Differentiation of Human Embryonic Stem Cells into Insulin-Positive Cells of a Single Hormone

Abstract

A method for promoting differentiation of pluripotent stem cells is provided. [Solution] An in vitro method for the stepwise differentiation of pluripotent cells into a population of cells of the pancreatic endoderm lineage is provided, comprising culturing the cells at each stage of differentiation in a medium containing 5 mM to 20 mM glucose, in which population more than 10% of the cells express markers characteristic of single-hormonal pancreatic beta cells.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of U.S. Provisional Patent Application No. 61 / 579,3 51, filed on December 22, 2011, and the entire disclosure of that application is incorporated herein by reference for all purposes.

[0002] (Field of the Invention) The present invention is in the field of cell differentiation. More specifically, the present invention provides single - hormone insulin - producing cells differentiated from pluripotent stem cells using conditions defined at each step of step - wise differentiation. More than 10% of the insulin - producing cells differentiated in the population express markers characteristic of single - hormone pancreatic β cells.

Background Art

[0003] Due to the progress of cell replacement therapy for type I diabetes and the shortage of transplantable islets of Langerhans, attention has been focused on the development of a source of insulin - producing cells, i.e., β cells, suitable for engraftment. As one approach, for example, functional β cells may be generated from pluripotent stem cells such as embryonic stem cells. In vertebrate embryonic development, pluripotent cells give rise to a group of cells containing three germ layers (ectoderm, mesoderm, and endoderm) through a process known as gastrulation. For example, tissues such as the thyroid, 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. Definitive endoderm cells express HNF3β, GATA4, MIXL1, CXCR4, and SO

[0004] tive endoderm cell) ​​​​​​​​​expresses a number of markers such as X17.

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

[0006] The migration of endodermal tissues brings the endoderm into proximity with different mesodermal tissues that help regionalize the gut tube. This is achieved by a number of secreted factors such as FGF, Wnt, TGF-B, retinoic acid (RA), and BMP ligands, as well as their antagonists. For example, FGF4 and BMP promote the expression of Cdx2 in the presumptive posterior endoderm and inhibit the expression of the anterior genes Hhex and SOX2 (Development 2000, 127:1563 - 1567). WNT signaling has also been shown to act in parallel with FGF signaling to promote hindgut development and inhibit foregut fate (Development 2007, 134:2207 - 2217). Finally, retinoic acid secreted by the mesenchyme regulates the foregut-hindgut boundary (Curr Biol 2002, 12:1215 - 1220).

[0007] The expression levels of specific transcription factors may be used to specify tissue identity. During the transformation of embryonic endoderm into the primitive gut tube, the gut tube is regionalized at the molecular level into broad domains that can be observed by restricted gene expression patterns. For example, the pancreatic domain regionalized in the gut tube shows very high expression of PDX-1 and CDX2 as well as S ​ shows very low expression of OX2. Similarly, the presence of high levels of Foxe1 is a marker of esophageal tissue. NKX2.1 is highly expressed in lung tissue. SOX2 / Odd1 (OSR1) is highly expressed in gastric tissue. The expression of PROX1 / Hhex / AFP is high in liver tissue. SOX17 is highly expressed in tissues of the bile duct structure. PDX1, NKX6.1 / PTf1a, and NKX2.2 are highly expressed in pancreatic tissue. CDX2 expression is high in intestinal tissue. The above summary is a citation from Dev Dyn 2009,238 :29 - 42 and Annu Rev Cell Dev Biol 2009,25:2 21 - 251. Pancreatic formation results from the differentiation of definitive endoderm into pancreatic endoderm (Annu Rev Cell Dev Biol 2009,25:221 - 251; Dev Dyn 2009 ,238:29 - 42). The dorsal and ventral pancreatic domains arise from the foregut epithelium. Also, the foregut gives rise to the esophagus, trachea, lung, thyroid, stomach, liver, pancreas, and biliary system. Cells of the pancreatic endoderm express the pancreatic - duodenal homeobox gene PDX1. In the absence of PDX1, the pancreas does not develop beyond the formation of ventral and dorsal buds. Therefore,

[0008] the expression of PDX1 marks an important step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine and endocrine tissues. The exocrine and endocrine tissues result from the differentiation of the pancreatic endoderm. D'Amour et al. derived human embryonic stem cells in the presence of high concentrations of activin and low serum.

[0009]

[0010] describes the production of enriched media for definitive endoderm of the embryo (Nature Biotechnol 2005, 23: 1534 - 1541; U.S. Patent No. 7,704,73 8). Transplantation of these cells under the kidney capsule of mice led to differentiation into more mature cells with characteristics of endodermal tissue (U.S. Patent No. 7,704,738). Definitive endoderm cells derived from human embryonic stem cells can further differentiate into 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 immunodeficient mice led to the formation of functional pancreatic endocrine cells after a maturation period of 3 - 4 months (U.S. Patent No. 7,993,920 and U.S. Patent No. 7,534,608).

[0011] Fisk et al. reported 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 differentiated into endoderm using a combination of sodium butyrate and activin (U.S. Patent No. 7,326,572). Next, the cells were cultured in combination with a BMP antagonist such as noggin and EGF or betacellulin to generate PDX1 - positive cells. Final differentiation was induced by nicotinamide.

[0012] Small molecule inhibitors have also been used for the induction of pancreatic endocrine progenitors. For example, small molecule inhibitors of TGF - B receptors and BMP receptors (Development 2011, 1 38: 861 - 871; Diabetes 2011, 60: 239 - 247) have significantly ... It has been used to increase the number of pancreatic endocrine cells. In addition, small molecule activators have also been used to generate definitive endoderm cells or pancreatic progenitor cells (Curr Opin Cell Biol 2009, 21: 727-732; Nature Chem Biol 2009, 5: 258-265). Previous attempts in the induction of pancreatic progenitor cells from human embryonic stem cells have revealed the importance of co-expression of PDX-1 and NKX6.1 in the correct identification of pancreatic endoderm. However, the art has identified a population of cells that are positive for the expression of PDX-1 and NKX6 but have low or no expression of CDX2, and previous reports have not yet reached the testing of the presence of markers immediately anterior to the developing pancreas. SOX2, which marks the anterior endoderm, is not expressed in adult islets and is expressed at very low levels in the developing pancreas (Diabetes 2005, 54: 3402-4309). In contrast, some of the examples of the present application disclose a cell population in which at least 30% of the pancreatic endoderm cells generated from human embryonic stem cells are positive for the expression of PDX-1 and NKX6.1 and negative for the expression of CDX2 and SOX2.

[0013] All previous attempts to generate functional pancreatic β cells have not led to the obtaining of cells with the characteristics of mature β cells. The characteristics of mature β cells include the expression of the single hormone insulin, the correct processing of proinsulin to insulin and C-peptide, the strong expression of PDX-1 and NKX6.1, the appropriate insulin release in response to glucose, the expression of glucose transporters, and the high expression of glucokinase. All previous reports have two

[0014] ​​​​​​​​​​​​​​​ resulted in endocrine cells that produce the pancreatic hormones described above. For example, D’Amour et al (Nature Biotech 2006,24:1392~1401) reported the generation of cell populations containing up to 10% insulin-positive cells and up to 20% endocrine cells as measured by synaptophysin . Other similar reports (Cell Res 2009, 19:429~438;Stem Cells 2007,25:1940~1953; Diabetes Obes Metab 2008,10:186~194) have also shown the differentiation of pluripotent cells into non-functional insulin-positive cells. In fact, recent studies have clearly shown that the transplantation of multiphor-monal cells in severe combined immunodeficient (SCID) mice did not result in the generation of functional β cells(Diabetes 2011,60: 239~247;Nature Biotech 2011,29:750~756). In the human fetal pancreas, a subset (up to 10-20%) of endocrine cells are multiphor-monal cells, but multiphor-monal cells are absent in the adult human pancreas(Histochem Cell Biol 1999,112:147~153;J Histochem Cytoch em 2009,57:811~824). As the rapidly growing field of regenerative medicine continues to mature, there is a strong need for methods for the formation of terminally differentiated, properly regulated pancreatic endocrine cells . It is demonstrated herein that human embryonic stem cells can be differentiated into functional pancreatic β cells in vitro using manipulation of defined culture conditions that are stringent and defined, and precise timing of the addition of activators / inhibitors of various pathways .

[0015] As the rapidly growing field of regenerative medicine continues to mature, there is a strong need for methods for the formation of terminally differentiated, properly regulated pancreatic endocrine cells . Suitable Using manipulation of defined culture conditions that are stringent and defined, and precise timing of the addition of activators / inhibitors of various pathways it is demonstrated herein that human embryonic stem cells can be differentiated into functional pancreatic β cells in vitro It is carried out. Specifically, the exact timing of BMP inhibition, along with the use of vitamin C and the gradient of retinoic acid, has been proven to be effective in the generation of single-hormone pancreatic endocrine cells. has been demonstrated.

Summary of the Invention

Means for Solving the Problems

[0016] The present invention provides a population of cells of the pancreatic endoderm lineage obtained in vitro by stepwise differentiation of pluripotent cells. Glucose is supplemented in the medium used in each step of differentiation. In some embodiments, the cells are cultured in a medium containing 5 mM to 20 mM glucose in each step of differentiation. In some embodiments, in the pancreatic endoderm cell population generated by the differentiation of pluripotent stem cells, more than 10% of the cells in the differentiated population express markers characteristic of single-hormone pancreatic β cells.

[0017] In some embodiments, in the pancreatic endoderm cell population generated by the differentiation of pluripotent stem cells, more than 30% of the differentiated population is positive for the expression of PDX-1 and NKX6.1, but negative for the expression of CDX2 and SOX2.

