Differentiation of human embryonic stem cells into single hormonal insulin positive cells

Abstract

To provide single hormonal insulin producing cells differentiated from pluripotent stem cells, and an in vitro method for differentiation.SOLUTION: The present invention provides an in vitro differentiated population of pancreatic endoderm cells obtained from the stepwise differentiation of pluripotent cells, wherein cells at each step of differentiation are cultured in medium comprising 5 mM to 20 mM glucose.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 61 / 579,313, filed December 22, 2011. 51, which application is incorporated herein in its entirety and is hereby incorporated by reference. This document is hereby incorporated by reference for any purpose.

[0002] FIELD OF THE INVENTION The present invention is in the field of cell differentiation. More specifically, the present invention relates to a method for producing a cell that differentiates cells at each step of stepwise differentiation. Single-hormone insulin-producing cells differentiated from pluripotent stem cells using defined conditions More than 10% of the differentiated insulin-producing cells in the population are single hormone receptors. They express markers characteristic of pancreatic beta cells. [Background technology]

[0003] Advances in cell replacement therapy for type 1 diabetes and the shortage of transplantable islets of Langerhans have led to a decline in engraftment. Attention is focused on developing a source of insulin-producing cells, i.e., β cells, suitable for the treatment of leukemia. One approach is to generate functional beta cells from pluripotent stem cells, such as embryonic stem cells. This sometimes happens.

[0004] During vertebrate embryonic development, pluripotent cells differentiate into three types in a process known as gastrulation. give rise to a group of cells comprising the germ layers (ectoderm, mesoderm, and endoderm). For example, the thyroid Tissues such as the thymus, pancreas, intestine, and liver develop from the endoderm through intermediate stages. The intermediate stage in this process is the formation of definitive endoderm cells. tive endoderm cells) express HNF3β, GATA4, MIXL1, CXCR4, and SO It expresses many markers, including X17.

[0005] By the end of gastrulation, the endoderm specifically marks the anterior, middle, and posterior regions of the endoderm. It is divided into anterior-posterior domains that can be recognized by the expression of a panel of factors that interact with it. For example, Hhex and Sox2 specify the anterior region of the endoderm, and Cdx1, 2, and 4 Identify the second half.

[0006] Migration of endoderm tissue brings it into close proximity with distinct mesodermal tissues that help to regionalize the gut tube. This includes, for example, FGF, Wnt, TGF-B, retinoic acid (RA), and BMP receptors. This is achieved by a number of secreted factors, such as ATP, ATPases, and their antagonists. For example, FGF4 and BMP promote the expression of Cdx2 in the presumptive hindgut endoderm and It inhibits the expression of the genes Hhex and SOX2 (Development 2000 WNT signaling also promotes hindgut development and It has been shown to act in parallel with FGF signaling to inhibit intestinal fate (Development 2007,134:2207~2217). Finally, the mesenchyme Retinoic acid secreted by the retinoic acid regulates the foregut-hindgut boundary (Curr Biol 2002,12:1215~1220).

[0007] Expression levels of specific transcription factors can be used to specify tissue identity During the transformation of the definitive endoderm into the gastrula, the gut undergoes restricted gene expression patterns. The turns are then regionalized into large domains that can be observed at the molecular level. For example, The intestinal-regionalized pancreatic domain exhibited very high expression of PDX-1 and CDX2 and S Similarly, the presence of high levels of Foxe1 indicates that esophageal tissue expresses very little OX2. NKX2.1 is highly expressed in lung tissue. SOX2 / Odd OSR1 is highly expressed in gastric tissue. SOX17 is highly expressed in bile duct tissue. PDX1, NKX6.1 / PTf1a and NKX2.2 are highly expressed in pancreatic tissue. DX2 expression is high in intestinal tissue. The above summary is from Dev Dyn 2009, 238 :29~42 and Annu Rev Cell Dev Biol 2009,25:2 This is a quote from 21-251.

[0008] Pancreas formation results from the differentiation of definitive endoderm into pancreatic endoderm (Annu Rev Ce ll Dev Biol 2009,25:221~251;Dev Dyn 2009 ,238:29-42). The dorsal and ventral pancreatic domains arise from the foregut epithelium. The intestine gives rise to the esophagus, trachea, lungs, thyroid gland, stomach, liver, pancreas, and biliary system.

[0009] Cells of the pancreatic endoderm express the pancreatic-duodenal homeobox gene PDX1. In the absence of 1, the pancreas does not develop beyond the formation of ventral and dorsal buds. Expression of PDX1 marks a critical step in pancreatic organogenesis. Among other cell types, exocrine and endocrine tissues include: It arises from differentiation of pancreatic endoderm.

[0010] D'Amour et al. reported that human embryonic stem cell-derived erythrocytes were cultured in the presence of high concentrations of activin and low serum. describe the production of an enriched medium for definitive endoderm from the mouse embryo (Nature Biotechnol 2005,23:1534~1541;US Patent No.7,704,73 8) Transplantation of these cells under the kidney capsule in mice resulted in the formation of more endodermal tissue-like structures. Human embryonic stem cells differentiated into mature cells (U.S. Patent No. 7,704,738). The definitive endoderm cells derived from the blastocysts were PDX1-positive cells after the addition of FGF-10 and retinoic acid. (U.S. Patent Publication No. 2005 / 0266554A1). Subsequent transplantation of these pancreatic progenitor cells under the kidney capsule of failing mice resulted in maturation within 3–4 months. After this period, functional pancreatic endocrine cells were formed (U.S. Pat. No. 7,993,920 and and U.S. Patent No. 7,534,608).

[0011] Fisk et al. report a system for the production of pancreatic islet cells from human embryonic stem cells. (U.S. Patent No. 7,033,831). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first transformed into endoderm cells using a combination of sodium butyrate and activin. The cells were then differentiated into BMP antigens such as noggin (U.S. Patent No. 7,326,572). PDX1-positive cells were cultured in combination with EGF or betacellulin, along with an agonist. Terminal differentiation was induced by nicotinamide.

[0012] Small molecule inhibitors have also been used to induce pancreatic endocrine progenitor cells. Small molecule inhibitors of FB receptors and BMP receptors (Development 2011, 1 38:861-871; Diabetes 2011,60:239-247) are significant In addition, small molecule activators have also been used to increase the number of pancreatic endocrine cells in embryonic bodies. It has been used to generate endoderm cells or pancreatic progenitor cells (Curr Opin C ell Biol 2009,21:727~732;Nature Chem Bio l 2009,5:258~265).

[0013] Previous attempts to derive pancreatic progenitor cells from human embryonic stem cells have not yielded the correct pancreatic endoderm. We have revealed the importance of co-expression of PDX-1 and NKX6.1 in the identification of new tumors. The art has reported that a population of cells positive for PDX-1 and NKX6 expression is CDX2 Although previous reports have identified the low or absent expression of The presence of immediately anterior markers has not yet been examined. SOX2, which marks the anterior endoderm, It is not expressed in adult islets and is expressed at very low levels in the developing pancreas. (Dia Betes 2005, 54:3402-4309). In contrast, some of the examples of this application At least 30% of the pancreatic endoderm cells generated from human embryonic stem cells are PDX-1 and The cells were positive for the expression of NKX6.1 and CDX2, and negative for the expression of CDX2 and SOX2. Disclosed are cell populations.

[0014] All previous attempts to generate functional pancreatic β-cells have failed to produce cells with the characteristics of mature β-cells. The mature β cells are characterized by the ability to react with a single hormone. expression of insulin, correct processing of proinsulin to insulin and C-peptide, PD Strong expression of X-1 and NKX6.1, appropriate insulin release in response to glucose, and These include the expression of glucose transporters and high expression of glucokinase. These cells produce endocrine cells that produce the above pancreatic hormones. (Nature Biotech 2006,24:1392~1401) Contains up to 10% insulin-positive cells and up to 20% endocrine cells as measured by physin. Other similar reports (Cell Res 2009, 19:429~438;Stem Cells 2007,25:1940~1953; Diabetes Obes Metab 2008,10:186-194) also In fact, recent studies have shown that pluripotent cells differentiate into non-functional insulin-positive cells. reported that transplantation of multihormonal cells in severe combined immunodeficient (SCID) mice resulted in functional β-cell proliferation. (Diabetes 2011, 60: 239~247;Nature Biotech 2011,29:750~756). In the human fetal pancreas, a portion of the endocrine cells (up to 10-20%) are multihormonal cells. Multihormonal cells disappear in the adult human pancreas (Histochem Cell Biol 1999,112:147~153;J Histochem Cytoch em 2009,57:811~824).

[0015] As the burgeoning field of regenerative medicine continues to mature, the development of terminally differentiated, well-regulated pancreatic Methods for the generation of secretory cells are highly desirable. suitable Appropriate and defined culture conditions Manipulation and precise timing of addition of various pathway activators / inhibitors in human embryonic stem cells It is demonstrated herein that cells can be differentiated in vitro into functional pancreatic beta cells. Specifically, the precise timing of BMP inhibition, the use of vitamin C, and the administration of retinoic acid The use of a phospholipid gradient has been shown to be effective in generating single-hormonal pancreatic endocrine cells. It was revealed. Summary of the Invention [Means for solving the problem]

[0016] The present invention provides a method for the development of pancreatic endoderm lineages (PACEs) obtained in vitro by stepwise differentiation of pluripotent cells. The medium used in each differentiation step provides a population of cells with a specific phenotype (increatic endoderm lineage). In some embodiments, glucose is supplemented at 5 mM to 20 mM at each step of differentiation. Cultivate cells in medium containing M glucose.

[0017] In some embodiments, differentiation of pluripotent stem cells generates a pancreatic endoderm cell population. More than 10% of the cells in the differentiated population express markers characteristic of single-hormonal pancreatic β cells. do.

[0018] In some embodiments, differentiation of pluripotent stem cells generates a pancreatic endoderm cell population. , more than 30% of the differentiated population was positive for PDX-1 and NKX6.1 expression, but C The expression of DX2 and SOX2 is negative.

[0019] In some embodiments, stepwise differentiation is performed in medium further supplemented with a TGF-B ligand. In some embodiments, the stepwise differentiation comprises culturing undifferentiated human embryonic stem cells. The method includes culturing undifferentiated human embryonic stem cells in a medium further supplemented with a WNT activator. In some embodiments, stepwise differentiation involves culturing definitive embryos in medium further supplemented with FGF ligands. In some embodiments, the stepwise differentiation comprises culturing the leaf cells in the presence of an shh inhibitor, FGF ligands, PKC activators, TGF-B ligands, retinoids, and BMP inhibitors In some embodiments, the method further comprises culturing the intestinal cells in a medium further supplemented with a gradient of Stepwise differentiation can be achieved by further treating with PKC activators, shh inhibitors, retinoids, and BMP inhibitors. The method includes culturing posterior foregut cells in a supplemented medium. In some embodiments, stepwise differentiation involves culturing cells in a medium further supplemented with ascorbic acid. The method includes the step of:

[0020] In one embodiment, the present invention relates to differentiation in a medium containing 5 mM to 20 mM glucose. The method comprises culturing the cells at each stage of the process to convert the pluripotent cells into a cell population of the pancreatic endoderm lineage. In some embodiments, the present invention provides an in vitro method for differentiating pluripotent cells into The in vitro method for stepwise differentiation further comprises the addition of a TGF-B ligand and a WNT activator. Pluripotent cells are transformed into definitive endoderm (DE) by culturing them in medium supplemented with In some embodiments, the method for stepwise differentiation of pluripotent cells includes differentiating the pluripotent cells into pluripotent cells. The in vitro method further comprises culturing DE cells in medium supplemented with FGF ligand. In some embodiments, the method comprises differentiating the DE cells into intestinal cells by The in vitro method for stepwise differentiation of the present invention further comprises the addition of an shh inhibitor, an FGF ligand, a PK Intestinal cells were cultured in medium supplemented with C activators, TGF-B ligands, retinoids, and BMP inhibitors. The method includes a step of differentiating the intestinal cells into posterior foregut endoderm cells by culturing the intestinal cells. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further comprises: h inhibitors, FGF ligands, PKC activators, TGF-B ligands, retinoids, and B By culturing intestinal cells in a medium supplemented with MP inhibitors, the intestinal cells were differentiated into posterior foregut endoderm. In some embodiments, the method for stepwise differentiation of pluripotent cells includes differentiating the pluripotent cells into pluripotent cells. In vitro methods also include the use of PKC activators, shh inhibitors, retinoids, and BMP inhibitors. Posterior foregut endoderm cells were cultured in a medium supplemented with an anti-inflammatory agent. In some embodiments, the stepwise differentiation of pluripotent cells includes differentiating the pluripotent cells into pancreatic foregut cells. The in vitro method for further comprises administering an shh inhibitor, a TGF-B inhibitor, and a retinoid. By culturing pancreatic foregut cells in a medium supplemented with In some embodiments, the in vitro differentiation of pluripotent cells comprises a step of differentiating the pluripotent cells. The Toro method further comprises differentiating the pancreatic endoderm cells into a pancreatic β cell population.

[0021] In one embodiment, at least one step of an in vitro method for stepwise differentiation of pluripotent cells is In some embodiments, the differentiation medium is further supplemented with ascorbic acid. More than 10% of the cells in the population are single hormone insulin positive cells. In some embodiments, greater than 30% of the pancreatic endoderm cells in the culture generated by the methods of the invention are PD X-1+, NKX6.1+, SOX2-, and CDX2-.

