v)% ITS-X, 50 ng / mL recombinant human fibroblast growth factor 10 (FGF10), 0.04 μM EC23, 0.2 μM LDN193189, 0.25 μM SANT1, 5 μM zinc sulfate, 55 μM 2-ME, and 0.25 mM ascorbic acid. Suspension culture was carried out in a 30 mL single-use bioreactor equipped with a six-channel magnetic stirrer in a 5% CO incubator at 37°C while stirring at 45 rpm.

[0216] Pancreatic progenitor cells (PP) were then induced to differentiate into pancreatic endocrine progenitor cells (EP) in suspension culture for 7 days in DMEM medium containing 0.15 (v / v)% BSA, 1x NEAA, 0.4x PS, 1 (v / v)% B-27 Supplement, 0.3 (v / v)% ITS-X, 20 ng / mL EGF, 50 ng / mL Exendin4, 0.01 μM EC23, 0.2 μM LDN193189, 0.25 μM SANT1, 10 μM RepSox, 0.5 μM DBZ, 10 μM Y-27632, 5 μM Zinc Sulfate, 10 μg / mL Heparin, and 5 mM Nicotinamide, or in a 4:1 mixture of DMEM and HAM'S F12 medium. Suspension culture was carried out in a 30 mL single-use bioreactor equipped with a six-channel magnetic stirrer in a 5% CO incubator at 37°C while stirring at 45 rpm.

[0217] Finally, pancreatic endocrine progenitor cells (EPs) were induced to differentiate into pancreatic β cells by suspension culture for 10–12 days in DMEM medium containing 0.15 (v / v)% BSA, 1× NEAA, 0.4× PS, 1 (v / v)% B-27 supplement, 0.3 (v / v)% ITS-X, 50 ng / mL exendin, 50 ng / mL human recombinant insulin-like growth factor (IGF), 5 μM forskolin, 5 μM zinc sulfate, 10 μM RepSox, 10 μg / mL heparin, and 5 mM nicotinamide, or in a 4:1 mixture of DMEM and HAM's F12 medium. Suspension culture was carried out in a 30 mL single-use bioreactor attached to a 6-channel magnetic stirrer, stirred at 45 rpm, in a 5% CO2 incubator at 37°C, or in a 100 mL single-use bioreactor (ABLE) attached to a microbial culture device (BMZ-P; ABLE), stirred at 50 rpm, in a 5% CO2 environment at 37°C.

[0218] [Example 1] (1) Differentiation induction Differentiation into endodermal cells was induced using the same method as in Comparative Example 1. Differentiation of these endodermal cells into primitive gut cells (PGT) was induced by suspension culture for 3 days in the medium used in Comparative Example 1 supplemented with 0.67 μM LDN193189, 1 μM EC23, or both. Furthermore, 2.5 μM Y-27632 was added to the medium to maintain the shape of the aggregates. Suspension culture was performed in a 30 mL single-use bioreactor equipped with a six-channel magnetic stirrer, stirring at 45 rpm in a 5% CO2 incubator at 37°C. Differentiation into posterior foregut cells (PFG) and beyond was induced using the same method as in Comparative Example 1.

[0219] (2) Quantitative RT-PCR Total RNA was isolated and purified from the cells obtained by differentiation induction in (1) using Isogen (Nippon Gene), and cDNA was synthesized from the obtained total RNA using PrimeScript II (Takara Bio). Quantitative PCR was performed using the obtained cDNA as a template with GoTaq qPCR master mix (Promega) on a MyiQ qPCR machine (Bio-Rad). Detection was performed using the SYBR Green intercalation method, and gene expression levels were compared using relative quantification methods such as the ΔCt method or ΔΔCt method. The expression level of each gene was normalized to the housekeeping gene OAZ1.

[0220] The base sequences of the primers used in quantitative PCR are as follows: OAZ1 F: GTC AGA GGG ATC ACA ATC TTT CAG (SEQ ID NO: 1) OAZ1 R: GTC TTG TCG TTG GAC GTT AGT TC (SEQ ID NO: 2) INS F :TTG TGA ACC AAC ACC TGT GC (Sequence number 3) INS R: GTG TGT AGA AGA AGC CTC GTT CC (SEQ ID NO: 4) GCG F :ACA TTC ACC AGT GAC TAC AGC AAG (SEQ ID NO. 5) GCG R : GGC AAT GTT ATT CCT GTT CCT C (SEQ ID NO: 6) NKX6.1 F:ATC TTC GCC CTG GAG AAG AC (Sequence number 7) NKX6.1 R:CGT GCT TCT TCC TCC ACT TG (SEQ ID NO: 8)