[0018] In some embodiments, the stepwise differentiation includes culturing undifferentiated human embryonic stem cells in a medium further supplemented with a TGF-β ligand. In some embodiments, the stepwise differentiation includes culturing undifferentiated human embryonic stem cells in a medium further supplemented with a WNT activator. In some embodiments, the stepwise differentiation includes culturing definitive endoderm in a medium further supplemented with an FGF ligand.

[0019] It includes the step of culturing leaf cells. In some embodiments, stepwise differentiation is carried out in a medium supplemented with a gradient of a shh inhibitor, an FGF ligand, a PKC activator, a TGF-B ligand, a retinoid, and a BMP inhibitor and includes the step of culturing intestinal cells. In some embodiments , stepwise differentiation further includes culturing posterior foregut cells in a medium supplemented with a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor . In some embodiments, stepwise differentiation includes culturing cells in a medium supplemented with ascorbic acid .

[0020] In one embodiment, the present invention provides an in vitro method for stepwise differentiating pluripotent cells into a cell population of the pancreatic endoderm lineage, which includes culturing cells at each stage of differentiation in a medium containing 5 mM to 20 mM glucose. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further includes differentiating pluripotent cells into definitive endoderm (DE) cells by culturing the pluripotent cells in a medium supplemented with a TGF-B ligand and a WNT activator. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further includes differentiating DE cells into intestinal cells by culturing the DE cells in a medium supplemented with an FGF ligand. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further includes differentiating intestinal cells into posterior foregut endoderm cells by culturing the intestinal cells in a medium supplemented with a shh inhibitor, an FGF ligand, a PK C activator, a TGF-B ligand, a retinoid, and a BMP inhibitor. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further includes sh h H inhibitor, FGF ligand, PKC activator, TGF-β ligand, retinoid, and B culturing intestinal cells in a medium supplemented with MP inhibitor to differentiate the intestinal cells into posterior foregut endoderm cells. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further comprises culturing posterior foregut endoderm cells in a medium supplemented with a PKC activator, shh inhibitor, retinoid, and BMP inhibitor to differentiate the posterior foregut endoderm cells into pancreatic foregut cells. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further comprises culturing pancreatic foregut cells in a medium supplemented with shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the pancreatic foregut cells into pancreatic endoderm cells. In some embodiments, the in vitro method for stepwise differentiation of pluripotent cells further comprises differentiating pancreatic endoderm cells into a pancreatic β cell population. In one embodiment, in at least one step of the in vitro method for stepwise differentiation of pluripotent cells, ascorbic acid is further supplemented to the medium. In some embodiments, more than 10% of the cells in the differentiated population are insulin-positive cells of a single hormone. In some embodiments, more than 30% of the pancreatic endoderm cells in the culture produced by the method of the present invention are PDX-1+, NKX6.1+, SOX2-, and CDX2-. In one embodiment, the present invention relates to an in vitro method for differentiating human embryonic stem cells into pancreatic β cells, the method comprising: a) generating a population of definitive endoderm (DE) by culturing undifferentiated human embryonic stem cells in a medium supplemented with glucocorticoid, TGF-β ligand, and WNT activator; b) culturing the DE cells in a medium supplemented with an FGF ligand, PKC activator, and BMP inhibitor to differentiate the DE cells into posterior foregut endoderm cells; c) culturing the posterior foregut endoderm cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the posterior foregut endoderm cells into pancreatic foregut cells; d) culturing the pancreatic foregut cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the pancreatic foregut cells into pancreatic endoderm cells; e) culturing the pancreatic endoderm cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the pancreatic endoderm cells into pancreatic β cell populations. In some embodiments, in at least one step of the in vitro method for stepwise differentiation of pluripotent cells, ascorbic acid is further supplemented to the medium. In some embodiments, more than 10% of the cells in the differentiated population are insulin-positive cells of a single hormone. In some embodiments, more than 30% of the pancreatic endoderm cells in the culture produced by the method of the present invention are PDX-1+, NKX6.1+, SOX2-, and CDX2-.

[0021] In one embodiment, in at least one step of the in vitro method for stepwise differentiation of pluripotent cells, ascorbic acid is further supplemented to the medium. In some embodiments, more than 10% of the cells in the differentiated population are insulin-positive cells of a single hormone. In some embodiments, more than 30% of the pancreatic endoderm cells in the culture produced by the method of the present invention are PDX-1+, NKX6.1+, SOX2-, and CDX2-. In one embodiment, the present invention relates to an in vitro method for differentiating human embryonic stem cells into pancreatic β cells, the method comprising: a) generating a population of definitive endoderm (DE) by culturing undifferentiated human embryonic stem cells in a medium supplemented with glucocorticoid, TGF-β ligand, and WNT activator; b) culturing the DE cells in a medium supplemented with an FGF ligand, PKC activator, and BMP inhibitor to differentiate the DE cells into posterior foregut endoderm cells; c) culturing the posterior foregut endoderm cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the posterior foregut endoderm cells into pancreatic foregut cells; d) culturing the pancreatic foregut cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the pancreatic foregut cells into pancreatic endoderm cells;

[0022] e) culturing the pancreatic endoderm cells in a medium supplemented with a shh inhibitor, TGF-β inhibitor, and retinoid to differentiate the pancreatic endoderm cells into pancreatic β cell populations. In some embodiments, in at least one step of the in vitro method for stepwise differentiation of pluripotent cells, ascorbic acid is further supplemented to the medium. In some embodiments, more than 10% of the cells in the differentiated population are insulin-positive cells of a single hormone. In some embodiments, more than 30% of the pancreatic endoderm cells in the culture produced by the method of the present invention are PDX-1+, NKX6.1+, SOX2-, and CDX2-. In one embodiment, the present invention relates to an in vitro method for differentiating human embryonic stem cells into pancreatic β cells, the method comprising: a) generating a population of definitive endoderm (DE) by culturing undifferentiated human embryonic stem cells in a medium supplemented with glucocorticoid, TGF-β ligand, and WNT activator; a) culturing cells, b) culturing DE cells in a medium supplemented with glucose and an FGF ligand to generate a population of intestinal cells c) culturing the intestinal cells in a medium supplemented with glucose, an shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a BMP inhibitor gradient to generate a population of posterior foregut endoderm cells that express PDX-1 and SOX2 d) culturing the posterior foregut cells in a medium supplemented with glucose, a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells that express PDX-1 and NKX6.1 e) culturing the pancreatic foregut cells in a medium supplemented with glucose, an shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells that express a higher level of NKX6.1 and a lower level of SOX2 compared to pancreatic foregut cells f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. e) culturing the pancreatic foregut cells in a medium supplemented with glucose, a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells that express PDX-1 and NKX6.1 e) culturing the pancreatic foregut cells in a medium supplemented with glucose, an shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells that express a higher level of NKX6.1 and a lower level of SOX2 compared to pancreatic foregut cells f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. e) culturing the pancreatic foregut cells in a medium supplemented with glucose, an shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells that express a higher level of NKX6.1 and a lower level of SOX2 compared to pancreatic foregut cells f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. In some embodiments, the pancreatic β-cell population generated by the method of the present invention is PDX-1+, NKX6.1+, SOX2−, and CDX2−. In some embodiments, ascorbic acid is further supplemented to the medium in at least one step of the stepwise differentiation method. In some embodiments, the pancreatic β-cells obtained by the method of the present invention are single-hormone insulin-producing cells and are also NKX6.1+ and PDX-1+. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 2E

Figure 2F

Figure 2G

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 3E

Figure 3F

Figure 3G

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4E

Figure 4F

Figure 4G

Figure 5A

Figure 5B

Figure 5C

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 6G

Figure 6H

Figure 6I

Figure 6J

Figure 6K

Figure 6L

Figure 6M

Figure 6N

Figure 6O

Figure 6P

Figure 6Q

Figure 6R

Figure 6S

Figure 6T

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 7E

Figure 7F

Figure 7G

Figure 8A

Figure 8B

Figure 8C

Figure 8D

Figure 8E

Figure 8F

Figure 8G

Figure 9A

Figure 9B

Figure 9C

Figure 9D

Figure 9E

Figure 9F

Figure 9G

Figure 9H

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 10E

Figure 10F

Figure 10G

Figure 10H

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 11E

Figure 11F

Figure 11G

Figure 11H

Figure 12A

Figure 12B

Figure 12C

Figure 12D

Figure 12E

Figure 12F

Figure 12G

Figure 13A

Figure 13B

Figure 13C

Figure 13D

Figure 13E

Figure 13F

Figure 13G

Figure 14A

Figure 14B

Figure 14C

Figure 14D

Figure 14E

Figure 14F

Figure 14G

Figure 14H

Figure 15A

Figure 15B

Figure 15C

Figure 15D

Figure 15E

Figure 15F

Figure 15G

Figure 16A

Figure 16B

Figure 16C

Figure 16D

Figure 16E

Figure 16F

Figure 17A

Figure 17B

Figure 17C

Figure 17D

Figure 17E

Figure 17F

Figure 17G

Figure 17H

Figure 17I

Figure 17J

Figure 18A

Figure 18B

Figure 18C

Figure 18D

Figure 18E

Figure 18F

Figure 18G

Figure 19A

Figure 19B

Figure 19C

Figure 19D

Figure 19E

Figure 19F

Figure 19G

Figure 20A

Figure 20B

Figure 20C

Figure 20D

Figure 20E

Figure 20F

Figure 20G

Figure 20H

Figure 20I

Figure 20J

Figure 21A

Figure 21B

Figure 21C

Figure 21D

Figure 21E

Figure 21F

Figure 21G

Figure 21H

Figure 21I

Figure 21J

Figure 22A

Figure 22B

Figure 22C

Figure 22D

Figure 22E

Figure 22F

Figure 22G

Figure 22H

Figure 22I

Figure 22J

Fig. 22K

Fig. 22L

Mode for Carrying Out the Invention

[0024] For the sake of clarity in the disclosure, and without being limiting, the "Mode for Carrying Out the Invention" is divided into the following sub-items that explain or exemplify specific features, embodiments, or uses of the present invention. The "Mode for Carrying Out the Invention" divides the specific features, embodiments, or uses of the present invention into the following sub-items for explanation or exemplification. Divide.