[0022] In one embodiment, the present invention provides an in vitro method for differentiating human embryonic stem cells into pancreatic β cells. With respect to the Toro method, this method comprises: a) culturing a group of embryos to generate a population of definitive endoderm (DE); Undifferentiated human embryonic stem cells were cultured in a medium supplemented with β-lactam, TGF-B ligands, and WNT activators. b) culturing the cells in the presence of glucose and FGF ligand to generate a population of intestinal cells; c) culturing the DE cells in a medium supplemented with PDX-1 and SOX2; To generate a population of foregut endoderm cells, we used glucose, an shh inhibitor, and an FGF ligand. , PKC activators, TGF-B ligands, retinoids, and BMP inhibitors were supplemented. d) culturing intestinal cells in the culture medium; and d) culturing intestinal cells in the culture medium. To generate a population of cells, glucose, PKC activators, shh inhibitors, and retinoids were used. e) culturing posterior foregut cells in a medium supplemented with PDX-1 and BMP inhibitors; Higher levels of NKX6.1 and lower levels of SOX2 compared to pancreatic foregut cells To obtain a population of pancreatic endoderm cells expressing α-glucose, α-glucose-shh inhibitors, TGF-B, and α-glucose-shh inhibitors, f) culturing pancreatic foregut cells in a medium supplemented with an inhibitor and a retinoid; and In some embodiments, the method of the present invention comprises differentiating endometrial cells into a pancreatic β cell population. The pancreatic β cell population generated by this method is PDX-1+, NKX6.1+, SOX2-, and and CDX2-. In some embodiments, in at least one step of the stepwise differentiation method, In some embodiments, the medium is further supplemented with ascorbic acid. The pancreatic β cells obtained from this method are insulin-producing cells that produce a single hormone, NKX6.1+ and PDX-1+. [Brief explanation of the drawings]

[0023] [Figure 1A] 1 shows FACS histogram expression profiles at S3 day 2 of isotype control markers in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1B] 1 shows FACS histogram expression profiles of chromogranin markers at S3 day 2 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1C] 1 shows FACS histogram expression profiles of markers for KI-67 at S3 day 2 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1D] Figure 1 shows FACS histogram expression profiles at day 2 of S3 for markers of NKX6.1 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1E] 1 shows FACS histogram expression profiles of markers for SOX2 at day 2 of S3 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1F] 1 shows FACS histogram expression profiles of markers for CDX2 at day 2 of S3 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 1G] 1 shows FACS histogram expression profiles of markers for PDX-1 at S3 day 2 in cells differentiated according to Example 1. The percentage expression of each marker is shown in each histogram. [Figure 2A] Figure 1 shows FACS histogram expression profiles of isotype control markers in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2B] Figure 1 shows FACS histogram expression profiles of chromogranin markers in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2C] 1 shows FACS histogram expression profiles of markers for KI-67 in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2D] Figure 1 shows the FACS histogram expression profile of the marker NKX6.1 in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2E] Figure 1 shows FACS histogram expression profiles of the marker SOX2 in cells differentiated according to Example 1 and harvested at S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2F] Figure 1 shows FACS histogram expression profiles of the marker CDX2 in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 2G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 in cells differentiated according to Example 1 and harvested on S4 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3A] Figure 1 shows FACS histogram expression profiles of isotype control markers in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3B] 1 shows FACS histogram expression profiles of chromogranin markers in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3C] Figure 1 shows FACS histogram expression profiles of markers for KI-67 in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3D] Figure 1 shows the FACS histogram expression profile of the marker NKX6.1 in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3E] Figure 1 shows FACS histogram expression profiles of the marker SOX2 in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3F] Figure 1 shows FACS histogram expression profiles of the marker CDX2 in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 3G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 in cells differentiated according to Example 1 and harvested on S5 day 2. The percentage expression of each marker is shown in each histogram. [Figure 4A] Figure 1 shows FACS histogram expression profiles of markers for isotype controls for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4B] Figure 1 shows FACS histogram expression profiles of chromogranin markers for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4C] Figure 1 shows the FACS histogram expression profile of the marker KI-67 for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4D] Figure 1 shows the FACS histogram expression profile of the marker NKX6.1 for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4E] Figure 1 shows the FACS histogram expression profile of the marker SOX2 for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4F] Figure 1 shows the FACS histogram expression profile of the marker CDX2 for cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 4G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 in cells differentiated according to Example 1 and harvested at S5 day 7. The percentage expression of each marker is shown in each histogram. [Figure 5A] Figure 1 shows FACS histogram expression profiles of chromogranin (y-axis) and CDX2 (x-axis) markers for cells differentiated according to Example 1 and harvested at S5 day 2. The percentage co-expression for each plot is indicated in each histogram. [Figure 5B] Figure 1 shows FACS histogram expression profiles of chromogranin (y-axis) and SOX2 (x-axis) markers for cells differentiated according to Example 1 and harvested at S5 day 2. The percentage co-expression for each plot is indicated in each histogram. [Figure 5C] Figure 1 shows FACS histogram expression profiles of chromogranin (y-axis) and NKX6.1 (x-axis) markers for cells differentiated according to Example 1 and harvested on S5 day 2. The percentage co-expression for each plot is indicated in each histogram. [Figure 6A] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6B] 1 shows data from real-time PCR analysis of CD142 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6C] 1 shows data from real-time PCR analysis of FOXE1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6D] 1 shows data from real-time PCR analysis of HNF4-alpha gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6E] 1 shows data from real-time PCR analysis of NKX2.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6F]1 shows data from real-time PCR analysis of NKX2.2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6G] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6H] 1 shows data from real-time PCR analysis of OSR1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6I] 1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6J] 1 shows data from real-time PCR analysis of the expression of the gene for PROX1 in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6K] 1 shows data from real-time PCR analysis of the expression of the gene for PTF1a in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6L] 1 shows data from real-time PCR analysis of SOX17 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6M] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6N] 1 shows data from real-time PCR analysis of inulin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6O]1 shows data from real-time PCR analysis of ZIC1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6P] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6Q] 1 shows data from real-time PCR analysis of glucagon gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6R] 1 shows data from real-time PCR analysis of Ngn3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6S] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 6T] 1 shows data from real-time PCR analysis of somatostatin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 1 and harvested at S2, S3, S4 and S5. [Figure 7A] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7B] 1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7C] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7D]1 shows data from real-time PCR analysis of NGN3 gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7E] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7F] 1 shows data from real-time PCR analysis of albumin gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 7G] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of the H1 cell line differentiated according to Example 2 and harvested on day 3 of S2, S3, or S4. [Figure 8A] 1 shows data from real-time PCR analysis of the expression of the marker NKX6.1 in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8B] 1 shows data from real-time PCR analysis of the expression of the marker PDX-1 in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8C] 1 shows data from real-time PCR analysis of the expression of markers for NGN3 in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8D] 1 shows data from real-time PCR analysis of the expression of markers of NeuroD in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8E] 1 shows data from real-time PCR analysis of the expression of chromogranin markers in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8F] 1 shows data from real-time PCR analysis of the expression of the marker CDX2 in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 8G]1 shows data from real-time PCR analysis of the expression of the marker SOX2 in H1 cells differentiated according to Example 3 and harvested at S2, S3, S4, or S5. [Figure 9A] 1 shows data from real-time PCR analysis of the expression of the marker NKX6.1 in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9B] 1 shows data from real-time PCR analysis of the expression of the marker PDX-1 in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9C] 1 shows data from real-time PCR analysis of the expression of chromogranin markers in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9D] 1 shows data from real-time PCR analysis of the expression of markers for NGN3 in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9E] 1 shows data from real-time PCR analysis of the expression of markers of NeuroD in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9F] 1 shows data from real-time PCR analysis of the expression of the marker CDX2 in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9G] 1 shows data from real-time PCR analysis of the expression of the marker albumin in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 9H] 1 shows data from real-time PCR analysis of the expression of the marker SOX2 in H1 cells differentiated according to Example 4 and harvested on day 4 at S3 and S4. [Figure 10A] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10B]1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10C] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10D] 1 shows data from real-time PCR analysis of NGN3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10E] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10F] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10G] 1 shows data from real-time PCR analysis of albumin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 10H] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 5 and harvested at stage 4. [Figure 11A] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11B] 1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11C] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11D]1 shows data from real-time PCR analysis of NGN3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11E] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11F] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11G] 1 shows data from real-time PCR analysis of albumin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 11H] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 6 and harvested on day 3 of S3 or S6. [Figure 12A] 1 shows data from real-time PCR analysis of the expression of the gene NKX6.1 in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12B] 1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12C] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12D] 1 shows data from real-time PCR analysis of NGN3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12E]1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12F] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 12G] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 7 and harvested on day 3 of S3, S4, or S5. [Figure 13A] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13B] 1 shows data from real-time PCR analysis of PDX-1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13C] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13D] 1 shows data from real-time PCR analysis of NGN3 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13E] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13F] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 13G]1 shows data from real-time PCR analysis of SOX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 8 and harvested at S3, S4, S5, or S6. [Figure 14A] Figure 1 shows FACS histogram expression profiles of markers for isotype control at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14B] Figure 1 shows FACS histogram expression profiles of chromogranin markers at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14C] Figure 1 shows FACS histogram expression profiles of markers for KI-67 at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14D] Figure 1 shows the FACS histogram expression profile of NKX6.1 markers at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14E] Figure 1 shows FACS histogram expression profiles of markers for SOX2 at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14F] Figure 1 shows FACS histogram expression profiles of markers for HNF3B at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14G] Figure 1 shows FACS histogram expression profiles of markers for CDX2 at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 14H] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 at day 4 of S3 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15A]Figure 1 shows FACS histogram expression profiles of markers for isotype control at S4 day 2 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15B] Figure 1 shows the FACS histogram expression profile of NKX6.1 markers at S4 day 2 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15C] Figure 1 shows FACS histogram expression profiles of markers for KI-67 at S4 day 2 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15D] Figure 1 shows FACS histogram expression profiles of chromogranin markers at S4 day 2 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15E] Figure 1 shows FACS histogram expression profiles of markers for SOX2 at day 2 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15F] Figure 1 shows the FACS histogram expression profile of the marker CDX2 at day 2 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 15G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 at day 2 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16A] Figure 1 shows FACS histogram expression profiles of markers for isotype control at S4 day 4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16B] Figure 1 shows the FACS histogram expression profile of NKX6.1 markers at day 4 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16C]Figure 1 shows FACS histogram expression profiles of chromogranin markers at day 4 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16D] Figure 1 shows FACS histogram expression profiles of markers for SOX2 at day 4 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16E] Figure 1 shows the FACS histogram expression profile of the marker CDX2 at day 4 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 16F] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 at day 4 of S4 for cells differentiated according to Example 9. The percentage expression of each marker is shown in each histogram. [Figure 17A] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17B] 1 shows data from real-time PCR analysis of HHex gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17C] 1 shows data from real-time PCR analysis of FOXE1 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17D] 1 shows data from real-time PCR analysis of IPF1 (PDX-1) gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17E] 1 shows data from real-time PCR analysis of NKX2.1 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17F] 1 shows data from real-time PCR analysis of NKX2.2 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17G] 1 shows data from real-time PCR analysis of the expression of the gene for NKX6.1 in cells of the human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17H] 1 shows data from real-time PCR analysis of the expression of the gene for PROX1 in cells of the human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17I] 1 shows data from real-time PCR analysis of SOX2 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 17J] 1 shows data from real-time PCR analysis of SOX9 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 9 and harvested at S1D3, S2D3, S3D4, S4D2, and S4D4. [Figure 18A] Figure 1 shows FACS histogram expression profiles of markers for isotype control at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18B] Figure 1 shows the FACS histogram expression profile of NKX6.1 markers at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18C] Figure 1 shows FACS histogram expression profiles of chromogranin markers at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18D]Figure 1 shows FACS histogram expression profiles of markers for SOX2 at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18E] Figure 1 shows FACS histogram expression profiles of markers for CDX2 at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18F] Figure 1 shows FACS histogram expression profiles of markers for KI-67 at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 18G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 at day 3 of S3 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19A] Figure 1 shows FACS histogram expression profiles of markers for isotype control at S4 day 5 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19B] Figure 1 shows the FACS histogram expression profile of NKX6.1 markers at day 5 of S4 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19C] Figure 1 shows FACS histogram expression profiles of chromogranin markers at S4 day 5 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19D] Figure 1 shows FACS histogram expression profiles of markers for SOX2 at day 5 of S4 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19E] Figure 1 shows the FACS histogram expression profile of the marker CDX2 at day 5 of S4 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19F]Figure 1 shows FACS histogram expression profiles of markers for KI-67 at S4 day 5 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 19G] Figure 1 shows FACS histogram expression profiles of markers for PDX-1 at day 5 of S4 for cells differentiated according to Example 10. The percentage expression of each marker is shown in each histogram. [Figure 20A] 10 shows data from real-time PCR analysis of somatostatin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20B] 10 shows data from real-time PCR analysis of PDX1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20C] 10 shows data from real-time PCR analysis of Pax6 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20D] 10 shows data from real-time PCR analysis of Pax4 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20E] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20F] 10 shows data from real-time PCR analysis of NGN3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20G] 10 shows data from real-time PCR analysis of glucagon gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20H] 1 shows data from real-time PCR analysis of the expression of the gene for NeuroD in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20I] 10 shows data from real-time PCR analysis of insulin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 20J] 10 shows data from real-time PCR analysis of chromogranin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 11. [Figure 21A] 1 shows data from real-time PCR analysis of somatostatin gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21B] 1 shows data from real-time PCR analysis of PDX1 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21C] 1 shows data from real-time PCR analysis of Pax6 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21D] 1 shows data from real-time PCR analysis of Pax4 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21E] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21F] 1 shows data from real-time PCR analysis of NGN3 gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21G] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21H] 1 shows data from real-time PCR analysis of insulin gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21I] 1 shows data from real-time PCR analysis of glucagon gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 21J] 1 shows data from real-time PCR analysis of chromogranin gene expression in cells of human embryonic stem cell line H1 differentiated according to Example 12 and harvested at S4 day 2, S5 day 2, and S5 day 9. [Figure 22A] 1 shows data from real-time PCR analysis of Pax4 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22B] 1 shows data from real-time PCR analysis of Pax6 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22C] 1 shows data from real-time PCR analysis of PDX1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22D] 1 shows data from real-time PCR analysis of the expression of the gene for PTF1a in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22E] 1 shows data from real-time PCR analysis of glucagon gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22F] 1 shows data from real-time PCR analysis of insulin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22G] 1 shows data from real-time PCR analysis of NeuroD gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22H]1 shows data from real-time PCR analysis of ngn3 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22I] 1 shows data from real-time PCR analysis of Zic1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22J] 1 shows data from real-time PCR analysis of CDX2 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22K] 1 shows data from real-time PCR analysis of albumin gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. [Figure 22L] 1 shows data from real-time PCR analysis of NKX6.1 gene expression in cells of the human embryonic stem cell line H1 differentiated according to Example 13 and harvested on day 3 of S5. DETAILED DESCRIPTION OF THE INVENTION

[0024] For clarity of disclosure, and without limitation, the term "modes for carrying out the invention" is used interchangeably with "modes for carrying out the invention." The "Aspects" are divided into the following subsections that describe or illustrate particular features, embodiments, or applications of the invention: Divide.

[0025] definition Stem cells are undifferentiated cells defined by both the ability to self-renew and the ability to differentiate at the single cell level. Stem cells include self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells can also give rise to multiple germ layers (endodermal, mesodermal, and ectodermal). They are characterized by their ability to differentiate in vitro from stem cells (e.g., stem cells) into functional cells of various cell lineages. The blastocysts also give rise to tissues of multiple germ layers after implantation, and after injection into blastocysts, virtually all (if not all) contributes to most (if not all) organizations.