[0221] (Measurement results) The results of measuring gene expression levels are shown in Figure 1. When the cells obtained under each condition of Example 1 were compared with the cells obtained by the method of Comparative Example 1, a decrease in the expression of the INS gene and the NKX6.1 gene, which are genes specific to pancreatic β cells, was observed under the condition where LDN193189 was added. On the other hand, an increase in the expression of the GCG gene, which is a gene specific to pancreatic α cells, was observed under the condition where EC23 was added. This demonstrates that when inducing differentiation of endodermal cells into primitive gut cells (PGT), the addition of LDN193189 suppresses differentiation induction into pancreatic β cells, while the addition of EC23 promotes differentiation induction into pancreatic α cells. Furthermore, under conditions in which both LDN193189 and EC23 were added, the expression of the INS gene and the NKX6.1 gene was decreased, and the expression of the GCG gene was increased, indicating that pancreatic α cells, not pancreatic β cells, were produced (Figure 1). Therefore, the cells obtained in this example were mainly pancreatic α cells, and when differentiation was induced from endodermal cells using primitive gut cells (PGT) by the method of Example 1, pancreatic α cells could be efficiently produced.

[0222] [Example 2] (1) Differentiation induction Primitive gut cells (PGT) were produced from human iPS cells in the same manner as in Example 1. The obtained primitive gut cells (PGT) were induced to differentiate into cells at the posterior foregut (PFG) stage and beyond using a medium in which ascorbic acid was removed from the medium in Comparative Example 1.

[0223] (2) Measurement of GCG secretion amount 2 × 10 cells obtained by differentiation induction in (1) 6 The cells were harvested and cultured for 30 minutes in DMEM medium (containing 2 mM glucose) containing 10 mM HEPES and 0.1% (v / v) BSA. The cells were then washed twice with glucose-free DMEM medium containing 10 mM HEPES and 0.1% (v / v) BSA, and then cultured for 1 hour in 1 mL of DMEM medium containing 2.0 mM glucose, 10 mM HEPES, and 0.1% (v / v) BSA. The GCG concentration in the culture supernatant was then measured using a Glucagon ELISA 10 μl Kit (Mercodia). For comparison, the amount of GCG secreted from the cells obtained in Example 1 and Comparative Example 1 was also measured by the above method.

[0224] (3) Quantitative RT-PCR Quantitative PCR was performed on the cells obtained by differentiation induction in (1) using the same method as that described in Example 1. In this example, the following primers were added to the primers in Example 1 and used for quantitative PCR. ARX ​​F: AAG GAG GTG TGC TAA AGG CTG (SEQ ID NO: 9) ARX ​​R:GCT GGT CCT CTG TTT CCA TTT G (SEQ ID NO: 10) GC F:CTG AGT GCT GCA CCA AAG AG (SEQ ID NO: 11) GC R:ATT TGT GGG TTC CAC GTA GGT AG (SEQ ID NO: 12)

[0225] (Measurement results) A significant difference was observed in the amount of glucagon secreted between the cells obtained by the method of Example 1 and the cells obtained by the method of Example 2. Specifically, the amount of glucagon secreted by the cells obtained by the method of Example 2 was greater than that of the cells obtained by the method of Example 1. In other words, it was found that in the differentiation induction step after primitive gut cells (PGT), removing ascorbic acid from the differentiation induction medium (or culturing in a differentiation induction medium not containing ascorbic acid) efficiently induced differentiation of primitive gut cells into pancreatic alpha cells (Figure 2). Similarly, when the cells obtained by the method of Example 2 are compared with the cells obtained by the method of Comparative Example 1 (FIG. 3), it is found that the cells obtained by the method of Example 2 secrete significantly more glucagon than the cells obtained by the method of Comparative Example 1. This indicates that pancreatic α-cells can be produced more efficiently from human iPS cells by producing primitive gut cells (PGT) by the method of Example 1 and then performing the subsequent differentiation induction step in the absence of ascorbic acid. Therefore, when human iPS cells are induced to differentiate by the method described in the present invention, differentiation into pancreatic β cells is suppressed while differentiation into pancreatic α cells is promoted, thereby enabling efficient production of pancreatic α cells. Next, the results of measuring the gene expression levels of cells obtained by the method of Example 2 and cells obtained by the method of Comparative Example 1 are shown in Figure 4. In the cells obtained by the method of Example 2, expression of the GCG gene, ARX gene, and GC gene, which are genes specific to pancreatic alpha cells, was high, while expression of the INS gene and NKX6.1 gene, which are genes specific to pancreatic beta cells, was low. This also demonstrates that pancreatic alpha cells can be efficiently produced by inducing differentiation of pluripotent stem cells using the method of the present invention.

[0226] [Example 3] In Example 3, the effect of oxygen concentration in the culture medium in the process of inducing differentiation from pancreatic endocrine precursor cells (EP) to pancreatic α cells was examined. (1) Differentiation induction Pancreatic endocrine precursor cells (EP) were produced in the same manner as in Comparative Example 1. For differentiation induction of pancreatic endocrine precursor cells (EP) to pancreatic α cells, the medium of Comparative Example 1 was used, and suspension culture was carried out for 12 days using a microorganism culture device (BMZ-P; Able) while controlling the oxygen concentration in the culture medium. The dissolved oxygen concentration in the culture medium was controlled so that it was the oxygen concentration shown in Table 1, where the saturated oxygen concentration in the culture medium at 1 atm is taken as 100%.