[0025] Definition Stem cells are undifferentiated cells defined by both self-renewal ability and differentiation ability at the single-cell level. Stem cells can generate progeny cells including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are also characterized by the ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm). Stem cells can also generate tissues of multiple germ layers after transplantation and, after injection into a blastocyst, substantially (not all) cells. generate progeny cells including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells can also generate tissues of multiple germ layers after transplantation and, after injection into a blastocyst, substantially (not all) cells. generate tissues of multiple germ layers after transplantation and, after injection into a blastocyst, substantially (not all) Contributes to most (if not all) of the organizations.

[0026] Stem cells are classified by developmental potential as: (1) totipotent, meaning capable of giving 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 occur within a particular 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 comparatively unspecialized cells e.g. For example, differentiation is the process by which cells acquire the characteristics of specialized cells, such as nerve cells or muscle cells. "Communicated cells" or "differentiated cells" are cells that have become more specialized ("communicated") 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 that 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 refers to the genetic makeup of a cell, i.e., which cell it came from and which cell it is derived from. defines whether it can occur. A cell lineage positions the cell within a given genetic system of development and differentiation. A lineage-specific marker refers to a feature specifically associated with the phenotype of cells of the target lineage and can be used to evaluate the differentiation of undifferentiated cells into the target lineage.

[0028] As used herein, a "marker" refers to a nucleic acid or polypeptide molecule that is differentially expressed in the target cell. In this context, differential expression means an increase in the level of a positive marker and a decrease in the level of a negative marker. The detectable level of a marker nucleic acid or polypeptide is sufficiently high or low in the target cell compared to other cells such that the target cell can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0029] As used herein, a cell is "positive" or "positive for" a specific marker when the specific marker is detected within the cell. Similarly, a cell is "negative" or "negative for" a specific marker when the specific marker is not detected within the cell.

[0030] As used herein, "Stage 1" and "S1" are used interchangeably to identify cells that express markers characteristic of definitive endoderm (DE).

[0031] As used herein, "definitive endoderm" refers to cells that arise from the epiblast during gastrulation and have the characteristics of cells that form the gastrointestinal tract and its derivatives. Definitive endoderm cells express at least one of the following markers: HNF3β, GATA4, SOX17, CXCR 4, Cerberus, OTX2, Goosecoid, C-Kit, CD99, and MIXL1.

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

[0033] As used herein, "stage 2" and "S2", which identify cells expressing markers characteristic of the primitive gut tube, are used interchangeably.

[0034] "Foregut endoderm" refers to endodermal cells that give rise to the esophagus, lung, stomach, liver, pancreas, gallbladder, and part of the duodenum.

[0035] "Posterior foregut" refers to endodermal cells that can give rise to the posterior stomach, pancreas, liver, and part of the duodenum.

[0036] "Midgut endoderm" refers to endodermal cells that can give rise to the intestine, part of the duodenum, appendix, and ascending colon.

[0037] "Hindgut endoderm" refers to endodermal cells that can give rise to the distal one-third of the transverse colon, descending colon, sigmoid colon, and rectum.

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

[0039] "Stage 4" and "S4" are used interchangeably to identify cells that express markers characteristic of pancreatic foregut progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic foregut progenitor cell lineage" refers to cells that express at least one of the following markers: PDX-1, NKX6.1, HNF6, FOXA2, PTF1a, Prox1, and HNF4α. As used herein, "Stage 5" and "S5" are used interchangeably to identify cells that express markers characteristic of pancreatic endoderm and pancreatic endocrine progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic endoderm lineage" refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF1β, PTF1α, HNF6, HNF4α, SOX9, HB9, or PROX1. Cells that express markers characteristic of the pancreatic endoderm lineage do not substantially express CDX2 or SOX2. As used herein, "Stage 6" and "S6" are used interchangeably to identify cells in which pancreatic endocrine cells are enriched. As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. "Pancreatic endocrine progenitor cells" or "pancreatic endocrine precursor cells"

[0040] As used herein, "Stage 4" and "S4" are used interchangeably to identify cells that express markers characteristic of pancreatic foregut progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic foregut progenitor cell lineage" refers to cells that express at least one of the following markers: PDX-1, NKX6.1, HNF6, FOXA2, PTF1a, Prox1, and HNF4α. As used herein, "Stage 5" and "S5" are used interchangeably to identify cells that express markers characteristic of pancreatic endoderm and pancreatic endocrine progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic endoderm lineage" refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF1β, PTF1α, HNF6, HNF4α, SOX9, HB9, or PROX1. Cells that express markers characteristic of the pancreatic endoderm lineage do not substantially express CDX2 or SOX2. As used herein, "Stage 6" and "S6" are used interchangeably to identify cells in which pancreatic endocrine cells are enriched. As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. "Pancreatic endocrine progenitor cells" or "pancreatic endocrine precursor cells" As used herein, "Stage 4" and "S4" are used interchangeably to identify cells that express markers characteristic of pancreatic foregut progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic foregut progenitor cell lineage" refers to cells that express at least one of the following markers: PDX-1, NKX6.1, HNF6, FOXA2, PTF1a, Prox1, and HNF4α. As used herein, "Stage 5" and "S5" are used interchangeably to identify cells that express markers characteristic of pancreatic endoderm and pancreatic endocrine progenitor cells. As used herein, "cells that express markers characteristic of the pancreatic endoderm lineage" refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF1β, PTF1α, HNF6, HNF4α, SOX9, HB9, or PROX1. Cells that express markers characteristic of the pancreatic endoderm lineage do not substantially express CDX2 or SOX2.

[0041] As used herein, "Stage 6" and "S6" are used interchangeably to identify cells in which pancreatic endocrine cells are enriched. As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.

[0042] As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. As used herein, "pancreatic endocrine cells" or "pancreatic hormone-expressing cells" or "cells that express markers characteristic of the pancreatic endocrine lineage" refers to cells that are capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. "Pancreatic endocrine progenitor cells" or "pancreatic endocrine precursor cells"

[0043] "Pancreatic endocrine progenitor cells" or "pancreatic endocrine precursor cells" , pancreas pancreatic hormone expressing cellrefers to pancreatic endoderm cells that can become such 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 β cells" are glucose-responsive and can be positive for PDX-1 and NKX6.1 and refer to single-hormone insulin-positive cells.

[0045] In this specification, "d1", "d 1", and "day 1", "d2", "d 2", and "day 2", "d3", "d 3" and "day 3", etc. are used interchangeably. These numerical combinations identify the days of incubation at different stages in the stepwise differentiation protocol of the present application.

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

[0047] "Glucose" and "D-glucose" are used interchangeably herein and refer to the sugar, dextrose, which is commonly found in nature.

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

[0049] In the present application, "chromogranin" and "CHGN" are used interchangeably to identify the gene encoding the acidic secreted glycoprotein chromogranin. ​​​​

[0050] Identify the proteins expressed in pancreatic endocrine progenitor cells and the genes encoding them "NeuroD" and "NeuroD1", which are used interchangeably herein,

[0051] "LDN" and "LDN-193189" are used interchangeably herein and refer to a BMP receptor inhibitor available from Stemgent, California, USA

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

[0053] Another desirable phenotype of proliferated pluripotent stem cells is the ability to differentiate into cells of all three germ layers, namely, endoderm germ layer, mesoderm, and ectoderm tissues. The pluripotency of stem cells can be demonstrated, for example by injecting the cells into SCID mice and using 4% paraformaldehyde to fix the formed teratomas ​​​​After fixing the tumors, they are histologically examined for traces of cell types from the three germ layers and can be confirmed thereby. Alternatively, pluripotency can be determined by forming embryoid bodies and evaluating these embryoid bodies for the presence of markers associated with the three germ layers.

[0054] The expanded pluripotent stem cell line can have its karyotype determined by using standard G-banding methods and comparing it to the published karyotype of the corresponding primate species. It is desirable for the cells to have a "normal karyotype", which means that the cells are euploid, all human chromosomes are present and there are no obvious changes. Pluripotent cells can be easily cultured and expanded using various feeder layers or containers coated with matrix proteins. Alternatively, a chemically defined surface combined with a defined medium such as mTesr™1 medium (StemCell Technologies, Vancouver, Canada) can be used for routine cell expansion. Pluripotent cells can be easily removed from culture plates enzymatically, mechanically, or using various calcium chelating agents such as EDTA (ethylenediaminetetraacetic acid). Alternatively, pluripotent cells can be grown in suspension in the absence of matrix proteins or feeder layers.

[0055] Sources of pluripotent stem cells Types of pluripotent stem cells that can be used include established lines of pluripotent cells derived from post-pregnancy formed tissues such as pre-embryonic tissues (e.g., blastocysts, etc.), embryonic tissues, or fetal tissues, collected at any time during pregnancy (although not necessarily, usually before about 10-12 weeks of gestation). is included. Non-limiting examples include the establishment of human embryonic stem cells (hESCs) or human embryonic germ cells lines, such as, for example, human embryonic stem cell lines H1, H7, and H9 (WiCell Research Institute, Madison, Wisconsin, USA ). Cells harvested from a pluripotent stem cell population that has already been cultured in the absence of feeder cells are also suitable. Induced pluripotent cells (iPS) or reprogrammed pluripotent cells that can be induced from adult somatic cells using the forced expression of a number of transcription factors related to pluripotency, such as OCT4, Nanog, Sox2, KLF4, and ZFP42, are also suitable (Annu Rev Genomics Hum Genet, 2011, 12:165-185). The human embryonic stem cells used in the method of the present invention may be 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. U.S.A., 1995:92:7844). The formation 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 being continuously identified. Examples of markers of pluripotent stem cells include, for example, the following, namely, ABC G2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OC T4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SS

[0056] EA-4, Tra 1-60, Tra 1-81, the expression of one or more of these.