[0026] Stem cells are defined by their developmental potential as (1) totipotent, meaning the ability to give rise to all embryonic and extraembryonic cell types; (2) pluripotency, meaning the ability to give rise to all embryonic cell types; and (3) giving rise to a small set of cell lineages. However, multipotency (which means the ability to develop all of these functions within a specific tissue, organ, or physiological system) For example, hematopoietic stem cells (HSCs) are oligopotent stem cells restricted to self-renewal, blood cell differentiation, and Contains progenitor cells, as well as all cell types and elements that are normal constituents of blood (e.g., platelets) (4) a more restricted subset of cell lineages compared to multipotent stem cells (5) oligopotency, meaning the ability to give rise to one cell lineage (e.g., spermatogenic stem cells); ) are classified as unipotent, meaning they have the ability to produce

[0027] Differentiation occurs when unspecialized ("neutral") or relatively unspecialized cells e.g. It is the process by which specialized cells acquire the characteristics of specialized cells, such as nerve cells or muscle cells. "Commonly differentiated cells" or "differentiated cells" are cells that have become more specialized ("commonly differentiated") within a cell lineage. The term "committed" when applied to the differentiation process " are cells that continue to differentiate into specific cell types or subsets of cell types under normal circumstances and to a point where they cannot differentiate into different cell types or revert to less differentiated cell types. "Dedifferentiation" refers to cells that have progressed in the differentiation pathway. Refers to the process of returning to an unspecialized (or unconstrained) state. In this case, cell lineage is the genetic information of the cell, i.e., which cell it came from and which cell it is derived from. The lineage of a cell defines the genetic makeup of that cell within a given genetic system of development and differentiation. Lineage-specific markers identify the phenotype of cells in a lineage of interest. refers to the characteristics specifically associated with differentiation of uncommitted cells into targeted lineages. can be used to evaluate

[0028] As used herein, a "marker" is a nucleic acid that is differentially expressed in a cell of interest. or polypeptide molecules. In this context, differential expression refers to the level of a positive marker. The level of a marker nucleic acid or polypeptide is increased and the level of a negative marker is decreased. A detectable level is either sufficiently high or low in the cells of interest relative to other cells. Therefore, the cells of interest can be isolated using any of a variety of methods known in the art. It is possible to identify and distinguish them from other cells.

[0029] As used herein, a cell is a cell that expresses a specific marker when the specific marker is detected within the cell. Similarly, cells are "positive for" or "positive for" specific markers detected intracellularly. If not present, the result is "negative for" or "negative" for the specific marker.

[0030] As used herein, "stage 1" and "S1" refer to the development of embryos characteristic of the definitive endoderm (DE). These are used interchangeably to identify cells that express a marker.

[0031] As used herein, "definitive endoderm" refers to the layer of the embryo that arises from the epiblast during gastrulation and is responsible for the development of the gastrointestinal tract. Definitive endoderm cells are cells that have the characteristics of cells that form the ducts and their derivatives. Expresses at least one of the following markers: HNF3β, GATA4, SOX17, CXCR 4, Cerberus, OTX2, Goosecoid, C-Kit, CD99, and MIXL1.

[0032] As used herein, the "gut" refers to a tract that expresses at least one of the following markers: refers to cells derived from the definitive endoderm that: HNF3-β, HNF1-β, or HNF4-α Intestinal cells can give rise to all endodermal organs, such as the lung, liver, pancreas, stomach, and intestine.

[0033] As used herein, a "stem" refers to a cell that expresses markers characteristic of the primitive gut. "Page 2" and "S2" are used interchangeably.

[0034] "Foregut endoderm" refers to the germ layer that gives rise to the esophagus, lungs, stomach, liver, pancreas, gallbladder, and part of the duodenum. It refers to endoderm cells that produce

[0035] The "posterior foregut" can give rise to the posterior stomach, pancreas, liver, and part of the duodenum Refers to endoderm cells.

[0036] The "midgut endoderm" can give rise to the intestine, part of the duodenum, appendix, and ascending colon. It refers to endoderm cells.

[0037] The "hindgut endoderm" gives rise to the distal one-third of the transverse colon, the descending colon, the sigmoid colon, and the rectum. It refers to endoderm cells that can be transformed into stem cells.

[0038] Both "stage 3" and "S3" are cells that express markers characteristic of foregut endoderm. These terms are used interchangeably to identify cells that express markers characteristic of the foregut lineage. As used herein, refers to a cell that expresses at least one of the following markers: PDX-1, FOXA2, CDX2, SOX2, and HNF4 α.

[0039] "Stage 4" and "S4" are cells that express markers characteristic of pancreatic foregut progenitor cells. As used herein, "pancreatic foregut progenitor cell lineage" refers to a cell lineage that is capable of expressing a pancreatic foregut progenitor cell lineage. A "cell expressing markers characteristic of a lineage" expresses at least one of the following markers: Cells refer to: PDX-1, NKX6.1, HNF6, FOXA2, PTF1a, Prox 1, and HNF4 α.

[0040] As used herein, "stage 5" and "S5" refer to the development of pancreatic endoderm and pre-pancreatic endocrine cells. The terms "progenitor cell" and "progenitor cell expressing markers" are used interchangeably to identify cells that express markers characteristic of progenitor cells. As used herein, "cells expressing markers characteristic of the pancreatic endoderm lineage" refers to the following markers: Refers to cells expressing at least one of the following markers: PDX1, NKX6.1, HNF1β , PTF1 α, HNF6, HNF4 α, SOX9, HB9, or PROX1. In the pancreas Cells expressing markers characteristic of germ layer lineages do not substantially express CDX2 or SOX2. do not have.

[0041] As used herein, "stage 6" and "S6" refer to a stage in which pancreatic endocrine cells are enriched. Used interchangeably to identify cells.

[0042] As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "pancreatic endocrine cells" refers to cells that express a pancreatic hormone. "Cells expressing markers characteristic of the secretory lineage" refers to cells expressing at least one of the following hormones: Refers to cells capable of expressing: insulin, glucagon, somatostatin, and pancreatic Polypeptide.

[0043] "Pancreatic endocrine precursor cells" or "pancreatic endocrine precursor cells" refer to pancreatic endoderm cells that can give rise to pancreatic hormone-expressing cells. Such cells can express at least one of the following markers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

[0044] As used herein, "functional pancreatic beta cells" refer to cells that are glucose responsive and PDX-1 and refers to single hormone insulin positive cells that may be positive for NKX6.1.

[0045] As used herein, "d1", "d 1", and "day 1", "d2", "d 2", and "Day 2", "d3", "d 3" and "day 3" are used interchangeably. The combination of letters represents the incubation time at different stages in the stepwise differentiation protocol of the present application. Identify the day of the session.

[0046] "Ascorbic acid" and "vitamin C" are used interchangeably herein and are used in humans and other Concerning essential nutrients for animal species.

[0047] "Glucose" and "D-glucose" are used interchangeably herein and refer to naturally occurring The term "dextrose" refers to the sugar found in

[0048] Cells that are "positive" or marker "+" for a particular marker (i.e., PDX-1+) are cells in which that particular marker can be detected. Marker "-" (i.e., NKX6.1-) cells are identified as being NKX6.1-positive cells, and are identified as being NKX6.1-negative cells, as taught herein. These are cells in which the marker is not detected by the method.

[0049] As used herein, "chromogranin" and "CHGN" refer to the acidic secreted glycoprotein chromogranin. Used interchangeably to identify the gene encoding lanin.

[0050] Identifying proteins expressed in pancreatic endocrine precursor cells and the genes encoding them The terms "NeuroD" and "NeuroD1" are used interchangeably herein.

[0051] "LDN" and "LDN-193189" are used interchangeably herein and are incorporated by reference in their entirety. 1 shows a BMP receptor inhibitor available from Stemgent, California.

[0052] Isolation, expansion and culture of pluripotent stem cells Pluripotent stem cells express stage-specific embryonic antigens (SSEA) 3 and 4, as well as Tra-1 and Tra-6. 0 and one or more of the markers detectable by antibodies called Tra-1-81. (Thomson et al., Science 282:1145, 1998). In vitro differentiation of pluripotent stem cells was achieved using SSEA-4, Tra-1-60, and Tra-1 This results in the loss of expression of -81. Undifferentiated pluripotent stem cells generally lack alkaline phosphatase This is available from the manufacturer (Vector Laboratories, California, USA). After fixing the cells with 4% paraformaldehyde as described by ries, , which can be detected by development using Vector Red as a substrate Undifferentiated pluripotent stem cells also generally express OCT4 as detected by RT-PCR. and TERT are also expressed.

[0053] Another desirable phenotype of expanded pluripotent stem cells is the ability to differentiate into all three germ layers, i.e., endoderm. The ability of stem cells to differentiate into cells of germinal, mesodermal, and ectodermal tissues. The cells were injected into SCID mice, and the malformations formed were analyzed using 4% paraformaldehyde. After fixing the tumors, they were examined histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency can be confirmed by the formation of embryoid bodies, which can be cultured for 3 days. This can be determined by assessing for the presence of markers associated with two germ layers.

[0054] The expanded pluripotent stem cell lines were analyzed using standard G-banding techniques and compared with published methods for the corresponding primate species. The karyotype can be determined by comparing it with the karyotype of the cell. A "normal karyotype" is defined as a cell that is euploid and has all human chromosomes. Pluripotent cells are the most common type of cells in the human body. They are easily grown in culture using a matrix layer or in matrix protein-coated vessels. Alternatively, mTesr™ 1 medium (Vancouver, Canada) can be used. Combined with defined media such as StemCell Technologies Such chemically defined surfaces may be used for routine cell growth. Pluripotent cells can be grown by enzymes. or mechanically, or with various calcium They can be easily removed from the culture plate using a chelating agent. Cells are grown in suspension in the absence of matrix proteins or feeder layers. Good too.

[0055] Sources of pluripotent stem cells The types of pluripotent stem cells that can be used include those derived from embryos at any time during pregnancy (not necessarily during pregnancy). Pre-embryonic tissue (e.g., blastocysts) collected before approximately 10-12 weeks of pregnancy (usually before 10-12 weeks of pregnancy) Established lines of pluripotent cells derived from tissues formed after conception, such as embryonic or fetal tissues. Non-limiting examples include the establishment of human embryonic stem cells (hESCs) or human embryonic germ cells. strains, such as human embryonic stem cell lines H1, H7, and H9 (Madison, Wisconsin, USA). (Dison's WiCell Research Institute) Also suitable are cells taken from a pluripotent stem cell population that has already been cultured in the absence of leader cells. In addition, many pluripotency-related genes, such as OCT4, Nanog, Sox2, KLF4, and ZFP42, have been implicated in pluripotency. Inducible pluripotency can be induced from adult somatic cells using forced expression of relevant transcription factors Also suitable are in vitro pluripotent stem cells (IPS) or reprogrammed pluripotent cells (Annu Rev Genomics Hum Genet, 2011, 12:165-185). The human embryonic stem cells used in this method were prepared as described by Thomson et al. (U.S. Patent No. 5,843,780; Science, 1998, 282:1 145;Curr.Top.Dev.Biol.,1998,38:133;Proc. Natl. Acad. Sci. USA, 1995:92:7844).

[0056] Generation of cells expressing markers characteristic of the pancreatic endoderm lineage from pluripotent stem cells The characteristics of pluripotent stem cells are well known to those skilled in the art, and further characteristics of pluripotent stem cells are continually being developed. Markers of pluripotent stem cells include, for example, the following: ABC G2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OC T4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SS Examples include expression of one or more of EA-4, Tra 1-60, and Tra 1-81.

[0057] Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9(N Human embryonic stem cell line H1 (NIH code: WA01), human embryonic stem cell line H1 (NIH code: WA09), human ... Stem cell line H7 (NIH code: WA07) and human embryonic stem cell line SA002 (Cell artis, Sweden). The following markers are characteristic of pluripotent cells: Wachi, ABCG2, cripto, CD9, FOXD3, CONNEXIN43, CON NEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42 , SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81 Cells expressing at least one of these are also suitable for use in the present invention.

[0058] Markers characteristic of the definitive endoderm lineage include SOX17, GATA4, HNF3 β, and GSC. , CER1, Nodal, FGF8, brachyury, Mix-like homeobox protein, F GF4, CD48, eomesodermin (EOMES), DKK4, FGF17, GATA 6, CXCR4, C-Kit, CD99, and OTX2. Cells expressing at least one of the markers characteristic of the endodermal lineage are suitable for use in the present invention. In one aspect of the present invention, the present invention provides a method for producing a cell lineage that expresses markers characteristic of the definitive endoderm lineage. In another aspect, the cells express markers characteristic of the definitive endoderm lineage. In another embodiment, the cells expressing markers characteristic of the definitive endoderm lineage are mesendoderm cells. Cells expressing β-actin are definitive endoderm cells.

[0059] Markers characteristic of the pancreatic endoderm lineage include PDX1, NKX6.1, HNF1 β, and PTF 1α, HNF6, HNF4α, SOX9, HB9, and PROX1. These cells express at least one of these markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, cells characteristic of the pancreatic endoderm lineage are suitable for use in the present invention. The cells expressing these markers are pancreatic endoderm cells, and express PDX-1 and NKX6.1. The expression is substantially higher than that of CDX2 and SOX2.

[0060] Markers characteristic of the pancreatic endocrine lineage include NGN3, NEUROD, ISL1, and PDX1. NKX6.1, PAX4, ARX, NKX2.2, and PAX6. In one embodiment, the pancreatic endocrine cells produce the following hormones: insulin, glucan, and expressing at least one of pancreatic polypeptide, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention are those containing a small number of markers characteristic of the pancreatic endocrine lineage. In one embodiment of the present invention, the cells express at least one marker characteristic of the pancreatic endocrine lineage. Pancreatic endocrine cells are cells that express pancreatic hormones. Alternatively, the pancreatic endocrine cells may be pancreatic hormone-secreting cells.

[0061] In one aspect of the present invention, the pancreatic endocrine cells express markers characteristic of the β-cell lineage. Cells expressing markers characteristic of the β-cell lineage are those expressing PDX1 and the following transcription factors: Factors, namely, NKX2.2, NKX6.1, NEUROD, ISL1, and HNF3 β The cells express at least one of MAFA, PAX4, and PAX6. In this manner, cells expressing markers characteristic of the beta cell lineage are beta cells.

[0062] The present invention provides a method for the detection of monohormonal insulin-positive cells that are also PDX-1 and NKX6.1 positive. The present invention describes an in vitro method and cell population capable of generating cells. The differentiation of human pluripotent cells into cells of a single hormone is induced stepwise through the following intermediate stages: It includes a series of stages. a) Pluripotent cells in a medium containing glucose, a TGF-B ligand, and a WNT activator 1. The generation of definitive endoderm (DE) cells from undifferentiated human embryonic stem cells, comprising culturing b) Culturing DE cells in a medium containing glucose, vitamin C, and an FGF ligand. Differentiation of DE cells into intestinal cells, including the steps. c) Differentiation of intestinal cells into posterior foregut endoderm cells that express PDX-1 and SOX2. Differentiation is mediated by shh inhibitors, BMP inhibitors, TGF-B ligands, FGF ligands, and retinoic acid. This was achieved by culturing intestinal cells in the presence of phosphate, vitamin C, and a PKC activator. will be done. d) Posterior foregut cells express PDX-1 and NKX6.1 and are compared with posterior foregut cells The differentiation of these cells into pancreatic foregut cells expressing low levels of SOX2. inhibitors, BMP inhibitors, low doses of retinoic acid, vitamin C, and PKC activators This is achieved by culturing posterior foregut cells under e) Pancreatic foregut cells express PDX-1 and higher levels of NKX6 compared to pancreatic foregut cells. The differentiation of pancreatic endoderm cells into pancreatic endoderm cells that express SOX1 and low levels of SOX2. , and supplemented with an shh inhibitor, a TGF-B inhibitor, low-dose retinoic acid, and vitamin C. This is achieved by culturing pancreatic foregut cells in culture medium. f) Differentiating pancreatic endoderm cells into pancreatic endocrine progenitor cells, followed by pancreatic endocrine secretion of a single hormone. The differentiation into cells is induced by the treatment with an shh inhibitor, low doses of retinoic acid, and vitamin This is achieved by culturing pancreatic endoderm cells in medium supplemented with C.