[0227] [Table 1]

[0228] (2) Oxygen concentration measurement The oxygen concentration in the culture medium was measured over time using a fluorescent dissolved oxygen meter. The measured values ​​show the amount of dissolved oxygen (%) when the saturated dissolved oxygen concentration in the culture medium at 1 atm is taken as 100%.

[0229] (3) Quantitative RT-PCR During the stage of inducing differentiation from pancreatic endocrine precursor cells (EP) to pancreatic α cells, the obtained cells were collected every two days, and quantitative RT-PCR was carried out by the method described in Example 1.

[0230] (Measurement results) The oxygen concentration during cultivation in #3-1 is shown in Figure 5. When the oxygen concentration was not controlled, the amount of dissolved oxygen was about 15% due to oxygen consumption by the cells. On the other hand, when the oxygen concentration was controlled using the cultivation device, the dissolved oxygen concentration could be controlled to within about ±10% of the target oxygen concentration. The gene expression levels of the obtained cells were measured, and the results are shown in Figures 6 and 7. Compared to the oxygen concentration non-controlled group (Comparative Example 1), the expression of the GCG gene, a gene specific to pancreatic α cells, was higher in the oxygen concentration controlled group (Example 3) (Figure 6), and the expression of the INS gene, a gene specific to pancreatic β cells, was lower in the oxygen concentration controlled group (Figure 7). This indicates that pancreatic α-cells can be produced more efficiently if the oxygen concentration is appropriately controlled during the differentiation induction process from pancreatic endocrine precursor cells (EP) to pancreatic α-cells.

Claims

1. (a) inducing differentiation of endodermal cells, which have been induced to differentiate from pluripotent stem cells, into primitive gut cells (PGT) by culturing them in the presence of a bone morphogenetic protein (BMP) signal inhibitor and retinoic acid or a retinoic acid analog; (b) culturing the primitive intestinal cells (PGT) to induce differentiation into pancreatic endocrine precursor cells (EP); and (c) culturing the pancreatic endocrine precursor cells (EPs) to induce differentiation into pancreatic α cells; Including, The endodermal cells are definitive endoderm cells, The steps (b) and (c) are carried out in the absence of ascorbic acid; The step (c) is carried out under oxygen supply conditions, The oxygen supply conditions are controlled so that the dissolved oxygen concentration in the culture solution is 20% or more, when the saturated dissolved oxygen concentration in the culture solution at 37°C under 1 atm is taken as 100%. A method for inducing differentiation into pancreatic alpha cells.

2. The method for inducing differentiation into pancreatic α cells according to claim 1, wherein the oxygen supply conditions are controlled so that the dissolved oxygen concentration in the culture solution is 20 to 50%, when the saturated dissolved oxygen concentration in the culture solution at 37°C under 1 atm is taken as 100%.

3. The method for inducing differentiation into pancreatic α cells according to claim 1 or 2, wherein the step (a) is carried out in the presence of a ROCK signal inhibitor.

4. The method for inducing differentiation into pancreatic alpha cells according to any one of claims 1 to 3, wherein the endodermal cells induced to differentiate from the pluripotent stem cells are endodermal cells induced to differentiate by culturing the pluripotent stem cells in a medium containing a TGFβ superfamily signal activator, and then culturing the pluripotent stem cells in a medium to which FGF2 and BMP4 are not added.

5. The step (b) (b1) inducing differentiation of the primitive gut cells (PGT) into posterior foregut cells (PFG) by culturing the PGT in the presence of a protein kinase C (PKC) activator; (b2) inducing differentiation of the posterior foregut cells (PFG) into pancreatic progenitor cells (PP) by culturing the PFG in the presence of retinoic acid or an analog thereof; and (b3) inducing differentiation of the pancreatic progenitor cells (PP) into pancreatic endocrine progenitor cells (EP) by culturing the pancreatic progenitor cells (PP) in the presence of a Notch signal inhibitor and a ROCK signal inhibitor: The method for inducing differentiation into pancreatic α cells according to any one of claims 1 to 4, comprising:

6. The method for inducing differentiation into pancreatic α cells according to any one of claims 1 to 5, wherein the step (c) comprises a step of inducing differentiation of the pancreatic endocrine precursor cells (EP) into pancreatic α cells by culturing the pancreatic endocrine precursor cells (EP) in the presence of an insulin receptor signal activator, transferrin, and selenious acid.

7. The method for inducing differentiation into pancreatic α cells according to any one of claims 1 to 6, wherein the culture is performed in suspension culture.

8. The method for inducing differentiation into pancreatic α cells according to any one of claims 1 to 7, wherein the oxygen supply is carried out under agitated culture.

Images