[0057] ​​​​​Suitable pluripotent stem cells for use in the present invention include, for example, human embryonic stem cell line H9 (N IH code: WA09), human embryonic stem cell line H1 (NIH code: WA01), human embryonic stem cell line H7 (NIH code: WA07), and human embryonic stem cell line SA002 (Cell artis, Sweden). Cells that express at least one of the following markers characteristic of pluripotent cells, namely, ABCG2, cripto, CD9, FOXD3, CONNEXIN43, CON NEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42 SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81 are also suitable for use in the present invention. Markers characteristic of the embryonic endoderm lineage are SOX17, GATA4, HNF3 β, GSC CER1, Nodal, FGF8, short tail malformation, Mix-like homeobox protein, F

[0058] GF4, CD48, eomesodermin (EOMES), DKK4, FGF17, GATA 6, CXCR4, C-Kit, CD99, and OTX2. Cells that express at least one of the markers characteristic of the embryonic endoderm lineage are suitable for use in the present invention. In one aspect of the present invention, cells that express markers characteristic of the embryonic endoderm lineage are primitive streak progenitor cells. In another aspect, cells that express markers characteristic of the embryonic endoderm lineage are mesendoderm cells. In another aspect, cells that express markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells. GF4, CD48, eomesodermin (EOMES), DKK4, FGF17, GATA 6, CXCR4, C-Kit, CD99, and OTX2 are selected from the group consisting of. Embryo Cells expressing at least one of the markers characteristic of the endoderm lineage are suitable for use in the present invention. In one aspect of the present invention, cells expressing markers characteristic of the embryonic endoderm lineage are primitive streak progenitor cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are mesendoderm cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells. Cells expressing markers characteristic of the embryonic endoderm lineage are suitable for use in the present invention. In one aspect of the present invention, cells expressing markers characteristic of the embryonic endoderm lineage are primitive streak progenitor cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are mesendoderm cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells. Cells expressing markers characteristic of the embryonic endoderm lineage are primitive streak progenitor cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are mesendoderm cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells. Cells expressing markers characteristic of the embryonic endoderm lineage are primitive streak progenitor cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are mesendoderm cells. In another aspect, cells expressing markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells. Cells expressing markers characteristic of the embryonic endoderm lineage are embryonic endoderm cells.

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

[0060] Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, and PAX6. In one embodiment, pancreatic endocrine cells can express at least one of the following hormones, namely insulin, glucagon, somatostatin, and pancreatic polypeptide. Those suitable for use in the present invention are cells expressing at least one marker indicative of the characteristics of the pancreatic endocrine lineage. In one aspect of the present invention, cells expressing a marker characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells. Pancreatic endocrine cells may be pancreatic hormone-expressing cells. Alternatively, pancreatic endocrine cells may be pancreatic hormone-secreting cells.

[0061] In one aspect of the present invention, pancreatic endocrine cells are cells expressing a marker characteristic of the β-cell lineage. Cells expressing a marker characteristic of the β-cell lineage express PDX1 and at least one of the following transcription factors, namely NKX2.2, NKX6.1, NEUROD, ISL1, HNF3β , MAFA, PAX4, and PAX6. In one aspect of the present invention, cells expressing a marker characteristic of the β-cell lineage are β-cells.

[0062] ​​​​​​​​​​​ The present invention describes an in vitro method and cell population capable of generating single hormone insulin-positive cells that are also PDX-1 and NKX6.1 positive. The method used in the present invention stepwise guides the differentiation of human pluripotent cells into single hormone cells through the following intermediate stages and includes a series of stages. a) Generation of definitive endoderm (DE) cells from undifferentiated human embryonic stem cells, including culturing the pluripotent cells in a medium containing glucose, a TGF-β ligand, and a WNT activator. b) Differentiation of DE cells into intestinal cells, including culturing the DE cells in a medium containing glucose, vitamin C, and an FGF ligand. c) Differentiation of intestinal cells into posterior foregut endoderm cells expressing PDX-1 and SOX2. This differentiation is achieved by culturing the intestinal cells in the presence of an shh inhibitor, a BMP inhibitor, a TGF-β ligand, an FGF ligand, retinoic acid, vitamin C, and a PKC activator. d) Differentiation of posterior foregut cells into pancreatic foregut cells expressing PDX-1 and NKX6.1 and a lower level of SOX2 compared to posterior foregut cells. This differentiation is achieved by culturing the posterior foregut cells in the presence of an shh inhibitor, a BMP inhibitor, a low dose of retinoic acid, vitamin C, and a PKC activator. e) Differentiation of pancreatic foregut cells into pancreatic endoderm cells expressing PDX-1 and a higher level of NKX6.1 and a lower level of SOX2 compared to pancreatic foregut cells. This differentiation is achieved by culturing the pancreatic foregut cells in a medium supplemented with an shh inhibitor, a TGF-β inhibitor, a low dose of retinoic acid, and vitamin C. f) Differentiating pancreatic endoderm cells into pancreatic endocrine progenitor cells and subsequently into single hormone pancreatic endocrine ​​​​​​​​​​​​​​The step of differentiating into cells. This differentiation is achieved by culturing pancreatic endoderm cells in a medium supplemented with an shh inhibitor, a low dose of retinoic acid, and vitamin C.

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

[0064] In some embodiments, the medium formulation used for all subsequent processes for generating intestinal stage cells contains ascorbic acid (also known as vitamin C). In one embodiment, the concentration of ascorbic acid is about 0.01 mM to about 1 mM. In one 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 by these examples.

Example

[0066] (Example 1) Differentiation of human embryonic stem cells of cell line H1 into pancreatic endocrine progenitor cells in the absence of fetal bovine serum - Regulation of the BMP / TGF - B pathway results in improved production of pancreatic endoderm population and a decrease in the percentage of the SOX2+ population. This example was carried out to show that a pancreatic endoderm culture can be generated that has low levels of expression of CDX2 and SOX2 while having very high expression levels of PDX - 1 and NKX6.1. This example shows that a pancreatic endoderm culture can be generated that has low levels of expression of CDX2 and SOX2 while having very high expression levels of PDX - 1 and NKX6.1. This was carried out to show that a pancreatic endoderm culture can be generated that has low levels of expression of CDX2 and SOX2 while having very high expression levels of PDX - 1 and

[0067] The human embryonic stem cell line H1 (hESC H1) was harvested at various passages (passage 40 to passage 52) Harvested and supplemented with 10 μM Y27632 (Rock inhibitor, catalog number Y0503, Sigma Aldrich, Missouri, USA) in mTeSR™1 medium (StemCell Technologies, Vancouver, Canada), seeded as single cells at a density of 100,000 cells per cm onto dishes coated with MATRIGEL® (1:30 dilution, BD Biosciences, New Jersey, USA). Forty-eight hours after seeding, the cultures were washed in incomplete PBS (phosphate-buffered saline without Mg or Ca) for approximately 30 seconds and incubated. The cultures were differentiated into the pancreatic endocrine lineage as follows: a. Stage 1 (definitive endoderm (DE) - day 3): Stage 1 medium (0.1% fatty acid-free BSA (catalog number 68700, Proliant, Iowa, USA), 0.001 2 g / mL sodium bicarbonate (catalog number S3187, Sigma Aldrich, Missouri, USA), 1X GlutaMax™ (catalog number 35050-079 2 , Invitrogen), 5 mM D-glucose (catalog number G8769, Sigma Aldrich, Missouri, USA) supplemented with 100 ng / mL GDF8 (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-heptaazatetracyclo[19.3.1.1~2,6~.1~8,12~]heptacosa-1(25 ),2(27),3,5,8(26),9,11,21,23-nonaene-16-one, US Patent Application No. 12 / 494,789, the entire contents of which are incorporated herein by reference)) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 2 medium. b. Stage 2 (pancreatic progenitor cells - day 4): Stage 2 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8 and 1 μM MCX compound) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 3 medium. c. Stage 3 (pancreatic endocrine cells - day 5): Stage 3 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8, 1 μM MCX compound and 10 ng / mL activin A (R&D Systems, Minnesota, USA)) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 4 medium. d. Stage 4 (pancreatic endocrine cells - day 6): Stage 4 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8, 1 μM MCX compound and 10 ng / mL activin A) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 5 medium. e. Stage 5 (pancreatic endocrine cells - day 7): Stage 5 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8, 1 μM MCX compound and 10 ng / mL activin A) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 6 medium. f. Stage 6 (pancreatic endocrine cells - day 8): Stage 6 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8, 1 μM MCX compound and 10 ng / mL activin A) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 7 medium. g. Stage 7 (pancreatic endocrine cells - day 9): Stage 7 medium (0.1% fatty acid-free BSA, 0.001 2 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose supplemented with 100 ng / mL GDF8, 1 μM MCX compound and 10 ng / mL activin A) and incubated for 24 hours. The cultures were then washed with incomplete PBS and incubated in Stage 8 medium. Cells were cultured for 1 day in MCDB-131 medium (Catalog No. 10372-019, Invitrogen, California, USA) containing . Then, cells were cultured for 1 day in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 100 ng / mL GDF8, and 100 nM MCX compound . Then, cells were cultured for 1 day in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, and 100 ng / mL GDF8. Cells were cultured for 1 day. b. Stage 2 (gastrula - 2 days): Cells from Stage 1 were treated for 2 days in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, and 25 ng / mL FGF7. Cells were treated for 2 days. c. Stage 3 (foregut - 2 days): Cells from Stage 2 were cultured in Stage 3 medium (1:200 dilution of ITS-X (Invitrogen)), 2.5 mM glucose, 1X Gl utaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BS A, 25 ng / mL FGF7, 10 ng / mL activin-A (R & D sys tems), 0.25 μM SANT-1 (shh inhibitor, SigmaAldrich) , 1 μM retinoic acid (RA) (SigmaAldrich), and 200 nM TP B (PKC activator, Catalog No. 565740; EMD, New Jersey, USA) supplemented with 100 nM LDN-193189 (BMP receptor inhibitor; Catalog No. 04 -0019; containing Stemgent) of California, USA, in MCDB-131 fold In the medium), cultured for 1 day. Then, these cells were cultured for another 1 day in the medium of stage 3 supplemented with 10 nM of LDN-193189. d. Stage 4 (pancreatic foregut progenitor cells - 2 days): The cells of stage 3 were cultured in MCDB-131 fold medium supplemented with 1: 200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 5 0 nM of RA, 200 nM of TPB, and 50 nM of LDN-193189 for 2 days. e. Stage 5 (pancreatic endoderm - 2 - 7 days): The cells of stage 4 were cultured in MCDB-131 fold medium supplemented with 1:2 00 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, and 50 nM of RA for 2 - 7 days.