[0063] In one embodiment, the cells are cultured in a medium containing less than 25 mM glucose at all stages of stepwise differentiation. In some embodiments, the glucose concentration is , in the range of about 8 mM to about 20 mM glucose.

[0064] In some embodiments, all steps for generating gut-stage cells and thereafter are The media formulations used in this study contain ascorbic acid (also known as vitamin C). In one embodiment, the concentration of ascorbic acid is from about 0.01 mM to about 1 mM. In this embodiment, the concentration of ascorbic acid is about 0.1 mM to about 0.5 mM.

[0065] The present invention will be further illustrated by the following examples, but the present invention is not limited to these examples. It is not something that can be done. [Example]

[0066] Example 1 Differentiation of human embryonic stem cells from cell line H1 into pancreatic endocrine progenitor cells in the absence of fetal bovine serum Modulation of the BMP / TGF-B pathway improves the production of pancreatic endoderm populations and increases the SOX2+ population. This results in a percentage decrease. This example shows that PDX-1 and PDX-2 have low levels of expression of CDX2 and SOX2. We demonstrate that pancreatic endoderm cultures with very high expression levels of NKX6.1 can be generated. This was carried out in order to

[0067] The human embryonic stem cell line H1 (hESC H1) was harvested at various passages (passages 40 to 52). The cells were harvested and treated with 10 μM Y27632 (ROC inhibitor, Cat. No. Y0503, Missouri, USA). The medium was supplemented with mTeSR®1 medium (Sigma-Aldrich, Canada). StemCell Technologies in Vancouver, As a cell, 1cm 2 At a concentration of 100,000 cells per 1000 cells, MATRIGEL™ ( 1:30 dilution, BD Biosciences, New Jersey, USA) 48 hours after seeding, the cultures were resuspended in incomplete PBS (without Mg or Ca). The cultures were washed for approximately 30 seconds in phosphate-buffered saline (PBS) and incubated. The cells were differentiated into the pancreatic endocrine lineage as described above. a. Stage 1 (Definitive Endoderm (DE) - 3 days): Stage 1 medium (0.1% fat-free Acid BSA (Cat. No. 68700, Proliant, Iowa, USA), 0.001 2 g / mL sodium bicarbonate (catalog no. S3187, Sigma, Missouri, USA) Aldrich), 1X GlutaMax™ (Cat. No. 35050-079 , Invitrogen), 5 mM D-glucose (Cat. No. G8769, Millipore, USA) 100 ng / mL GDF8 (Sigma-Aldrich, Zurich) supplemented with 100 ng / mL GDF8 (Sigma-Aldrich, Zurich). R&D Systems, Minnesota, USA) and 1 μM MCX compound (GSK3B inhibitor, 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaa Thetetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa-1(25 ),2(27),3,5,8(26),9,11,21,23-nonane-16-one, No. 12 / 494,789, which is incorporated herein by reference in its entirety. MCDB-131 medium (Cat. No. 10372-019, San Diego, CA, USA) containing The cells were cultured in 0.1% fatty acid-free HCl (Invitrogen, USA) for 1 day. BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 100 ng / mL GDF8, and 100 nM MCX compound The cells were cultured for 1 day in MCDB-131 medium supplemented with 0.1% fatty acid-free HCl. BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, MCDB-131 supplemented with 5 mM D-glucose and 100 ng / mL GDF8 The cells were cultured in the medium for 1 day. b. Stage 2 (gastrulation -2 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM The mice were incubated in MCDB-131 medium supplemented with D-glucose and 25 ng / mL FGF7 for 2 days. It was processed for a while. c. Stage 3 (foregut -2 days): Stage 2 cells were cultured in Stage 3 medium (ITS-X (Invitrogen) at a 1:200 dilution), 2.5 mM glucose, 1X Glu utaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BS A, 25 ng / mL FGF7, 10 ng / mL activin-A (R&D system) tems), 0.25 μM SANT-1 (shh inhibitor, SigmaAldrich) , 1 μM retinoic acid (RA) (SigmaAldrich), and 200 nM TP B (PKC activator, Cat. No. 565740; EMD, New Jersey, USA) Supplemented with 100 nM LDN-193189 (BMP receptor inhibitor; Catalog No. 04 -0019; Stemgent, California, USA) containing MCDB-131x The cells were cultured in PBS for 1 day. Then, the cells were treated with 10 nM LDN-193189. The cells were cultured for an additional day in supplemented Stage 3 medium. d. Stage 4 (Pancreatic foregut progenitor cells - day 2): Stage 3 cells were cultured in ITS-X 1: 200 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 5 M supplemented with 0 nM RA, 200 nM TPB, and 50 nM LDN-193189 The cells were cultured in CDB-131 medium for 2 days. e. Stage 5 (pancreatic endoderm - days 2 to 7): Dilute stage 4 cells with ITS-X at 1:2 ratio. 00 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, and The cells were cultured in MCDB-131 medium supplemented with 50 nM RA for 2 to 7 days.

[0068] At specific stages, real-time PCR, immunohistochemistry, or fluorescence-activated cell sorting (F Samples were collected and analyzed by the ACS.

[0069] For FACS analysis, hESC-derived cells were incubated with TrypLE Express (catalyst). Incubate at 37°C for 3-5 minutes in a 500µL PBS (log number 12604, Invitrogen). The cells were then released into a single cell suspension by centrifugation. The cells were then soaked in staining buffer (0.2% lipid-free). PBS containing fatty acid BSA) (Cat. No. 554657, New Jersey, USA) The cells were washed twice in a 500-well plate (BD Biosciences). The cells were first stained with Green Fluorescent LIVE / DEAD cell dye (Inv Incubate at 4°C for 20 minutes in a 5000kJ / mL syringe (Protein Genetics catalog number L23101). The cells were then washed once with cold PBS. μL of Cytofix / Cytoperm Buffer (BD Bioscience The cells were fixed in BD Perm / W (Cat. No. 554722) for 20 minutes at 4°C, followed by BD Perm / W Ash Buffer Solution (BD Biosciences catalog number The cells were washed twice with 5% PBS (554723). The cells were then supplemented with 2% normal serum (of the appropriate species for the secondary antibody). Resuspend in 100 μL of staining / blocking solution consisting of Perm / Wash buffer. The cells were then incubated with primary antibodies at empirically predetermined dilutions for 30 minutes at 4°C. The cells were incubated and then washed twice with Perm / Wash buffer. Cells were incubated with antibody for 30 min at 4°C, then washed with Perm / Wash buffer. After washing twice with the solution, the cells were analyzed using a BD FACS Canto II.

[0070] The following dilutions of primary antibodies were used: rabbit anti-insulin (1:100; Cat. No. C 27C9, Cell Signaling, Massachusetts, USA), mouse anti-insulin Phosphorus (1:100; catalogue no. ab6999, Abcam, Massachusetts, USA) , mouse anti-glucagon (1:1250; catalog no. G2654; Sigma-Aldrich ich), rabbit anti-synaptophysin (1:100; Cat. No. A0010, California, USA) (Dako, California), rabbit anti-chromogranin A (1:800; Dako), mouse anti- Antibody to NKX6.1 (1:50; DSHB, University of Iowa, USA) Iowa), mouse anti-CDX2 (1:250; Invitrogen), goat anti-Neu roD (1:500; R&D Systems), mouse anti-SOX2 ( mouse anti-NKX2.2 (DSHB), mouse anti-Pax6 (BD, USA); mouse anti-PDX-1 (BD, California, USA); mouse anti-PDX-1 (BD, California, USA); The following antibodies were used: goat anti-mouse Alexa 647 (1:500; Invitrogen) n), goat anti-rabbit PE (1:200; Invitrogen), donkey anti-goat (1:80 After adding the secondary antibody, the samples were incubated at 4°C for 30 minutes. The cells were then washed a final time with Perm / Wash buffer. Cells were analyzed on a BD FACS Canto II using software at least Won 30,000 events.

[0071] 1A to 1G show isotopic images of cells differentiated according to Example 1 and analyzed on day 2 of S3. Type control (Figure 1A), chromogranin (Figure 1B), KI-67 (Figure 1C), NK X6.1 (Figure 1D), SOX2 (Figure 1E), CDX2 (Figure 1F), PDX-1 (Figure 1G) The FACS histogram expression profiles of the markers are shown. On day 2 of stage 3, more than 95% of the cells expressed PDX-1 (Figure 1G). Approximately 60% of the cells in the population were positive for SOX2 expression (Figure 1E). However, CDX2 (Fig. 1F) or NKX6.1 (Fig. 1D), or chromogranin Fewer than 10% of the cells were positive for KI-67 (Figure 1B). At stage 3, a significant percentage of cells are active, as indicated by the high percentage were in the cell cycle (Fig. 1C).

[0072] 2A-2G show FAC of cells differentiated according to Example 1 and harvested on day 2 of S4. Isotype control determined by S staining (Figure 2A), chromogranin (Figure 2B) , KI-67 (Fig. 2C), NKX6.1 (Fig. 2D), SOX2 (Fig. 2E), CDX2 (Fig. The expression profiles of PDX-1 (Fig. 2G) and PDX-2 (Fig. 2F) are shown. The histograms show the PDX-1 expression levels. As in stage 3, more than 95% of the cells were PDX-1 (Figure 2G ) expression, whereas approximately 10% of the cells were positive for CDX2 expression (Fig. 2F). were positive, and approximately 40% of the cells were positive for NKX6.1 expression (Fig. 2D). Approximately 45% of cells were positive for SOX2 expression (Fig. 2E), which was 6% of the time from S3. 0% low. Chromogranin expression was approximately 3% (Fig. 2B). KI-67 positive cells (Fig. At stage 4, a significant percentage of cells were found to be fibroblasts, as indicated by the high percentage of cells in stage 2C. The cells were in an active cell cycle.

[0073] 3A-3G show stage 5 differentiation following the differentiation protocol outlined in this example. Relative expression profiles determined by FACS analysis of cells harvested on day 2 are depicted. Figure 3A: Isotype control; Figure 3B: Chromogranin; Figure 3C: KI-67; 3D: NKX6.1; Fig. 3E: SOX2; Fig. 3F: CDX2; Fig. 3G: PDX-1. The percentage of expression of the markers is shown in each histogram. As in stages 3 and 4, 95% of the cells The above were positive for PDX-1 expression, but approximately 1 in 10 cells were positive for CDX2 expression. 0% were positive, and approximately 67% or more of the cells were positive for NKX6.1 expression. SOX2 expression was approximately 50%, lower than in stage 3, but similar to that in S4. It was.

[0074] 4A-4G show the results of stage 5 differentiation according to the protocol outlined in this example. PDX-1 as measured by FACS staining of cells harvested and analyzed on day 7 (Figure 4G). , NKX6.1 (Figure 4D), CDX2 (Figure 4F), SOX2 (Figure 4E), Ki-67 (increased The expression of chromogranin (a marker for pancreatic endocrine gland; Figure 4B) and chromogranin (a marker for pancreatic endocrine gland; Figure 4C) was shown. Similar to stages 3 and 4, >90% of cells were positive for PDX-1. However, CDX2 expression was less than 10% and NKX6.1 expression was significantly increased to >70%. , SOX2 expression was dramatically reduced to approximately 2%.

[0075] Furthermore, the majority of cells expressing SOX2, CDX2, and NKX6.1 were chromophoric. The results were negative for the expression of cytokinin (see Figures 5A to 5C). In a population of cells resulting from S5 cultures prepared according to the protocol outlined above, At least 50% of the cells express PDX-1 and NKX6.1, but not CDX-2, S The cells were negative for OX2 and chromogranin. Table I shows the various endoderms from S3 to S5. The percentage expression of the markers is summarized.

[0076] [Table 1] * Total days since differentiation began

[0077] Figures 6A-6T show S2, S3, and S4 mice differentiated according to the protocol outlined in this example. , and S5 cells, and expression in undifferentiated H1 cells measured by real-time PCR The mRNA expression profile reported as fold change is shown. At stage 3, FOXe Very low expression of anterior foregut markers such as NKX1.1 (Figure 6C) and NKX2.1 (Figure 6E) However, SOX2 (Fig. 6M) and OSR1 (Fig. 6C) that mark the stomach region of the intestine were not detected. H) were significantly upregulated in stage 3, and their expression decreased in stages S4-S5. Pancreatic endogenous embryos such as 1a (Fig. 6K), NKX6.1 (Fig. 6G), and PDX-1 (Fig. 6I). Leaf markers reached maximum expression levels on day 2 of culture at S5. Transition through the PDX-1+ SOX2+ population at stage 3, followed by PDX at stages 4-5 -1+ NKX6.1+ SOX2- CDX2- (Figure 6I, Figure 6G, 6M, and 6A). Endocrine markers (chromogranin, insulin, The expression of leukagon, lucagon, and somatosteine ​​reached its maximum expression level at the end of S5. Expression of endocrine progenitor cell markers NKX2.2, NeuroD, and NGN3 increased during S4-S The maximum expression level was reached at 5. The expression of other lineage markers such as ZIC1 and SOX17 Currently, it remains low at S4 to S5.

[0078] In conclusion, stage 5 day 2 cells differentiated according to the protocol outlined in this example The cells express low levels of CDX2 and SOX2, while expressing NKX6.1 and PDX-1. Maintain high expression levels of BMP. Timely BMP inhibition and the use of low-dose RA in S4-S5 The unique combination of high glucose in S1-S2 and S3 is described in Example 1. This is thought to have resulted in a cell population of

[0079] Example 2 Effects of BMP inhibition and PKC activation on SOX2 expression during S3-S4 The protocol outlined in this example involves the inhibition of BMP, the addition of FGF7, and the activation of PKC. This study was conducted to clarify the effect of aging on SOX2 expression during S3-S4.

[0080] Cells of the human embryonic stem cell line H1 were harvested at various passages (passage 40 to passage 52) and cultured for 10 μL. As single cells, in mTesr®1 medium supplemented with Y27632, 1cm 2 MATRIGEL™ (1:30 dilution) was used at a concentration of 100,000 cells per well. After 48 hours from seeding, the cultures were subjected to pancreatic fractionation as follows: They differentiated into secretory lineage cells. a. Stage 1 (Definitive Endoderm (DE) - 3 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8. MCDB-131 medium supplemented with 1.5 μM MCX compound (GSK3B inhibitor) The cells were treated for 1 day. Then, from the second to third day, the cells were treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml Treated with MCDB-131 medium supplemented with 100 mg of glucose and 100 ng / mL of GDF8. I understood. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -3 days): Stage 2 cells were cultured in 50 ng / mL FGF7, 5 In the presence of 0 nM or 200 nM LDN-193189 and / or 200 nM TPB 1:200 dilution of ITS-X in the absence or presence of 2.5 mM glucose, 1x G lutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free B SA, 0.25 μM SANT-1, 20 ng / mL activin-A, 2 μM RA The treatment was performed using the supplemented MCDB-131 medium. The cells were incubated in the medium for 3 days.

[0081] [Table 2]

[0082] d. Stage 4 (Pancreatic foregut progenitor cells - 3 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 2 00 nM TPB, 400 nM LDN-193189, 2 μM ALk5 inhibitor (SD -208, Molecular Pharmacology 2007,72:152~ 161), and 100 nM of a CYP26A inhibitor (N-{4-[2-en-1-yl]-2-methyl-2-propanol). 1,3-(1H-1,2,4-triazol-1-yl)butyl)phenyl MCDB-13 supplemented with benzothiazol-2-amine (Belgium, Janssen, Belgium) The treatment was carried out for 3 days in 1x soil.

[0083] For all of the above conditions, mRNA was collected in steps S2 to S4 and analyzed using real-time PCR. The control condition for S3 was FGF7, AA, SANT, RA, and 200 nM L Refers to cultures in which DN-193189 was used at the concentrations described in step c above. As evidenced by the PCR data shown in Figure 7G, the The ablation resulted in the expression of endocrine markers such as NGN3 (Figure 7D) and pancreatic endocrine components such as chromogranin. Addition of PKC activators at S3 resulted in a significant decrease in secretory markers (see Figure 7C). and removal of LDN-193189 further reduced endocrine markers while NKX6. 1 (see Figures 7A to 7G). The addition of 200 nM L The efficacy of LDN-193189 was comparable to that of LDN-193189. Addition of TPB and IL-14 enhanced the expression of CDX2 (Fig. 7E) and albumin (Fig. 7F). Furthermore, both FGF7 and LDN-193189 suppressed the expression of SOX2 (Fig. 7G). Depletion of LDN-193189 significantly reduced FGF7 levels compared to cultures in which LDN-193189 was depleted but FGF7 was maintained. The expression of SOX2 (Fig. 7G) was increased and the expression of albumin (Fig. 7F) was decreased. Data show that precise regulation of BMP inhibition, FGF activation, and PKC activation contributes to the development of PDX. Endodermal dorsal membranes are rich in NKX6.1 and NKX6.1, but low in CDX2, SOX2, and albumin. Finally, we demonstrate that sustained inhibition of BMP during S3–S4 can lead to a decrease in the number of tumors. Injury increased the expression of pro-endocrine genes and up-regulated SOX2 expression. While secretory genes are increased, they are absent or low in the developing pancreas but are expressed in pre-existing organs such as the stomach. Inhibition of BMP was performed to prevent upregulation of SOX2 expression in the foregut endoderm. This confirms the need for precise adjustment.

[0084] Example 3 Early inhibition of BMPs at the foregut stage is required for subsequent induction of endocrine markers. do. This example demonstrates that early BMP signaling at S3 is essential for subsequent induction of endocrine markers. However, sustained inhibition of BMP at stage 3 also This results in strong expression of SOX2. High expression of endocrine markers and low expression of SOX2 To obtain both, we used a pancreatic endocrine marker with low expression of SXO2 and CDX2. A gradient of BMP inhibition is required to induce CAR.

[0085] Human embryonic stem cell line H1 cells were cultured at various passages (passages 40 to 52) with 10 μM Y 1 cm as single cells in mTesr™ 1 medium supplemented with 27632. 2 Current The cells were coated with MATRIGEL™ (1:30 dilution) at a concentration of 100,000 cells per well. Forty-eight hours after seeding, the cultures were cultured for the pancreatic endocrine lineage cells as follows: differentiated into cells. a. Stage 1 (Definitive Endoderm (DE) - Day 4): Prior to the initiation of DE, cells were washed and Incubate in complete PBS (without Mg or Ca) for 30 seconds, then transfer to S1 medium. Human cells were cultured as single cells on MATRIGEL™ coated dishes and incubated. Embryonic stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate , 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL MCDB-131 supplemented with GDF8 and 1.5 μM of MCX compound (GSK3B inhibitor) The cells were then treated with 0.1% fatty acid-free BSA, 0.0012 g / m 1L sodium bicarbonate, 1X GlutaMax™, 2.5 mM glucose, and and MCDB-131 medium supplemented with 100 ng / mL GDF8 for 2 days (days 2-4). Processed. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -3 days): Stage 2 cells were diluted 1:200 in ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight Sodium carbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 20 ng / mL Activin-A, 2 μM RA, 50 ng / mL FGF7, 100 nM LDN-1 93189 (on day 1 only or during stage 3), and 200 nM TPB were supplemented. In some media, LDN-193189 was added to the stem cells. Removed from Di3. d. Stage 4 (Pancreatic foregut progenitor cells - 3 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 1 00 nM TPB, 200 nM LDN-193189, 2 μM ALk5 inhibitor, and The cells were treated with MCDB-131 medium supplemented with 100 nM of a CYP26A inhibitor for 3 days. e. Stage 5 (Pancreatic endoderm / endocrine - 4 days): Stage 4 cells were cultured in the ITS-X 1 :200 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.001 5g / mL sodium bicarbonate, 2% fatty acid-free BSA, 200nM LDN-1931 The cells were treated for 4 days with MCDB-131 medium supplemented with 8, 9, and 2 μM of ALk5 inhibitor.

[0086] As is clear from the PCR results shown in Figures 8A to 8G, LDN- Removal of 193189 (a BMP inhibitor) is associated with pro-endocrine genetic disorders at stages 4 and 5. The expression of NGN3 (Fig. 8C), NeuroD (Fig. 8D), and chromogranin (Fig. 8E) was suppressed. However, the expression of PDX-1 (Fig. 8B) and NKX6.1 (Fig. 8A) was significantly increased at stage 4. It is not significantly downregulated compared to NGN3 and NeuroD at stages 1 to 5. Complete removal of LDN-193189 at 3 results in a significant increase in the expression of CDX2 ( (Figure 7F). After the addition of LDN-193189 on the first day of stage 3, the cells were cultured for 2–3 days after stage 3. Its removal in the eyes significantly increased the expression of NGN3 and NeuroD, while suppressing the expression of S It decreased the expression of CDX2 and SOX2 at stage 4. During stage 3, LDN-1 Cultures carrying 93189 showed very high expression of SOX2 at S3–S4 ( Figure 8 G) These data show that inhibition of BMP on day 1 of stage 3 triggers pancreatic endocrine markers. These results show that the expression of SOX2 and CDX2 is sufficient to suppress the expression of SOX2 while suppressing the expression of CDX2.

[0087] In summary, the formation of endocrine progenitor cells was induced to stages 4-5, and PDX-1 and N cells were expressed. Inhibition of BMP suppresses SOX2 expression while maintaining KX6.1 expression. is required on day 1 of stage 3. Furthermore, the addition of PKC activators at stage 3 significantly inhibits PD The expression of X-1 and NKX6.1 was further enhanced.

[0088] Example 4 Inhibition of BMP signaling at day 1 of stage 3 results in the generation of pancreatic progenitor cells at stage 4. whereas inhibition of BMP signaling at the end of stage 3 is sufficient to induce This results in a significant decrease in the expression of endocrine markers. This example demonstrates that early inhibition of BMP signaling at stage 3 inhibits pancreatic endocrine progenitor cell proliferation. While inhibition of BMP signaling at late stage 3 allows induction of CAR, These results show that the expression levels of endocrine precursor cell markers in the 4-cell group are significantly reduced.

[0089] Cells of the human embryonic stem cell line H1 at various passages (passages 40 to 52) were cultured in 10 μM Y2 1 cm as single cells in mTesr™ 1 medium supplemented with 7632. 2 Hit The cells were coated with MATRIGEL™ (1:30 dilution) at a concentration of 100,000 cells. Forty-eight hours after seeding, the cultures were cultured for the pancreatic endocrine lineage cells as follows: It differentiated into. a. Stage 1 (Definitive Endoderm (DE) - 4 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF MCDB-131 medium supplemented with 8 and 1.5 μM MCX compound (GSK3B inhibitor) The cells were then treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1× GlutaMax™, 2.5 mM glucose, and The cells were treated with MCDB-131 medium supplemented with 100 ng / mL of GDF8 for 3 days. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -3 days): Stage 2 cells were diluted 1:200 in ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight Sodium carbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 ng / mL MCDB-1 supplemented with 0.05% FGF7, 2 μM RA, and the mixtures listed in Table III below. The soil was treated with 31 times the amount of soil for 3 days.

[0090] [Table 3]

[0091] d. Stage 4 (Pancreatic foregut progenitor cells - 3 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 1 00 nM TPB, 200 nM LDN-193189, 2 μM ALk5 inhibitor, and The cells were treated with MCDB-131 medium supplemented with 100 nM of a CYP26A inhibitor for 3 days.

[0092] 9A to 9H show the results of the culture of pancreatic endoderm and endocrine precursor cells for the above combinations of culture conditions. The gene expression profiles of blastocyst and foregut endoderm markers are shown. Furthermore, blockade of the BMP pathway on day 1 of stage 3 significantly reduced the expression of the pancreatic endocrine marker, chromogranin. This is important for the induction of the subsequent endocrine program as measured by expression (see Figure 9C). However, the addition of BMP inhibitors on day 1 of stage 3 significantly suppressed endocrine markers in subsequent stages. Furthermore, the addition of BMP inhibitors at day 1 of stage 3 also triggers the expression of markers. At stages 3 and 4, the expression of the foregut marker SOX2 was reduced (Figure 9H). Adding BMP inhibitors only on the last day of stage 3 results in BMP only on day 1 of stage 3. At the end of stage 3, significantly higher levels of SOX2 were observed compared with cells treated with MP inhibitors. The expression levels shown in Figures 9A to 9H indicate very high expression levels of SOX2. The expression level of this gene is relative to that of undifferentiated H1 cells. In addition, SOX2 is a well-known transcription factor that is important in maintaining the pluripotency of ES cells. The examples demonstrate the sensitivity of stage 3 cultures to the duration and kinetics of BMP signaling and This further supports the results mentioned above, which revealed the influence of SOX2 on pancreatic endocrine induction and expression. I'm wearing it.

[0093] Example 5 Optimal dose of BMP inhibition at the pancreatic foregut stage (stage 4) The previous example described the optimal duration of BMP inhibition at stage 3. In the example, the optimal dose of BMP inhibitor in S4 medium is identified.

[0094] Human embryonic stem cell line H1 cells at various passages (passages 40 to 52) were cultured in mTesr (commercially available). In the target medium, 10 μM Y27632 and 1 cm 2 100,000 cells per The cells were plated as single cells on dishes coated with MATRIGEL™ (1:30 dilution) at a concentration of Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows: Ta. a. Stage 1 (Definitive Endoderm (DE) - 4 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8. MCDB-131 medium supplemented with 1 μM MCX compound (GSK3B inhibitor) for 1 day Then, from day 2 to day 4, the cells were treated with 0.1% fatty acid-free BSA, 0. 0.012 μg / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM Treated with MCDB-131 medium supplemented with glucose and 100 ng / mL GDF8. Ta. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -4 days): Stage 2 cells were diluted 1:200 in ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight Sodium carbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 ng / mL 0.05% FGF7, 2 μM RA, 20 ng / mL activin-A, 100 nM LDN-1 The cells were treated for one day with MCDB-131 medium supplemented with 93189 and 100 nM TPB. The cells were then incubated in a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X Glu taMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA , 0.25 μM SANT-1, 50 ng / mL FGF7, 2 μM RA, 20 ng / 3 mL of MCDB-131 medium supplemented with activin-A and 100 nM TPB Treatment was carried out for days. d. Stage 4 (Pancreatic foregut progenitor cells - 4 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 200 nM LDN-19318 9, 2 μM ALk5 inhibitor, 100 nM CYP26A inhibitor, and MCDB-13 supplemented with LDN-193189 (LDN-193189-193189) at the concentrations listed. Treatment was carried out in 1x1 medium on days 1 to 4 of stage 4.

[0095] [Table 4]

[0096] d. Stage 5 (Pancreatic endoderm / endocrine progenitor cells - 3 days): Stage 4 cells were cultured in the ITS 1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 0.0015g / mL sodium bicarbonate, 2% fatty acid-free BSA, 50nM LDN-1 Treatment with MCDB-131 medium supplemented with 93189 and 1 μM of ALk5 inhibitor for 3 days did.

[0097] The results of real-time PCR analysis of cells harvested after the above treatment are shown in Figures 10A to 10H. This figure shows the results of the treatment of 50 nM or 100 nM of LDN-193189 on day 1 or 2 of S4. Addition of the cells on days 1, 2, or 3 prolongs the expression of endocrine markers, whereas addition of the cells on days 3 or 4 prolongs the expression of endocrine markers. This indicates that low SOX2 expression can be maintained from S4 to S5 (Figure 10A-B). See 10H).

[0098] Example 6 Optimal dose of BMP inhibition at the foregut stage (stage 3) This example demonstrates the optimal dose of BMP inhibition at stage 3 and subsequent distribution at stage 6. Identify effects on secretory markers.