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

[0069] For FACS analysis, cells derived from hESCs were released into a single-cell suspension by incubating with TrypLE Express (Catalog number 12604, Invitrogen) at 37 °C for 3 - 5 minutes. Then, the cells were washed twice in staining buffer (PBS containing 0.2% fatty acid-free BSA) (Catalog number 554657, BD Biosciences, New Jersey, USA). For intracellular antibody staining, the cells The cells were first incubated with Green Fluorescent LIVE / DEAD cell dye (Inv itrogen catalog number L23101) at 4°C for 20 minutes to enable the distinction between live and dead during analysis, and then washed once with cold PBS. The cells were fixed in 250 μL of Cytofix / Cytoperm Buffer (BD Bioscience s catalog number 554722) at 4°C for 20 minutes, and then washed twice with BD Perm / W ash Buffer Solution (BD Biosciences catalog number 554723). The cells were resuspended in 100 μL of staining / blocking solution consisting of Perm / Wash buffer supplemented with 2% normal serum of the appropriate species of secondary antibody . Then, the cells were incubated with the primary antibody at an empirically pre-determined dilution at 4°C for 30 minutes, and then washed twice with Perm / Wash buffer. Finally, the cells were incubated with the appropriate secondary antibody at 4°C for 30 minutes, and then washed twice with Perm / Wash buffer and analyzed on a BD FACS Canto II. The following diluted primary antibodies were used. Rabbit anti-insulin (1:100; catalog number C 27C9, Cell Signaling, Massachusetts, USA), mouse anti-ins ulin (1:100; catalog number ab6999, Abcam, Massachusetts, USA)

[0070] , mouse anti-glucagon (1:1250; catalog number G2654; Sigma-Aldr ich), rabbit anti-synaptophysin (1:100; catalog number A0010, Dako, California, USA), rabbit anti-chromogranin A (1:800; Dako), mouse anti- Antibody NKX6.1 (1:50; DSHB, University of Iowa, USA) Iowa), mouse anti-CDX2 (1:250; Invitrogen), and goat anti-Neu roD (1:500; R&D Systems), mouse anti-SOX2 ( (BD, USA), mouse anti-NKX2.2 (DSHB), mouse anti-Pax6 (California, USA), mouse anti-PDX-1 (BD, California, USA) and 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 min. 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 isotypes 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). The population was positive for expression of β-lactamase inhibitors (C-lactamase inhibitors), with approximately 60% of the cells in the population positive for expression of SOX2 (Figure 1E). However, CDX2 (Fig. 1F) or NKX6.1 (Fig. 1D), or chromogranin Fewer than 10% of cells were positive for KI-67 (Figure 1B). As shown by the high percentage, at stage 3, a significant percentage of cells were active in the cell cycle (Figure 1C).

[0072] Figures 2A - 2G illustrate the expression profiles of isotype control (Figure 2A), chromogranin (Figure 2B) , KI - 67 (Figure 2C), NKX6.1 (Figure 2D), SOX2 (Figure 2E), CDX2 (Figure 2F), and PDX - 1 (Figure 2G) of cells differentiated according to Example 1 and harvested on the second day of S4 . The expression percentages of each marker are shown in each histogram. Similar to stage 3, more than 95% of the cells were positive for the expression of PDX - 1 (Figure 2G), but only about 10% of the cells were positive for the expression of CDX2 (Figure 2F), and about 40% of the cells were positive for the expression of NKX6.1 (Figure 2D). About 45% of the cells were positive for the expression of SOX2 (Figure 2E), which is 60% lower than that in S3 . The expression of chromogranin was about 3% (Figure 2B). As shown by the high percentage of KI - 67 positive cells (Figure 2C), at stage 4, a significant percentage of cells were in the active cell cycle .

[0073] Figures 3A - 3G illustrate the relative expression profiles determined by FACS analysis of cells harvested on the second day of stage 5 following the differentiation protocol outlined in this example . Figure 3A: Isotype control; Figure 3B: Chromogranin; Figure 3C: KI - 67; Figure 3D: NKX6.1; Figure 3E: SOX2; Figure 3F: CDX2; Figure 3G: PDX - 1. The expression percentages of each marker are shown in each histogram. Similar to stages 3 and 4, 95% of the cells ​​The above was positive for the expression of PDX-1, but about 1% of the cells were positive for the expression of CDX2, and about 67% or more of the cells were positive for the expression of NKX6.1. The expression of SOX2 was about 50%, which was lower compared to stage 3 but the same as the expression in S4.

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

[0075] Furthermore, most of the cells expressing SOX2, CDX2, and NKX6.1 were negative for the expression of chromogranin (see Figures 5A - 5C). Therefore, in the cell population resulting from the S5 culture prepared according to the protocol outlined in this example, at least 50% of the cells express PDX-1 and NKX6.1 but are negative for CDX-2, SOX2, and chromogranin. Table I summarizes the percentage expression of various endoderm markers in S3 - S5.

[0076]

Table 1

[0077] Figures 6A - 6T illustrate the mRNA expression profiles measured by real - time PCR of cells of S2, S3, S4 , and S5, which were differentiated according to the protocol outlined in this example, and reported as fold - changes in expression relative to undifferentiated H1 cells. At stage 3, very low expression of foregut markers such as FOXe 1 (Figure 6C) and NKX2.1 (Figure 6E) was observed. However, SOX2 (Figure 6M) and OSR1 (Figure 6 H), which mark the gastric region of the gut tube, were significantly up - regulated at stage 3 and their expression decreased at S4 - S5. Pancreatic endoderm leaf markers such as PTF1a (Figure 6K), NKX6.1 (Figure 6G), and PDX - 1 (Figure 6I) reached their maximum expression levels on day 2 of S5 in culture. PDR data indicate that cells transit through a PDX - 1+ SOX2+ population at stage 3 and then become PDX -1+ NKX6.1+ SOX2 - CDX2 - at S4 - S5 (see Figures 6I, Figure 6G, Figure 6M, and Figure 6A). The expression of endocrine markers (chromogranin, insulin, glucagon, and somatostatin) reached their maximum expression levels at the end of S5. The expression of pancreatic endocrine progenitor cell markers NKX2.2, NeuroD, and NGN3 reached their maximum expression levels at S4 - S 5. The expression of markers of other lineages such as ZIC1 and SOX17 remained low at S4 - S5. As a conclusion, cells on day 2 of stage 5 differentiated according to the protocol outlined in this example express low levels of CDX2 and SOX2 while maintaining high expression levels of NKX6.1 and PDX - 1. Timely BMP inhibition, use of low - dose RA at S4 - S5 ... ... ... ... ... ...

[0078] ... ... ... The unique combination of use and the use of high glucose at S1 to S2 is described in Example 1 and is thought to have resulted in the cell population.

[0079] (Example 2) The effects of BMP inhibition and PKC activation on SOX2 expression at S3 to S4 The protocol outlined in this example was performed to clarify the effects of BMP inhibition, addition of FGF7, and PKC activation on SOX2 expression at S3 to S4.

[0080] Cells of the human embryonic stem cell line H1 were harvested at various passages (passage 40 to passage 52) and as single cells in mTesr™ 1 medium supplemented with 10 μM Y27632, at a concentration of 100,000 cells per 1 cm 2 and seeded onto dishes coated with MATRIGEL® (1:30 dilution). Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine lineage as follows. a. Stage 1 (definitive endoderm (DE) - day 3): Before the start of DE, the cultures were washed and incubated for 30 seconds with incomplete PBS (without Mg or Ca), and then the medium for stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL®-coated dishes were treated for 1 day with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8, and 1.5 μM MCX compound (GSK3B inhibitor). Then, from day 2 to day 3, the cells were cultured with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 m MCDB-131 medium supplemented with 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8, and 1.5 μM MCX compound (GSK3B inhibitor). Treated with MCDB-131 medium supplemented with glucose of M and 100 ng / mL of GDF8. Treated. b. Stage 2 (gastrula - 3 days): The cells of stage 1 were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL of sodium bicarbonate, 1X GlutaMax™, 2.5 mM of D-glucose of M, and 50 ng / mL of FGF7 for 3 days. Treated for 3 days. c. Stage 3 (foregut - 3 days): The cells of stage 2 were treated with MCDB-131 medium supplemented with 50 ng / mL of FGF7, 5 0 nM or 200 nM of LDN-193189, and / or in the presence or absence of 200 nM of TPB, a 1:200 dilution of ITS-X, 2.5 mM of glucose, 1X G lutaMax™, 0.0015 g / mL of sodium bicarbonate, 2% fatty acid-free B SA, 0.25 μM of SANT-1, 20 ng / mL of activin-A, 2 μM of RA Supplemented and treated with MCDB-131 medium. Incubated the cells for 3 days in the medium using the combinations described in Table II below. Treated for 3 days.