[0099] Human embryonic stem cell line H1 cells were cultured at various passages (passages 40 to 52) with 10 μM Y 1 cm as single cells in mTesr™ 1 medium supplemented with 27632. 2 Current The cells were coated with MATRIGEL™ (1:30 dilution) at a concentration of 100,000 cells per well. Forty-eight hours after seeding, the cultures were cultured for the pancreatic endocrine lineage cells as follows: differentiated into cells. a. Stage 1 (Definitive Endoderm (DE) - 4 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF MCDB-131 medium supplemented with 8 and 1.5 μM MCX compound (GSK3B inhibitor) The cells were treated with 0.1% fatty acid-free BS for 1 day. Then, from the second to fourth day, the cells were treated with 0.1% fatty acid-free BS for 1 day. A, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2. MCDB-131 medium supplemented with 5 mM glucose and 100 ng / mL GDF8 It was processed. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -3 days): Stage 3 cells were cultured in ITS-X at a 1:200 dilution. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight Sodium carbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 ng / mL 0.05% FGF7, 2 μM RA, 20 ng / mL activin-A, 100 nM TPB, and and 10-50 nM LDN-193189 in MCDB-131 medium for 1 day. The cells were then incubated in a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X G lutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free B SA, 0.25 μM SANT-1, 50 ng / mL FGF7, 2 μM RA, 20 n MCDB-131 medium supplemented with 100 μg / mL activin-A and 100 nM TPB The treatment was carried out for 2 days. d. Stage 4 (Pancreatic foregut progenitor cells - 3 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 20 nM LDN-193189 , 2 μM ALk5 inhibitor, 100 nM CYP26A inhibitor, and 100 nM TPB The plants were treated for 3 days in MCDB-131 medium supplemented with 100mg of methyl ... e. Stage 5 (Pancreatic endoderm / endocrine progenitor cells - 3 days): Stage 4 cells were cultured in the ITS 1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 0.0015g / mL sodium bicarbonate, 2% fatty acid-free BSA, + / - 25nM LD MCDB-131 medium supplemented with N-193189 and / or 2 μM of ALk5 inhibitor for 3 days. f. Stage 6 (Pancreatic endocrine hormone production - 3 days): Stage 5 cells were cultured in ITS-X. 1:200 dilution of 2.5 mM glucose, 1X GlutaMax™, 0.0 MCDB-1 supplemented with 0.15 g / mL sodium bicarbonate and 2% fatty acid-free BSA The soil was treated with 31 times the amount of soil for 3 days.

[0100] Figures 11A-11H show that SOX2 expression is suppressed while triggering the expression of endocrine markers. We show that low-to-moderate inhibition of BMP on day 1 of stage 3 is required to maintain Furthermore, inhibiting BMP at stage 5 while increasing endocrine markers also This led to upregulation of SOX2 expression.

[0101] The data in this example further confirm the results presented in the previous examples. To trigger the induction of pancreatic endocrine markers while suppressing SOX2 expression, These findings support the need for precise regulation of the BMP pathway in 5.

[0102] Example 7 Optimal time frame for BMP inhibition at S3 (foregut stage) This example shows that the induction of endocrine secretion at later stages is maintained and SOX2 expression is not reduced. To identify the optimal time window in stage 3 for inhibiting BMP signaling, do.

[0103] Human embryonic stem cell line H1 cells at various passages (passages 40 to 52) were cultured in mTesr (commercially available). In the target medium, 10 μM Y27632 and 1 cm 2 100,000 cells per The cells were plated as single cells on dishes coated with MATRIGEL™ (1:30 dilution) at a concentration of Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows: Ta. a. Stage 1 (Definitive Endoderm (DE) - 4 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF The cells were treated with MCDB-131 medium supplemented with 8 and 1.5 μM of MCX compound for one day. From the second to fourth day, the cells were incubated in 0.1% fatty acid-free BSA, 0.0012 g / ml mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM glucose, and treated with MCDB-131 medium supplemented with 100 ng / mL GDF8. b. Stage 2 (gastrulation -3 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 3 days. c. Stage 3 (foregut -3 days): Stage 2 cells were isolated for only the first 2 hours of Stage 3. Or for 6 hours only or for 24 hours only, 1:200 dilution of ITS-X, 2.5 mM glucose 1X GlutaMax™, 0.0015g / mL sodium bicarbonate, 2% Fatty acid-free BSA, 0.25 μM SANT-1, 50 ng / mL FGF7, 2 μM Supplemented with RA, 20 ng / mL activin-A, and 100 nM TPB. The plants were treated with MCDB-131 medium containing M. LDN-193189. d. Stage 4 (Pancreatic foregut progenitor cells - 3 days): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 25 nM LDN-193189 , 2 μM ALk5 inhibitor, 100 nM CYP26A inhibitor, and 100 nM TPB The plants were treated for 3 days in MCDB-131 medium supplemented with 100mg of methyl ... e. Stage 5 (Pancreatic endoderm / endocrine progenitor cells - 3 days): Stage 4 cells were cultured in the ITS 1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, and 2 μM ALk5 The cells were treated with MCDB-131 medium supplemented with inhibitors for 3 days.

[0104] 12A-12G show real-time PCR analysis of the data collected in this example. Treatment with BMP inhibitors for at least 2 hours at stage 3 significantly reduced the expression of Ngn3 (Fig. 12D) and While triggering the expression of pro-endocrine transcription factors such as ATP and NeuroD (Figure ​(Figure12E), S It was possible to maintain very low expression of OX2 ( Figure 12G ) and NKX6 at S4–S5. 12A) and PDX-1 (Fig. 12B). On day 3 of the 5th trimester, CDX2 expression was significantly higher in the cells treated with BMP inhibitors than in the cells treated with BMP inhibitors for 24 hours. It was higher in cells treated with the inhibitor for 2 or 6 hours (Fig. 12F).

[0105] The data in this example demonstrate that the expression of CDX2 and SOX2 can be maintained at low levels. , and B to initiate endocrine differentiation while maintaining high expression of pancreatic endoderm markers. This suggests that 24-hour inhibition of the MP pathway is optimal.

[0106] Example 8 Optimal duration of stage 3 (foregut stage) and stage 4 (pancreatic foregut progenitor cell stage) This example demonstrates the S3 and S5 differentiation of pluripotent cells into cell populations of the pancreatic endocrine lineage. 4 was carried out to determine the optimal duration.

[0107] Cells of the human embryonic stem cell line H1 at various passages (passages 40 to 52) were cultured in 10 μM Y2 1 cm as single cells in mTesr™ 1 medium supplemented with 7632. 2 Hit The cells were coated with MATRIGEL™ (1:30 dilution) at a concentration of 100,000 cells. Forty-eight hours after seeding, the cultures were cultured for the pancreatic endocrine lineage cells as follows: It differentiated into. a. Stage 1 (Definitive Endoderm (DE) - 4 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8. MCDB-131 medium supplemented with 1.5 μM MCX compound (GSK3B inhibitor) The cells were treated for 1 day. Then, from the second to fourth days, the cells were treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml Treated with MCDB-131 medium supplemented with 100 mg of glucose and 100 ng / mL of GDF8. I understood. b. Stage 2 (gastrulation -2 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 ml MCDB-131 medium supplemented with 50 ng / mL of D-glucose and 50 ng / mL of FGF7. The treatment was carried out for 2 days. c. Stage 3 (foregut - 2-3 days): Stage 2 cells were diluted 1:200 in ITS-X. Dilution: 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 ng / mL FGF7, 2 μM RA, 20 ng / mL activin A, 100 nM TPB The mice were incubated for 1 day in MCDB-131 medium containing 100 nM LDN-193189. The cells were then treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1 X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fat-free Fatty acid BSA, 0.25 μM SANT-1, 50 ng / mL FGF7, 2 μM RA, MCDB-131 supplemented with 20 ng / mL activin-A and 100 nM TPB Treated with double soil. d. Stage 4 (pancreatic foregut progenitor cells - 2-3 days): Stage 3 cells were cultured in the ITS-X system. 1:200 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.00 15g / mL sodium bicarbonate, 2% fatty acid-free BSA, 25nM LDN-1931 89, 100 nM CYP26A inhibitor, and 100 nM TPB supplemented MCDB- The soil was treated with 131 times the amount of soil for 2-3 days. e. Stage 5 (Pancreatic endoderm / endocrine progenitor cells - 2 days): Stage 4 cells were cultured in the ITS 1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, and 1 μM ALk5 The cells were treated with MCDB-131 medium supplemented with inhibitors for 2 days. f. Stage 6 (Pancreatic endocrine progenitor cells / hormones - 2 days): Stage 5 cells were cultured using the ITS 1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 MCDB supplemented with 0.0015 g / mL sodium bicarbonate and 2% fatty acid-free BSA The soil was treated with -131 fold water for 2 days.

[0108] Real-time analysis of samples harvested at Stage 3, Stage 4, Stage 5, or Stage 6 The PCR analysis data are shown in Figures 13A to 13G. The data show that S3 and S4 were Extending S3 and S4 to 3 days increased NKX6.1 expression compared to culture without S3. The cells treated for 3 days at stage 3 showed a significant increase in the number of cells in the S3 and S4 periods (Fig. 13A). shows downregulation of the expression of pro-endocrine markers when compared to cultures with only 2 days ( (Figures 13D and 13E). Furthermore, extending stage 4 to 3 days significantly increased SOX2 expression. significantly increased (Fig. 13G).

[0109] The data obtained in this example, like the data generated in the previous examples, This shows that BMP inhibition promotes the foregut toward a high SOX2 population. Based on data from the example, the optimal duration of Stage 3 and Stage 4 is 2 days. It can be concluded that the ideal protocol would be one that induces high levels of expression of pro-endocrine markers. , differentiated cells with high expression of NKX6.1, low expression of CDX2, and low expression of SOX2. will bring about the cell.

[0110] Example 9 Prolonged exposure to BMP inhibition in the presence of high glucose and B27 supplements , significantly increase the expression of SOX2 in S3 and S4. This protocol covers the S3 and S4 stages during the stepwise differentiation of pluripotent cells into hormone-producing cells. This study was conducted to determine factors affecting SOX2 expression in mice.

[0111] Cells of the human embryonic stem cell line H1 were cultured on MATRIGEL™ (1:30 dilution) coated dishes. The cells were cultured in mTesr™ 1 medium until they reached 70% confluence and then incubated as follows: It differentiated into. a. Undifferentiated cells were cultured in 0.2% FBS, 100 ng / mL activin A, 20 ng / mL The cells were cultured for 1 day in RPMI medium (Invitrogen) supplemented with 1 mL of WNT-3α. The cells were then plated in 0.5% FBS, 100 ng / mL activin A-supplemented PBS. The cells were treated with RPMI medium for another 2 days (Stage 1). b. Stage 1 cells were cultured in DMEM supplemented with 2% FBS and 50 ng / mL FGF7. / F12 medium for 3 days (stage 2). c. Stage 2 cells were cultured in 1% B27, 0.25% SANT-1, 2 μM RA, Supplemented with 100 ng / mL Noggin (R & D Systems, Minnesota, USA). The cells were cultured in DMEM-high glucose medium for 4 days (stage 3). d. Stage 3 cells were cultured in 1% B27, 100 ng / mL Noggin, and 1 μM ALK. 5 inhibitor II (Axxora, CA, USA), and 50 nM TPB were added. The cells were treated with DMEM-high glucose medium containing 100% ethanol for 4 days (stage 4).

[0112] Figures 14A-14H show the results of the 4-day harvest of stage 3 markers. FACS histograms of the cells are shown: isotype control (Figure 14A), Romogranin (Figure 14B), KI-67 (Figure 14C), NKX6.1 (Figure 14D), SO X2 (Figure 14E), HNF3B (Figure 14F), CDX2 (Figure 14G), PDX-1 (Figure 1 4H). The percentage of expression of each marker is shown in each histogram. The majority of cells at stage 3 were positive for the expression of PDX-1 (Fig. 14H) and HNF3B (Fig. 14F), and N They were negative for the expression of KX6.1 (Fig. 14D) and chromogranin (Fig. 14B) and C DX2 (Fig. 14G). However, over 90% of the cells expressed SOX2. (Fig. 14E) were also strongly positive. This is because at stage 3, most of the cells were P Pancreatic PDX positive for DX-1 and SOX2 and negative for NKX6.1 This suggests the establishment of an endoderm population consistent with the anterior foregut population in the -1 domain.

[0113] Furthermore, in cell populations generated using the protocol outlined in this example, The percentage of cells that were SOX2+ at day 3 was determined using the protocol outlined in Example 1. This difference was significantly higher than the percentage of cells that were SOX2+ in the resulting cell population. is the response to a BMP antagonist (noggin) in the culture medium at stage 3 in this example. This may be due to prolonged exposure and the absence of FGF7 and PKC activators.

[0114] 15A to 15G show the results of differentiation according to Example 9 on day 2 of S4 for the following markers: FACS histogram expression profiles are shown. Figure 15A: Isotype control, Figure 15B: NKX6.1, Figure 15C: KI-67, Figure 15D: chromogranin, Figure 15E : SOX2, Fig. 15F: CDX2, Fig. 15G: PDX-1. The expression percentage of each marker was Shown in the histogram.

[0115] 16A-16F show the following markers of cells differentiated according to Example 9 on day 4 of S4: Figure 16A shows the FACS histogram expression profile of the isotype control. 16B: NKX6.1, 16C: chromogranin, 16D: SOX2, 16E: SOX2, 16F: SOX2, 16G: SOX2, 16H: SOX2, 16I: SOX2, 16J ... 6E: CDX2, Fig. 16F: PDX-1. The expression percentage of each marker is shown in each histogram. vinegar.

[0116] Table V below shows the S3 and S4 populations of cells differentiated according to the protocol outlined in this example. This figure summarizes the data obtained on the expression rate (%) of endoderm markers in the oocytes.

[0117] [Table 5]

[0118] 17A-17J show the results of the differentiation of human embryonic stem cell line H1 cells according to Example 9. Figure 17 shows the results of real-time PCR analysis of the expression of the following genes: 17B: HHex, Fig. 17C: FOXE1, Fig. 17D: IPF1 (PDX-1), Fig. 17 E:NKX2.1, Fig. 17F:NKX2.2, Fig. 17G:NKX6.1, Fig. 17H:PR OX1, Fig. 17I:SOX2, Fig. 17J:SOX9.

[0119] As can be seen in Figures 14 to 17 and Table V, on days 2 to 4 of stage 4, NKX There was a significant increase in PDX-1 expression from stage 3 to stage 6.1, while high PDX-1 expression was maintained. Although SOX2 expression decreased towards stage 4, up to 75% of cells were still SOX2+. As in Figure 5, CDX2+ cells, SOX2+ cells, and NKX6.1+ cells were This was achieved by the protocol outlined in Example 9. NKX6.1+ SOX2+ PDXs from stage 4 day 4 cell populations generated using This suggests that the proportion of -1+ CDX2-chromogranin negative cases is up to 50%. This is PDX-1+ NKX6.1+ SOX2-, CDX2-, chromosomes in S4-S5. The proportion of granin-negative cells was approximately 40-70%, and PDX-1+ NKX6.1+ SOX2+ This is in contrast to the cell population generated in Example 1, which was 2-25%. The cells generated using this protocol were PDX-1+ and NXK6.1+. The cells also showed low or negative populations for SOX2 and CDX2 compared to the cells generated in Example 9. The blastocysts, as defined as clusters, had a much higher percentage of pancreatic endoderm.

[0120] The data obtained in this example demonstrate that BMP inhibition in the presence of high glucose and B27 supplements Long-term exposure to toxins significantly increased SOX2 expression at stages 3 and 4 differentiation. This provides evidence that:

[0121] Example 10 Previously published protocols have not shown significant numbers of SOX2+ cells in stages 3–4. Brings about formation. Kroon et al. have developed a protocol for preparing cells of the pancreatic endoderm lineage from human embryonic stem cells. The col has been published (Nature Biotech 2008,26:443~452 , hereafter referred to as "Kroon"). In the examples provided herein Human embryonic stem cells were differentiated according to the Kroon protocol and characterized at different stages of differentiation. Expression of characteristic markers was assayed.

[0122] Cells of the human embryonic stem cell line H1 were cultured on MATRIGEL™ (1:30 dilution) coated dishes. The cells were seeded and cultured in mTesr™ medium until 70% confluence and then transferred to Kroon. Differentiation was performed using previously published protocols as follows. a) Undifferentiated cells were cultured in 0.2% FBS, 100 ng / mL activin A, 20 ng / mL After 1 day of exposure to RPMI medium supplemented with 1 mL of WNT-3a, 0.5% FBS, 1 mL of WNT-3a, and 1 mL of WNT-3a, ... and 1 mL of WNT-3a, 1 mL of WNT-3a, and 1 mL The cells were treated with RPMI medium supplemented with 100 ng / mL of activin A for an additional 2 days (step Page 1). b) Stage 1 cells were cultured in RPM supplemented with 2% FBS and 50 ng / mL FGF7. The cells were treated with I medium for 3 days (stage 2). c) Stage 2 cells were cultured in 1% B27, 0.25 μM SANT-1, and 2 μM RA. , supplemented with 50 ng / mL Noggin (R & D Systems, Minnesota) The cells were treated with MEM-high glucose medium for 3 days (stage 3). d) Stage 3 cells were cultured in DMEM-high glucose medium supplemented with 1% B27. The cells were cultured for 3 days (stage 4). e) Stage 4 cells were scraped from the wells and cultured in DMEM-high supplemented with 1% B27. They were resuspended as clusters in glucose medium for 2 days.

[0123] 18A-18G show the following markers on day 3 of S3 differentiated according to Example 10: The FACS histogram expression profile of the isotype control (Figure 18A) is shown. ), NKX6.1 (Figure 18B), chromogranin (Figure 18C), SOX2 (Figure 18D)C DX2 (Fig. 18E), KI-67 (Fig. 18F), PDX-1 (Fig. 18G). Percent expression is shown in each histogram.

[0124] 19A to 19G show the following mammograms of cells differentiated according to Example 10 on day 5 of S4. The FACS histogram expression profiles of the markers are shown. 19A), NKX6.1 (Fig. 19B), chromogranin (Fig. 19C), SOX2 (Fig. 19 D), CDX2 (Fig. 19E), KI-67 (Fig. 19F), and PDX-1 (Fig. 19G). The percentage expression of the marker is shown in each histogram.

[0125] As shown in Figures 18 and 19, by the end of stage 4 (day 5), Up to 20% of the clusters of cells are NKX6.1+ PDX-1+ SOX2- Furthermore, up to 20% were PDX-1+ NKX6.1+ SOX2+. A significant proportion of the cell population generated according to Example 10 was NKX6.1+ at stage 4. This indicates that the patients were SOX2+.

[0126] Table VI below shows the percentage of endoderm markers at S3-S4 in the cells generated in this example. This is a summary.

[0127] [Table 6] * Final 2 days in suspension culture

[0128] Example 11 The addition of ascorbic acid significantly reduced the number of multihormonal cells and the number of monohormonal insulins. The number of phosphorus-positive cells was increased at the same time.

[0129] Ascorbic acid affects the expression of markers during differentiation of pluripotent cells into hormone-producing cells The effects of glucose supplementation on differentiation were tested at all stages, and at stage 3 as follows: In the formation of , 4 and 5, cells were cultured in medium supplemented with ascorbic acid.

[0130] Human embryonic stem cell line H1 cells at various passages (passages 40 to 52) were cultured in mTesr (commercially available). In the target medium, 10 μM Y27632 and 1 cm 2 100,000 cells per The cells were seeded as single cells onto MATRIGEL™ (1:30 dilution) coated dishes at a concentration of Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows. a. Stage 1 (Definitive Endoderm (DE) - 3 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 100 ng / mL GDF8, and MCDB-131 medium supplemented with 1 μM MCX compound (GSK3B inhibitor) for 1 day. The cells were then treated with 0.1% fatty acid-free BSA, 0.0012 g / mL bicarbonate Sodium, 1X GlutaMax™, 5mM glucose, 100ng / mL 100 nM of GDF8 and 100 nM of MCX compound in MCDB-131 medium for 2 days. followed by an additional day of 0.1% fatty acid-free BSA, 0.0012 g / mL bicarbonate sodium phosphate, 1X GlutaMax™, 5 mM glucose, and 100 ng The cells were treated with MCDB-131 medium supplemented with 1 / mL of GDF8. b. Stage 2 (gastrulation -2 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM The mice were incubated in MCDB-131 medium supplemented with D-glucose and 25 ng / mL FGF7 for 2 days. It was processed for a while. c. Stage 3 (foregut -2 days): Stage 2 cells were diluted 1:200 in ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight sodium carbonate, 2% fatty acid-free BSA, 10 ng / mL activin-A, 25 ng / mL of FGF7, 0.25 μM SANT-1, 1 μM RA, 200 nM TPB (P KC activator), and 100 nM LDN-193189 (BMP receptor inhibitor) The cells were treated with CDB-131 medium for 1 day. Then, the cells were treated with a 1:200 dilution of ITS-X, 2 0.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL bicarbonate sodium phosphate, 2% fatty acid-free BSA, 10 ng / mL activin-A, 25 ng / mL 1 μM FGF7, 0.25 μM SANT-1, 1 μM RA, 200 nM TPB (PK C activator), MCDB-131 medium supplemented with 10 nM LDN-193189 was further Some cultures were treated with 0.25 mM ascorbic acid during stage 3. (Cat. No. A4544, Sigma, Missouri, USA). d. Stage 4 (Pancreatic foregut progenitor cells - day 2): Stage 3 cells were cultured in 0.25 mM ATP. 1:200 dilution of ITS-X, 2.5 mM glucose, with or without scorbic acid, 1X GlutaMax™, 0.0015g / mL sodium bicarbonate, 2% Fatty acid BSA, 0.25 μM SANT-1, 50 nM RA, 200 nM TPB, 5 The cells were treated with MCDB-131 medium supplemented with 0 nM LDN-193189 for 2 days. e. Stage 5 (pancreatic endoderm - days 2 to 7): Stage 4 cells were cultured in 0.25 mM Aspergillus oryzae. 1:200 dilution of ITS-X, 2.5 mM glucose, with or without corbic acid X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fat-free MCDB-131 supplemented with fatty acid BSA, 0.25 μM SANT-1, and 50 nM RA The plants were treated in double-stranded soil for 2 to 7 days.

[0131] 20A-20J show the results of differentiation of human embryonic stem cell line H1 cells according to Example 11. Figure 20A shows real-time PCR analysis of the expression of the following genes in guinea pigs: Figure 20B: PDX1, Figure 20C: Pax6, Figure 20D: Pax4, Figure 20E: NKX6. 1, Figure 20F: NGN3, Figure 20G: Glucagon, Figure 20H: NeuroD, Figure 20I: Insulin, Figure 20J: Chromogranin. This figure shows the effect of stage 3, or stages 3 and 4. The addition of ascorbic acid at the early stage increased the expression of somatostatin and glucagon at stages 4 and 5. The results show that the expression of insulin is significantly reduced while the expression of insulin is increased (Fig. 20A, (See Figures 20G and 20I.) Furthermore, at stages 4 and 5, PDX-1 and NKX6 were expressed. Expression of pancreatic endoderm markers such as 1 was increased by the addition of 0.25 mM ascorbic acid. There was no significant change (see Figures 20B and 20D). Expression was downregulated, while Pax4 expression was maintained (see Figures 20C and 20D). Cultures treated with + / - ascorbic acid from stage 3 to stage 5 developed insulin at the end of stage 5. Immunostaining for the hormones glucagon, somatostatin, and erythrocytes was performed. threonine-positive cells, glucagon- and somatostatin-positive cells, and multihormonal cells (1 The mean percentage of cells expressing two or more hormones is summarized.

[0132] [Table 7]

[0133] Example 12 Optimal Dosage of Ascorbic Acid in Stage 3 This example shows a single hormone, PDX-1 positive, and NKX6.1 positive insulin positive A study was carried out to determine the optimal dose of ascorbic acid to use for generating cells.

[0134] Human embryonic stem cell line H1 cells at various passages (passages 40 to 52) were cultured in mTesr (commercially available). In the target medium, 10 μM Y27632 and 1 cm 2 100,000 cells per The cells were seeded as single cells onto MATRIGEL™ (1:30 dilution) coated dishes at a concentration of Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows. a. Stage 1 (Definitive Endoderm (DE) - 3 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, and 100 ng / mL GDF The cells were cultured in MCDB-131 medium supplemented with 8 and 1 μM MCX compound (GSK3B inhibitor) for 1 day. The cells were then treated with 0.1% fatty acid-free BSA, 0.0012 g / mL bicarbonate Sodium, 1X GlutaMax™, 5mM glucose, 100ng / mL 100 nM of GDF8 and 100 nM of MCX compound in MCDB-131 medium for 2 days. followed by an additional day of 0.1% fatty acid-free BSA, 0.0012 g / mL bicarbonate sodium phosphate, 1X GlutaMax™, 5 mM glucose, and 100 ng The cells were treated with MCDB-131 medium supplemented with 1 / mL of GDF8. b. Stage 2 (gastrulation -2 days): Stage 1 cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM MCDB-131 medium supplemented with D-glucose and 0.25 mM ascorbic acid and The cells were treated with or without 25 ng / mL FGF7 for 2 days. c. Stage 3 (foregut -2 days): Stage 2 cells were diluted 1:200 in ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight sodium carbonate, 2% fatty acid-free BSA, 10 ng / mL activin A, 25 ng / m L FGF7, 0.25 μM SANT-1, + / - 0.25 mM ascorbic acid, 1 1 μM RA, and 200 nM TPB, and 100 nM LDN-1 on day 1. 93189 was treated with supplemented MCDB-131 medium, followed by 1:20 ITS-X. 0 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 10 ng / mL activin A, 25 ng / mL FGF7, 0.25 μM SANT-1, + / - 0.25 mM ascorbate phosphate, 1 μM RA, and 200 nM TPB, and for one day, 10 nM LDN-1 93189 was treated in supplemented MCDB-131 medium. d. Stage 4 (Pancreatic foregut progenitor cells - day 2): Stage 3 cells were cultured in ITS-X 1: 200% dilution, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 5 M supplemented with 0 nM RA, 200 nM TPB, and 50 nM LDN-193189 CDB-131 medium with or without the addition of 0.25mM to 1mM ascorbic acid , and treated for 2 days. e. Stage 5 (pancreatic endoderm - days 2 to 9): Stage 4 cells were cultured in 0.25 mM Aspergillus oryzae. A 1:200 dilution of ITS-X, 2.5 mM glucose, with or without the addition of corbic acid was used. 1X GlutaMax™, 0.0015g / mL sodium bicarbonate, 2% MCD supplemented with 0.05% fatty acid-free BSA, 0.25 μM SANT-1, and 50 nM RA. The seeds were treated with B-131 medium for 2 to 9 days.

[0135] 21A-21J show the results of differentiation of human embryonic stem cell line H1 cells according to Example 12. Figure 21A shows data from real-time PCR analysis of the expression of the following genes in the cerebrospinal fluid: Tatin, Fig. 21B: PDX1, Fig. 21C: Pax6, Fig. 21D: Pax4, Fig. 21E: N KX6.1, Fig. 21F: NGN3, Fig. 21G: NeuroD, Fig. 21H: Insulin, Fig. 21I: glucagon, Fig. 21J: chromogranin. Consistent with the data from Example 10, Addition of ascorbic acid at stages 2–4 significantly increased the levels of somatostatin, glucagon, and Pax6. significantly reduced the expression of insulin and Pax4 at stage 5, while maintaining the expression of insulin and Pax4. Furthermore, compared to 0.25 mM ascorbic acid, 0.5–1 mM ascorbic acid in S4 Finally, there was no significant benefit from using ascorbic acid in Stage 2. Addition of α-glucagon also reduced the expression of glucagon and somatostatin in stages S3-5. It has been proven that ascorbic acid is effective in maintaining insulin expression. Rubicic acid acts to regulate the expression of single hormones in a stage-specific manner. The addition of ascorbic acid is important in the early stages of the differentiation protocol, but not in the later stages. has not proven effective in reducing polyhormonal cell counts.

[0136] Example 13 The combination of retinoic acid and ascorbic acid inhibits the activity of single hormone insulin-positive cells. is required to generate This example demonstrates the use of pluripotent cells to generate single-hormone insulin-positive cells during differentiation. This was carried out to clarify the requirements for

[0137] Human embryonic stem cell line H1 cells at various passages (passages 40 to 52) were cultured in mTesr (commercially available). In the target medium, 10 μM Y27632 and 1 cm 2 100,000 cells per The cells were seeded as single cells onto MATRIGEL™ (1:30 dilution) coated dishes at a concentration of Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows. a. Stage 1 (Definitive Endoderm (DE) - 3 days): Prior to the initiation of DE, the cultures were washed and Incubate in incomplete PBS (without Mg or Ca) for 30 seconds, then stage 1 Human embryonic stem cells cultured as single cells on MATRIGEL™ coated dishes were added. Stem cells were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 100 ng / mL GDF8, Treatment with MCDB-131 medium supplemented with 1 μM MCX compound (GSK3B inhibitor) for 1 day The cells were then incubated in 0.1% fatty acid-free BSA, sodium bicarbonate, GlutaMatrix, and 1% ethanol. x™, plus 5 mM glucose, 100 ng / mL GDF8, and 100 nM Treatment with MCDB-131 medium supplemented with MCX compounds was performed on the second day, followed by another day of treatment. 0.1% Fatty Acid-Free BSA, 0.0012g / mL Sodium Bicarbonate, 1X Glut Supplemented with aMax™, 5 mM glucose, and 100 ng / mL GDF8 The plants were treated in MCDB-131 medium. b. Stage 2 (gastrulation -2 days): Cells were incubated in 0.1% fatty acid-free BSA, 0.0012 100 mg / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose MCD supplemented with 0.25 mM ascorbic acid and 25 ng / mL FGF7. The plants were treated with B-131 medium for 2 days. c. Stage 3 (foregut -2 days): Cells were incubated in a 1:200 dilution of ITS-X, 2.5 mM Glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate 2% fatty acid-free BSA, 10 ng / mL activin A, 25 ng / mL FGF7 , 0.25 mM ascorbic acid, 0.25 μM SANT-1, 1 μM RA, 200 nM TPB, and 100 nM LDN-193189 on day 1 were supplemented. Treatment with CDB-131 medium followed by a 1:200 dilution of ITS-X, 2.5 mM glutamic acid, and 2.5 mM glutamic acid were performed. course, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 10 ng / mL activin A, 25 ng / mL FGF7, 0 0.25 mM ascorbic acid, 0.25 μM SANT-1, 1 μM RA, and 200 nM TPB, and 10 nM LDN-193189 for an additional 1 day. Treated with MCDB-131 medium. d. Stage 4 (pancreatic foregut progenitor cells - day 2): Cells were cultured in a 1:200 dilution of ITS-X. 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL weight Sodium carbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 nM RA , 200 nM TPB, 50 nM LDN-193189, and 0.1 mM ascorbate The plants were treated for 2 days in MCDB-131 medium supplemented with phosphate. e. Stage 5 (pancreatic endoderm - 3 days): Cells were cultured in a 1:200 dilution of ITS-X, 2.5 0.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate The following cultures were performed in MCDB-131 medium supplemented with thorium and 2% fatty acid-free BSA. The treatment was carried out under the conditions for 3 days. +0.1mM ascorbic acid 0.1 mM ascorbic acid + 50 nM RA 0.1 mM ascorbic acid + 50 nM RA + 0.25 μM SANT-1 0.1 mM ascorbic acid + 50 nM RA + 0.25 μM SANT-1 + 5 0nM LDN-193189 0.1 mM ascorbic acid + 50 nM RA + 0.25 μM SANT-1 + 1 μM Alk5 inh 0.1 mM ascorbic acid + 50 nM RA + 0.25 μM SANT-1 + 1 μM Alk5 inhibitor + 50nM LDN-193189

[0138] 22A-22L show embryonic stem cells differentiated according to Example 13 and harvested on day 3 of S5. In cells of the cell line H1, Pax4 (Fig. 22A), Pax6 (Fig. 22B), and PDX1 (Fig. 22C), PTF1a (Fig. 22D), glucagon (Fig. 22E), insulin (Fig. 22F) , NeuroD (Figure 22G), ngn3 (Figure 22H), Zic1 (Figure 22I), CDX2 (Fig. 22J), albumin (Fig. 22K), and NKX6.1 (Fig. 22L) expression in real time. Data from PCR analysis are shown.