[0081]

Table 2

[0082] d. Stage 4 (pancreatic foregut progenitor cells - 3 days): The cells of stage 3 were treated with a 1: 200 dilution of ITS-X, 2.5 mM of glucose, 1X GlutaMax™, 0.0015 g / mL of sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM of SANT-1, 2 00 nM of TPB, 400 nM of LDN-193189, 2 μM of ALk5 inhibitor (SD -208, Molecular Pharmacology 2007,72:152~ disclosed in 161), and MCDB-13 supplemented with 100 nM of a CYP26A inhibitor (N-{4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl}-1,3-benzothiazol-2-amine, Belgium, Janssen) was treated with 1× medium for 3 days. ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl}-1,3- benzothiazol-2-amine, Belgium, Janssen) and cultured in 1× medium for 3 days. For all the above conditions, mRNA was collected at S2 - S4 and analyzed using real-time PCR. The control condition for S3 refers to the culture using FGF7, AA, SANT, RA, and 200 nM of LDN-193189 at the concentrations described in step c above. As is clear from the PCR data shown in FIGS. 7A - 7G, the removal of LDN-193189 at S3 resulted in a significant decrease in endocrine markers such as NGN3 (FIG. 7D) and intrapancreatic endocrine markers such as chromogranin (see FIG. 7C). The addition of a PKC activator and the removal of LDN-193189 at S3 further decreased the endocrine markers while increasing the expression of NKX6.1 (see FIGS. 7A - 7G). Furthermore, the addition of 50 nM of LDN-193189 had efficacy comparable to 200 nM of LDN-193189 in the induction of endocrine markers (chromogranin and NGN3). The removal of LDN-193189 and the addition of TPB at S3 increased the expression of CDX2 (FIG. 7E) and albumin (FIG. 7F) while suppressing the expression of SOX2 (FIG. 7G). Furthermore, the removal of both FGF7 and LDN-193189 significantly increased the expression of SOX2 (FIG. 7G) and decreased the expression of albumin (FIG. 7F) compared to the culture with LDN-193189 removed and FGF7 retained. These data indicate that precise regulation of BMP inhibition, FGF activation, and PKC activation is

[0083] For all the above conditions, mRNA was collected at S2 - S4 and analyzed using real-time PCR. The control condition for S3 refers to the culture using FGF7, AA, SANT, RA, and 200 nM of LDN-193189 at the concentrations described in step c above. As is clear from the PCR data shown in FIGS. 7A - 7G, the removal of LDN-193189 at S3 resulted in a significant decrease in endocrine markers such as NGN3 (FIG. 7D) and intrapancreatic endocrine markers such as chromogranin (see FIG. 7C). As is clear from the PCR data shown in FIGS. 7A - 7G, the removal of LDN-193189 at S3 resulted in a significant decrease in endocrine markers such as NGN3 (FIG. 7D) and intrapancreatic endocrine markers such as chromogranin (see FIG. 7C). The addition of a PKC activator and the removal of LDN-193189 at S3 further decreased the endocrine markers while increasing the expression of NKX6.1 (see FIGS. 7A - 7G). The addition of a PKC activator and the removal of LDN-193189 at S3 further decreased the endocrine markers while increasing the expression of NKX6.1 (see FIGS. 7A - 7G). Furthermore, the addition of 50 nM of LDN-193189 had efficacy comparable to 200 nM of LDN-193189 in the induction of endocrine markers (chromogranin and NGN3). Furthermore, the addition of 50 nM of LDN-193189 had efficacy comparable to 200 nM of LDN-193189 in the induction of endocrine markers (chromogranin and NGN3). The removal of LDN-193189 and the addition of TPB at S3 increased the expression of CDX2 (FIG. 7E) and albumin (FIG. 7F) while suppressing the expression of SOX2 (FIG. 7G). The removal of LDN-193189 and the addition of TPB at S3 increased the expression of CDX2 (FIG. 7E) and albumin (FIG. 7F) while suppressing the expression of SOX2 (FIG. 7G). Furthermore, the removal of both FGF7 and LDN-193189 significantly increased the expression of SOX2 (FIG. 7G) and decreased the expression of albumin (FIG. 7F) compared to the culture with LDN-193189 removed and FGF7 retained. Furthermore, the removal of both FGF7 and LDN-193189 significantly increased the expression of SOX2 (FIG. 7G) and decreased the expression of albumin (FIG. 7F) compared to the culture with LDN-193189 removed and FGF7 retained. These data indicate that precise regulation of BMP inhibition, FGF activation, and PKC activation is These data indicate that precise regulation of BMP inhibition, FGF activation, and PKC activation is required for PDX -1 and rich in NKX6.1 while low in CDX2, SOX2 and albumin, the endoderm do has been demonstrated to be able to bring about the main. Finally, the continuous inhibition of BMP at S3-S4 enhanced the expression of pro-endocrine genes and the up-regulation of SOX2 expression. This indicates that while increasing pancreatic endocrine genes, it does not up-regulate the expression of SOX2 that is absent or low in pancreatic development but present in anterior foregut endoderm organs such as the stomach, thus verifying that it is necessary to precisely regulate the inhibition of BMP. while increasing pancreatic endocrine genes, it does not up-regulate the expression of SOX2 that is absent or low in pancreatic development but present in anterior foregut endoderm organs such as the stomach, thus verifying that it is necessary to precisely regulate the inhibition of BMP. while increasing pancreatic endocrine genes, it does not up-regulate the expression of SOX2 that is absent or low in pancreatic development but present in anterior foregut endoderm organs such as the stomach, thus verifying that it is necessary to precisely regulate the inhibition of BMP. while increasing pancreatic endocrine genes, it does not up-regulate the expression of SOX2 that is absent or low in pancreatic development but present in anterior foregut endoderm organs such as the stomach, thus verifying that it is necessary to precisely regulate the inhibition of BMP.

[0084] (Example 3) Subsequently, to induce endocrine markers, early inhibition of BMP at the foregut stage is necessary. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required. This example shows that to subsequently induce endocrine markers, it is necessary to inhibit BMP signaling early at S3. However, continuous inhibition of BMP at stage 3 also results in strong expression of SOX2. To obtain both high expression of endocrine markers and low expression of SOX2, a gradient of BMP inhibition that induces pro-pancreatic endocrine markers while having low expression of SXO2 and CDX2 is required.

[0085] Cells of the human embryonic stem cell line H1 were seeded as single cells or at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM Y27632 at various passages (passage 40 - passage 52). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. Cells of the human embryonic stem cell line H1 were seeded as single cells or at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM Y27632 at various passages (passage 40 - passage 52). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. 2 each or at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM Y27632 at various passages (passage 40 - passage 52). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. or at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM Y27632 at various passages (passage 40 - passage 52). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. or at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM Y27632 at various passages (passage 40 - passage 52). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - 4 days): Before the start of DE, the cells were washed and Incubate for 30 seconds with complete PBS (without Mg or Ca), then transfer to the medium of S1 and incubated. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark)-coated dishes were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate , 1X GlutaMax (trademark), 2.5 mM D-glucose, 100 ng / mL GDF8, and 1.5 μM MCX compound (GSK3B inhibitor) for 1 day. Then, the cells were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM glucose, and 100 ng / mL GDF8 for 2 days (days 2 - 4). b. Stage 2 (gastrula - 3 days): The cells of stage 1 were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 m M D-glucose, and 50 ng / mL FGF7 for 3 days. c. Stage 3 (foregut - 3 days): The cells of stage 2 were treated with MCDB-131 medium supplemented with 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax (trademark), 0.0015 g / mL sodium bicarbonate, 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 (only on day 1 or during stage 3), and 200 nM TPB. In some media, LDN-193189 was removed from stage 3. d. Stage 4 (pancreatic foregut progenitor cells - 3 days): The cells of stage 3 were treated with 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 100 nM CYP26A inhibitor-supplemented MCDB-131 medium for 3 days. e. Stage 5 (pancreatic endoderm / endocrine - 4 days): Cells at stage 4 were treated with ITS-X at a 1 :200 dilution, 2.5 mM glucose, 1X GlutaMax™, 0.001 5 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 200 nM LDN-1931 89, and 2 μM ALk5 inhibitor-supplemented MCDB-131 medium for 4 days.

[0086] As is clear from the PCR results shown in FIGS. 8A - 8G, removal of LDN- 193189 (BMP inhibitor) from stage 3 halted the expression of the pro-endocrine genes NGN3 (FIG. 8C), NeuroD (FIG. 8D), and chromogranin (FIG. 8E) at stages 4 and 5. However, the expression of PDX-1 (FIG. 8B) and NKX6.1 (FIG. 8A) was not significantly downregulated compared to NGN3 and NeuroD at stages 4 ~5. Furthermore, complete removal of LDN-193189 at stage 3 resulted in a significant increase in the expression of CDX2 ( FIG. 7F). After addition of LDN-193189 on the first day of stage 3, removing it on days 2 - 3 of stage 3 significantly increased the expression of NGN3 and NeuroD while decreasing the expression of CDX2 and SOX2 at stage 4. Cultures that retained LDN-193189 throughout stage 3 showed very high expression of SOX2 at S3 - S4 (FIG. 8 ​​​​G). This data indicates that inhibition of BMP on the first day of stage 3 is sufficient to trigger pancreatic endocrine markers while suppressing the expression of SOX2 and CDX2. while showing that it is sufficient to suppress the expression of SOX2 and CDX2 while triggering pancreatic endocrine markers.

[0087] In summary, inhibition of BMP is required on the first day of stage 3 to induce the formation of endocrine progenitor cells in stages 4 - 5, maintain the expression of PDX-1 and NKX6.1, and suppress the expression of SOX2 on the other hand. Furthermore, addition of a PKC activator at stage 3 further enhanced the expression of PDX-1 and NKX6.1. while suppressing the expression of SOX2. Furthermore, addition of a PKC activator at stage 3 further enhanced the expression of PDX-1 and NKX6.1. while suppressing the expression of SOX2.