[0139] Cultures treated with the above combinations showed increased production of insulin, glucagon, and erythrocytes at the end of stage 5. Table VIII shows the immunostaining for insulin and somatostatin hormones. Sex cells, glucagon and somatostatin positive cells, and multihormonal cells (within one cell) The mean percentage of individuals expressing two or more hormones in the 100% genotype group is summarized.

[0140] As shown in Figure 22 and Table VIII below, low doses of retinoic acid at stage 5 The addition of ascorbic acid significantly increased the hormone production compared to cultures treated with vitamin C alone at S5. The total number of insulin-positive cells was significantly reduced, while the number of insulin-positive cells was significantly reduced. In addition, retinoic acid, ascorbic acid, and sonic hedgehog inhibitors , and ALK5 inhibitor combinations were treated with ascorbic acid (vitamin C) alone. Compared to cultures containing only insulin, the number of single-hormone insulin-positive cells was further increased. A unique combination of factors is required to generate single-hormone insulin-positive cells. This indicates that

[0141] [Table 8] The present invention may include the following aspects. [1] An in vitro differentiated population of pancreatic endoderm cells obtained from stepwise differentiation of pluripotent cells. Pancreatic cells at each stage of differentiation are cultured in a medium containing 5 mM to 20 mM glucose. A differentiated population of visceral endoderm cells. [2] More than 30% of the differentiated pancreatic endoderm cells are PDX-1+NKX6.1+, SOX2 - and CDX2-. [3] More than 10% of the cells in the differentiated population are single hormone insulin-positive cells. ] or [2]. A differentiated population of pancreatic endoderm cells. [4] The stepwise differentiation is carried out by culturing undifferentiated human embryonic stem cells in a medium further supplemented with a TGF-B ligand. The pancreatic endoderm cells according to any one of [1] to [3] above, which further comprises a step of culturing the cells underground. Differentiated populations of. [5] The stepwise differentiation is carried out by culturing undifferentiated human embryonic stem cells in a medium further supplemented with a WNT activator. A method for the differentiation of pancreatic endoderm cells according to any one of [1] to [4] above, comprising the step of culturing the cells in a culture medium containing the pancreatic endoderm cells. Formation group. [6] The stepwise differentiation is achieved by culturing definitive endoderm cells in a medium further supplemented with an FGF ligand. A differentiated population of pancreatic endoderm cells according to [1] above, comprising the step of: [7] The stepwise differentiation is performed by administering an shh inhibitor, an FGF ligand, a PKC activator, a TGF-B ligand, or Intestinal cells were cultured in a medium further supplemented with a gradient of amide, retinoid, and BMP inhibitor. A differentiated population of pancreatic endoderm cells according to any one of [1] to [4] above, comprising the step of: [8] The stepwise differentiation is mediated by a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor. The method for producing the pancreatic endothelial cells according to [5] above, further comprising culturing the posterior foregut cells in a supplemented medium. A differentiated population of germ layer cells. [9] The stepwise differentiation comprises culturing the cells in a medium further supplemented with ascorbic acid. A differentiated population of pancreatic endoderm cells according to any one of [1] to [6] above.

[10] In vitro method for stepwise differentiation of pluripotent cells into populations of cells of the pancreatic endoderm lineage Cultivate cells at each stage of differentiation in a medium containing 5mM to 20mM glucose. A method comprising the steps of:

[11] Culturing the pluripotent cells in a medium supplemented with a TGF-B ligand and a WNT activator. The method further comprises the step of differentiating the pluripotent cells into definitive endoderm (DE) cells by The in vitro method described in

[10] .

[12] The DE cells were cultured in a medium supplemented with an FGF ligand. The in vitro method described in

[11] above, further comprising a step of differentiating the alveoli into intestinal cells.

[13] shh inhibitors, FGF ligands, PKC activators, TGF-B ligands, retinoids, and a BMP inhibitor, and culturing the intestinal cells in a medium supplemented with the BMP inhibitor. The in vitro method according to

[12] above, further comprising a step of differentiating the cells into posterior foregut endoderm cells. .

[14] After treatment with PKC activators, shh inhibitors, retinoids, and BMP inhibitors, By culturing the posterior foregut endoderm cells, the posterior foregut endoderm cells are differentiated into pancreatic foregut cells. The in vitro method according to

[13] above, further comprising the step of:

[15] The pancreatic foregut cells were cultured in medium supplemented with an shh inhibitor, a TGF-B inhibitor, and a retinoid. The method further comprises the step of differentiating the pancreatic foregut cells into pancreatic endoderm cells by culturing the cells. The in vitro method described in

[14] above.

[16]

[15] above, further comprising the step of differentiating the pancreatic endoderm cells into a pancreatic β cell population. In vitro methods.

[17]

[10] to

[11] above, wherein the medium is further supplemented with ascorbic acid in at least one step.

[16] An in vitro method according to any one of claims 16 to 18.

[18] An in vitro method described in any one of

[0010] to

[17] above, wherein more than 10% of the cells in the differentiated population are single-hormone insulin-positive cells.

[19] Over 30% of the pancreatic endoderm cells in culture were PDX-1+, NKX6.1+, SOX2-, and and CDX2-.

[20] 1. An in vitro method for differentiating human embryonic stem cells into pancreatic beta cells, comprising: a) Undifferentiated human embryonic stem cells were cultured in a graft to generate a population of definitive endoderm (DE) cells. culturing the cells in a medium supplemented with glucose, a TGF-B ligand, and a WNT activator; , b) In order to generate a population of intestinal cells, the DE cells were cultured in a culture medium containing glucose and FGF ligands. Culturing the cells in a medium supplemented with c) To generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2, Glucose, shh inhibitor, FGF ligand, PKC activator, TGF-B ligand, culturing the intestinal cells in a medium supplemented with a gradient of a cytochrome P450 inhibitor and a BMP inhibitor; d) express PDX-1 and NKX6.1, and at lower levels compared to the posterior foregut cells 1. Glucose-, PKC-Activated Pancreatic Foregut Cells to Generate a Population of SOX2-Expressing Pancreatic Foregut Cells The posterior foregut cells were cultured in medium supplemented with an anti-inflammatory drug, an shh inhibitor, a retinoid, and a BMP inhibitor. and culturing the e) High levels of NKX6.1 and low levels compared to PDX-1 and pancreatic foregut cells To obtain a population of pancreatic endoderm cells expressing SOX2, glucose, and shh inhibitors Culturing the pancreatic foregut cells in a medium supplemented with a TGF-B inhibitor and a retinoid. The degree, f) differentiating said pancreatic endoderm cells into a pancreatic beta cell population. [twenty one] the pancreatic beta cell population is PDX-1+, NKX6.1+, SOX2-, and CDX2- The method according to

[20] above. [twenty two]

[20] wherein the medium is further supplemented with ascorbic acid in at least one step. Or the method described in

[21] . [twenty three] The pancreatic beta cells are stimulated with a single hormone, insulin, which is also NKX6.1+ and PDX-1+. The method according to

[22] above, wherein the cells are erythropoietin-producing cells.

Claims

1. An in vitro differentiated population of pancreatic endoderm cells obtained from stepwise differentiation of pluripotent cells. , cells at each stage of differentiation are cultured in a medium containing 5 mM to 20 mM glucose; A differentiated population of visceral endoderm cells.

2. More than 30% of the differentiated pancreatic endoderm cells are PDX-1+NKX6.1+, SOX2 2. The differentiated population of pancreatic endoderm cells according to claim 1, wherein the cells are CDX2-, CDX3-, and CDX4-.

3. 10. The method of claim 1, wherein greater than 10% of the cells in the differentiated population are single hormone insulin positive cells. or 2. A differentiated population of pancreatic endoderm cells.

4. The stepwise differentiation is carried out by culturing undifferentiated human embryonic stem cells in a culture medium further supplemented with a TGF-B ligand. A method for differentiation of pancreatic endoderm cells according to any one of claims 1 to 3, comprising culturing the cells in soil. Group.

5. The stepwise differentiation is performed by culturing undifferentiated human embryonic stem cells in a medium further supplemented with a WNT activator. The differentiated population of pancreatic endoderm cells according to any one of claims 1 to 4, comprising a step of culturing the differentiated population of pancreatic endoderm cells in a culture medium containing 2000 cells / mL of pancreatic endoderm cells. 。

6. The stepwise differentiation is achieved by culturing definitive endoderm cells in a medium further supplemented with an FGF ligand. The differentiated population of pancreatic endoderm cells according to claim 1, comprising the step of:

7. The stepwise differentiation is carried out by using an shh inhibitor, an FGF ligand, a PKC activator, a TGF-B ligand, or the like. Intestinal cells were cultured in a medium further supplemented with a gradient of amide, retinoid, and BMP inhibitor. A differentiated population of pancreatic endoderm cells according to any one of claims 1 to 4, comprising the step of:

8. The stepwise differentiation is mediated by a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor. The pancreatic endogenous embryo of claim 5, further comprising culturing posterior foregut cells in a supplemented medium. A differentiated population of leaf cells.

9. The stepwise differentiation comprises culturing the cells in a medium further supplemented with ascorbic acid. A differentiated population of pancreatic endoderm cells according to any one of claims 1 to 6.

10. In vitro method for stepwise differentiation of pluripotent cells into populations of cells of the pancreatic endoderm lineage and culturing the cells at each stage of differentiation in a medium containing 5 mM to 20 mM glucose. A method comprising the steps of:

11. Culturing the pluripotent cells in a medium supplemented with a TGF-B ligand and a WNT activator. The method further comprises differentiating the pluripotent cells into definitive endoderm (DE) cells by 11. The in vitro method of claim 10.

12. The DE cells are cultured in a medium supplemented with an FGF ligand. The in vitro method of claim 11 , further comprising the step of differentiating the alveoli into intestinal cells.

13. shh inhibitors, FGF ligands, PKC activators, TGF-B ligands, retinoids, and culturing the intestinal cells in a medium supplemented with a BMP inhibitor, The in vitro method of claim 12 , further comprising the step of differentiating into posterior foregut endoderm cells.

14. After treatment in medium supplemented with PKC activators, shh inhibitors, retinoids, and BMP inhibitors, By culturing the posterior foregut endoderm cells, the posterior foregut endoderm cells are differentiated into pancreatic foregut cells.

14. The in vitro method of claim 13, further comprising the step of:

15. The pancreatic foregut cells were cultured in a medium supplemented with an shh inhibitor, a TGF-B inhibitor, and a retinoid. The method further comprises the step of differentiating the pancreatic foregut cells into pancreatic endoderm cells by culturing the cells. The in vitro method of claim 14.

16. 16. The method of claim 15, further comprising differentiating the pancreatic endoderm cells into a pancreatic beta cell population. In vitro methods.

17. 10 to 1, wherein the medium is further supplemented with ascorbic acid in at least one step.

7. An in vitro method according to any one of claims 6 to 6.

18. 10. The method of claim 1, wherein greater than 10% of the cells in the differentiated population are single hormone insulin positive cells.

18. An in vitro method according to any one of claims 0 to 17.

19. More than 30% of the pancreatic endoderm cells in culture were PDX-1+, NKX6.1+, SOX2-, and and CDX2-.

20. 1. An in vitro method for differentiating human embryonic stem cells into pancreatic beta cells, comprising: a) To generate a population of definitive endoderm (DE) cells, undifferentiated human embryonic stem cells are cultured in a graft. culturing the cells in a medium supplemented with glucose, a TGF-B ligand, and a WNT activator; 、 b) In order to generate a population of intestinal cells, the DE cells are cultured in a medium containing glucose and FGF ligands. Culturing the cells in a medium supplemented with c) to generate a population of posterior foregut endoderm cells that express PDX-1 and SOX2; Glucose, shh inhibitor, FGF ligand, PKC activator, TGF-B ligand, Culturing the intestinal cells in a medium supplemented with a gradient of a cytochrome P450 inhibitor and a BMP inhibitor; d) express PDX-1 and NKX6.1 and at lower levels compared to the posterior foregut cells To generate a population of pancreatic foregut cells expressing SOX2, glucose, PKC activity, The posterior foregut cells were cultured in a medium supplemented with an agent, an shh inhibitor, a retinoid, and a BMP inhibitor. and culturing the e) High levels of NKX6.1 and low levels compared to PDX-1 and pancreatic foregut cells To obtain a population of pancreatic endoderm cells expressing SOX2, we used glucose, an shh inhibitor, and and culturing the pancreatic foregut cells in a medium supplemented with a TGF-B inhibitor and a retinoid. The degree, f) differentiating said pancreatic endoderm cells into a pancreatic beta cell population.

21. the pancreatic beta cell population is PDX-1+, NKX6.1+, SOX2-, and CDX2- The method of claim 20.

22. 20. The method of claim 20, wherein the medium is further supplemented with ascorbic acid in at least one step.

21. The method according to claim 21.

23. The pancreatic beta cells are induced by a single hormone, insulin, which is also NKX6.1+ and PDX-1+.

23. The method of claim 22, wherein the cell is an agonist-producing cell.

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