[0088] (Example 4) Inhibition of BMP signaling on the first day of stage 3 is sufficient to generate pancreatic progenitor cells at stage 4, while inhibition of BMP signaling on the last day of stage 3 results in a significant decrease in the expression of endocrine markers. while inhibition of BMP signaling on the last day of stage 3 results in a significant decrease in the expression of endocrine markers. while inhibition of BMP signaling on the last day of stage 3 results in a significant decrease in the expression of endocrine markers. This example shows that early inhibition of BMP signaling at stage 3 enables the induction of pancreatic endocrine progenitor cell markers, while inhibition of BMP signaling in the late stage of stage 3 significantly reduces the expression level of endocrine progenitor cell markers at stage 4. while inhibition of BMP signaling in the late stage of stage 3 significantly reduces the expression level of endocrine progenitor cell markers at stage 4. while inhibition of BMP signaling in the late stage of stage 3 significantly reduces the expression level of endocrine progenitor cell markers at stage 4.

[0089] Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM of Y27632. Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM of Y27632. 2 per Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM of Y27632. Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells at a concentration of 100,000 cells per cm² on dishes coated with MATRIGEL (trademark) (1:30 dilution) in mTesr (trademark) 1 medium supplemented with 10 μM of Y27632. Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - 4 days): Before the start of DE, the cultures were washed and Incubate for 30 seconds with incomplete PBS (without Mg or Ca), then stage 1 medium was added. Human embryonic stem cells cultured as single cells on MATRIGEL™-coated dishes were treated for 1 day with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, 100 ng / mL GDF 8, and 1.5 μM MCX compound (GSK3B inhibitor). Then the cells were treated for 3 days with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM glucose, and 100 ng / mL GDF8. b. Stage 2 (gastrula - 3 days): The cells from stage 1 were treated for 3 days with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, and 50 ng / mL FGF7. c. Stage 3 (foregut - 3 days): The cells from stage 2 were treated for 3 days with MCDB-131 medium supplemented with 1:200 dilution of ITS-X, 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, and the mixture described in Table III below. d. Stage 4 (pancreatic foregut progenitor cells - 3 days): The cells from stage 3 were treated with 1: of ITS-X, 3 days with MCDB-131 medium supplemented with 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, and the mixture described in Table III below.

[0090]

Table 3

[0091] d. Stage 4 (pancreatic foregut progenitor cells - 3 days): The cells from stage 3 were treated with 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 100 nM CYP26A inhibitor supplemented MCDB-131 medium for 3 days.

[0092] Figures 9A - 9H illustrate the gene expression profiles of pancreatic endoderm, endocrine progenitor cells, and foregut endoderm markers for the combinations of the above culture conditions. Similar to the previous examples, blockade of the BMP pathway on day 1 of stage 3 is important for the subsequent induction of the endocrine program as measured by the expression of the pancreatic endocrine marker, chromogranin (see Figure 9C). However, addition of the BMP inhibitor on day 1 of stage 3 triggers the expression of endocrine markers at subsequent stages. Furthermore, addition of the BMP inhibitor on day 1 of stage 3 also decreased the expression of SOX2, a foregut marker, at stages 3 - 4 (Figure 9H). However, adding the BMP inhibitor only on the last day of stage 3 shows a significantly higher expression of SOX2 at the end of stage 3 compared to cells treated with the BMP inhibitor only on day 1 of stage 3. The expression levels shown in Figures 9A - 9H are relative to the expression levels of undifferentiated H1 cells with very high expression levels of SOX2. In addition to being a marker of the anterior foregut, SOX2 is a well-known transcription factor important for maintaining the pluripotency of ES cells. This example further supports the aforementioned results that clarify the sensitivity of stage 3 cultures to the duration and kinetics of BMP signaling and its impact on subsequent pancreatic endocrine induction and expression of SOX2.

[0093] (Example 5) Optimal dosage of BMP inhibition at the pancreatic foregut stage (stage 4) In the foregoing examples, the optimal period of BMP inhibition at stage 3 was described. In this example, the optimal dosage of the BMP inhibitor in the S4 medium is identified. In this example, the optimal dosage of the BMP inhibitor in the S4 medium is identified.

[0094] Cells of the human embryonic stem cell line H1 of various passages (passage 40 to passage 52) were placed in mTesr (trade mark) 1 medium at a concentration of 10 μM of Y27632 and 100,000 cells per cm 2 and seeded as single cells on dishes coated with MATRIGEL (trademark) (1:30 dilution). After 48 hours from seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows and seeded as single cells on dishes coated with MATRIGEL (trademark) (1:30 dilution). After 48 hours from seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows . a. Stage 1 (definitive endoderm (DE) - 4 days): Before the start of DE, the cultures were washed and incubated for 30 seconds with incomplete PBS (without Mg or Ca), and then the medium for stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark)-coated dishes were treated for 1 day with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM D-glucose, 100 ng / mL GDF 8, and 1 μM of the MCX compound (GSK3B inhibitor). Then, from day 2 to day 4, the cells were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0. 0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM glucose, and 100 ng / mL GDF8. 0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM glucose, and 100 ng / mL GDF8. . b. Stage 2 (gastrula - 3 days): The cells at stage 1 were treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 2.5 mM D-glucose, and MCDB-131 medium supplemented with 50 ng / mL FGF7 were treated for 3 days. c. Stage 3 (foregut - 4 days): Cells at stage 2 were treated with a 1:200 dilution of ITS-X, 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 LDN-1 93189, and 100 nM TPB supplemented MCDB-131 medium for 1 day. Then, the cells were treated in MCDB-131 medium supplemented with 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 / mL activin-A, and 100 nM TPB for 3 days. d. Stage 4 (pancreatic foregut progenitor cells - 4 days): Cells at stage 3 were treated with a 1: 200 dilution of ITS-X, 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 the concentration of LDN-193189 (LDN-193189-193189) described in Table IV (below) were supplemented in MCDB-13 1 medium and treated from days 1 to 4 of stage 4.

[0095]

Table 4

[0096] d. Stage 5 (Pancreatic endoderm / Endocrine progenitor cells - 3 days): The cells of Stage 4 were cultured in ITS -X diluted 1:200, 2.5 mM glucose, 1X GlutaMax™, 0 .0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 50 nM LDN-1 93189, and 1 μM ALk5 inhibitor-supplemented MCDB-131 medium for 3 days .

[0097] The results of real-time PCR analysis of the cells harvested after the above treatment are shown in FIGS. 10A to 10H . This figure shows that adding 50 nM or 100 nM of LDN-193189 on the 1st or 2nd nd or 3rd or 4th day of S4 can extend the expression of endocrine markers while maintaining low expression of SOX2 in S 4 to S5 (see FIGS. 10A to 10H).

[0098] (Example 6) Optimal dosage of BMP inhibition at foregut stage (Stage 3) This example identifies the optimal dosage of BMP inhibition at Stage 3 and its subsequent effect on endocrine markers at Stage 6 .

[0099] Cells of the human embryonic stem cell line H1 were cultured as single cells or at a concentration of 100,000 cells in mTesr™1 medium supplemented with 10 μM Y 27632 at various passages (passage 40 to passage 52) and seeded on dishes coated with MATRIGEL™ (1:30 dilution) at 1 cm 2 per well or at a concentration of 100,000 cells. 48 hours after seeding, the cultures were differentiated into pancreatic endocrine system cells as follows . . a. Stage 1 (embryonic endoderm (DE) - 4 days): Before the start of DE, the culture was washed and incubated with incomplete PBS (without Mg or Ca) for 30 seconds, and then the medium for stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark) - coated dishes were treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM D - glucose, 100 ng / mL GDF 8, and 1.5 μM MCX compound (GSK3B inhibitor) for 1 day. Then, from day 2 to day 4, the cells were treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2. 5 mM glucose, and 100 ng / mL GDF8. b. Stage 2 (gastrula - 3 days): The cells of stage 1 were treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 m M D - glucose, and 50 ng / mL FGF7 for 3 days. c. Stage 3 (foregut - 3 days): The cells of stage 3 were treated with MCDB - 131 medium supplemented with 1:200 dilution of ITS - X, 2.5 mM glucose, 1X GlutaMax (trademark), 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, and 10 - 50 nM LDN - 193189 for 1 day. Then, the cells were treated with 1:200 dilution of ITS - X, 2.5 mM glucose, 1X G ​​​lutaMax (trademark), 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 g / mL activin-A, and 100 nM TPB supplemented MCDB-131 medium for 2 days. d. Stage 4 (pancreatic foregut progenitor cells - 3 days): Cells at stage 3 were treated with 1: 200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax (trademark), 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 supplemented MCDB-131 medium for 3 days. e. Stage 5 (pancreatic endoderm / endocrine progenitor cells - 3 days): Cells at stage 4 were treated with ITS -X 1:200 dilution, 2.5 mM glucose, 1X GlutaMax (trademark), 0 .0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, + / - 25 nM LD N-193189, and / or 2 μM ALk5 inhibitor supplemented MCDB-131 medium for 3 days. f. Stage 6 (pancreatic endocrine hormone production - 3 days): Cells at stage 5 were treated with ITS-X 1:200 dilution, 2.5 mM glucose, 1X GlutaMax (trademark), 0.0 015 g / mL sodium bicarbonate, and 2% fatty acid-free BSA supplemented MCDB-1 31 medium for 3 days.

[0100] Figures 11A - 11H show that low to moderate inhibition of BMP on day 1 of stage 3 is required to trigger the expression of endocrine markers while maintaining low expression of SOX2 indicating that Furthermore, inhibiting BMP at stage 5 while enhancing endocrine markers also led to upregulation of SOX2 expression.

[0101] The data of this example further confirm the results presented in the previous examples. This data supports that precise regulation of the BMP pathway at stages 3 - 5 is required to trigger the induction of pancreatic endocrine markers while suppressing SOX2 expression.

[0102] (Example 7) Optimal time frame of BMP inhibition at S3 (foregut stage) This example identifies the optimal time frame at stage 3 for inhibiting BMP signaling while maintaining endocrine induction at later stages and not reducing SOX2 expression.

[0103] Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells on dishes coated with MATRIGEL (trademark) (1:30 dilution) at a concentration of 100,000 cells per cm and 10 μM of Y27632 in mTesr (trademark) 1 medium. 2 Forty-eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - day 4): Before the start of DE, the cultures were washed and incubated for 30 seconds with incomplete PBS (without Mg or Ca), and then the medium for stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark)-coated dishes were cultured in 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM D-glucose, 100 ng / mL GDF 8. And treated for 1 day with MCDB-131 medium supplemented with 1.5 μM of the MCX compound. Next Then, from day 2 to day 4, the cells were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL of sodium bicarbonate, 1X GlutaMax™, 2.5 mM glucose, and 100 ng / mL of GDF8. b. Stage 2 (gastrula - 3 days): The cells of stage 1 were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL of sodium bicarbonate, 1X GlutaMax™, 2.5 m M of D-glucose, and 50 ng / mL of FGF7 for 3 days. c. Stage 3 (foregut - 3 days): The cells of stage 2 were treated with MCDB-131 medium containing 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL of sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM of SANT-1, 50 ng / mL of FGF7, 2 μM of RA, 20 ng / mL of activin-A, and 100 nM of TPB, and supplemented with 100 n M of LDN-193189 for only the first 2 hours or only 6 hours or only 24 hours of stage 3. d. Stage 4 (pancreatic foregut progenitor cells - 3 days): The cells of stage 3 were treated with MCDB-131 medium containing 1: 200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL of sodium bicarbonate, 2% fatty acid-free BSA, 25 nM of LDN-193189 2 μM of ALk5 inhibitor, 100 nM of CYP26A inhibitor, and 100 nM of TPB for 3 days. e. Stage 5 (pancreatic endoderm / endocrine progenitor cells - 3 days): The cells of stage 4 were treated with ITS ​​-1:200 dilution of -X, 2.5 mM glucose, 1X GlutaMax™, 0 .0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, and 2 μM ALk5 Cells were treated for 3 days with MCDB-131 medium supplemented with an inhibitor.

[0104] Figures 12A - 12G show the real-time PCR analysis of the data collected in this example. Treatment with a BMP inhibitor for at least 2 hours at stage 3 triggers the expression of pro-endocrine transcription factors such as Ngn3 (Figure 12D) and NeuroD (Figure 12E), while maintaining a very low expression of SOX2 (Figure 12G) and significantly increasing the expression of NKX6.1 (Figure 12A) and PDX-1 (Figure 12B) at S4 - S5. However, on day 3 of stage 5, the expression of CDX2 was higher in cells treated with the BMP inhibitor for 2 hours or 6 hours than in cells treated with the inhibitor for 24 hours (Figure 12F).

[0105] The data of this example suggest that a 24-hour inhibition of the BMP pathway is optimal to maintain low levels of CDX2 expression and SOX2 expression and to initiate endocrine differentiation while maintaining high expression of pancreatic endoderm markers.

[0106] (Example 8) Optimal periods at stage 3 (foregut stage) and stage 4 (pancreatic foregut progenitor cell stage) This example was carried out to determine the optimal periods of S3 and S4 in the stepwise differentiation of pluripotent cells into cell populations of the pancreatic endocrine system.

[0107] Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were treated with 10 μM Y2 In the mTesr (trademark) 1 medium supplemented with 7632, as single cells, at a concentration of 100,000 cells per 1 cm 2 hit cells were seeded on dishes coated with MATRIGEL (trademark) (1:30 dilution). 48 hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows a. Stage 1 (definitive endoderm (DE) - 4 days): Before the start of DE, the cultures were washed and incubated with incomplete PBS (without Mg or Ca) for 30 seconds, then the medium for Stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark)-coated dishes were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 mM D-glucose, 100 ng / mL GDF 8, and 1.5 μM MCX compound (GSK3B inhibitor) for 1 day. Then, from day 2 to day 4, the cells were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 m M glucose, and 100 ng / mL GDF8 b. Stage 2 (gastrula - 2 days): The cells from Stage 1 were treated with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 2.5 m M D-glucose, and 50 ng / mL FGF7 for 2 days c. Stage 3 (foregut - 2 - 3 days): The cells from Stage 2 were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax (trademark), 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 supplemented, and cultured for 1 day in MCDB-131 medium containing 100 nM LDN-193189 Then, the cells were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1 X 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, and 100 nM TPB supplemented MCDB-131 medium . d. Stage 4 (pancreatic foregut progenitor cells - 2 - 3 days): The cells of stage 3 were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.00 15 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 25 nM LDN-1931 89, 100 nM CYP26A inhibitor, and 100 nM TPB supplemented MCDB- 131 medium for 2 - 3 days. e. Stage 5 (pancreatic endoderm / endocrine progenitor cells - 2 days): The cells of stage 4 were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0 .0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, and 1 μM ALk5 inhibitor supplemented MCDB-131 medium for 2 days. f. Stage 6 (pancreatic endocrine progenitor cells / hormones - 2 days): The cells of stage 5 were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0 .0015 g / mL sodium bicarbonate, and 2% fatty acid-free BSA supplemented MCDB -131 medium ​​Treated at -131-fold for 2 days.

[0108] Real-time PCR analysis data of samples harvested at stage 3, stage 4, stage 5, or stage 6 are shown in FIGS. 13A to 13G. This data shows that extending S3 and S4 to 3 days increases the expression of NKX6.1 compared to the culture where S3 and S4 are 2 days (FIG. 13A). Cells treated for 3 days at stage 3 show downregulation of the expression of proendocrine markers when compared to the culture where the periods of S3 and S4 are only 2 days ( FIGS. 13D and 13E). Furthermore, extending stage 4 to 3 days significantly increased the expression of SOX2 ( FIG. 13G). The data obtained in this example, like the data generated in previous examples, shows that extended BMP inhibition promotes the foregut towards a higher population of SOX2. Based on the data from this example and the previous

[0109] examples, it can be concluded that the optimal periods for stage 3 and stage 4 are 2 days . An ideal protocol will result in differentiated cells with high levels of expression of proendocrine markers , high expression of NKX6.1, low expression of CDX2, and low expression of SOX2.

[0110] (Example 9) Long-term exposure to BMP inhibition in the presence of high glucose and B27 supplement significantly increases the expression of SOX2 in S3 and S4. This protocol was performed to determine the factors that affect the expression of SOX2 in S3 and S4 during the stepwise differentiation of pluripotent cells into hormone-producing cells.

[0111] The cells of the human embryonic stem cell line H1 were cultured on MATRIGEL (trademark) (1:30 dilution)-coated dishes and cultured in mTesr (trademark) 1 medium until 70% confluence, and differentiated as follows . a. Undifferentiated cells were cultured for 1 day in RPMI medium (Invitrogen) supplemented with 0.2% FBS, 100 ng / mL of activin A, and 20 ng / mL of WNT-3a. Then, the cells were further treated with RPMI medium supplemented with 0.5% FBS and 100 ng / mL of activin A for another 2 days (stage 1). b. The cells at stage 1 were treated with DMEM / F12 medium supplemented with 2% FBS and 50 ng / mL of FGF7 for 3 days (stage 2). c. The cells at stage 2 were cultured for 4 days in DMEM-high glucose medium supplemented with 1% of B27, 0.25% of SANT-1, 2 μM of RA, 100 ng / mL of noggin (R & D systems, Minnesota, USA) (stage 3). d. The cells at stage 3 were treated with DMEM-high glucose medium supplemented with 1% of B27, 100 ng / mL of noggin, 1 μM of ALK 5 inhibitor II (Axxora, California, USA), and 50 nM of TPB for 4 days (stage 4). e. Figures 14A to 14H illustrate FACS histograms of the cells harvested on the 4th day of stage 3 obtained for the following markers: isotype control (Figure 14A), chromogranin (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 expression percentages of each marker are shown in each histogram. Most of the cells at stage 3

[0112] were harvested and the expression percentage of each marker is shown in each histogram. Most of the cells at stage 3 was positive for the expression of PDX-1 (Figure 14H) and HNF3B (Figure 14F), and N was negative for the expression of NKX6.1 (Figure 14D), and showed low expression for chromogranin (Figure 14B) and C DX2 (Figure 14G). However, more than 90% of the cells were also strongly positive for SOX2 (Figure 14E). This indicates that at stage 3, most of the cells were positive for PDX -1 and SOX2 and negative for NKX6.1, suggesting the establishment of an endodermal population consistent with the anterior foregut population in the pancreatic PDX -1 domain.

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

[0114] Figures 15A - 15G show the FACS histogram expression profiles of the following markers on day 2 of S4 of cells differentiated according to Example 9. Figure 15A: Isotype control, Figure 15B: NKX6.1, Figure 15C: KI-67, Figure 15D: Chromogranin, Figure 15E : SOX2, Figure 15F: CDX2, Figure 15G: PDX-1. The expression percentages of each marker are shown in each histogram.

[0115] Figures 16A - 16F show the FACS histogram expression profiles of the following markers on day 4 of S4 of cells differentiated according to Example 9. Figure 16A: Isotype control - Rule, Figure 16B: NKX6.1, Figure 16C: Chromogranin, Figure 16D: SOX2, Figure 1 6E: CDX2, Figure 16F: PDX-1. The expression percentages of each marker are shown in each histogram as follows.

[0116] Table V below summarizes the data obtained for the expression rates (%) of endoderm markers in S3 and S4 of the cells differentiated according to the protocol outlined in this example as follows.

[0117]

Table 5

[0118] Figures 17A to 17J show the results of real-time PCR analysis of the expression of the following genes in the cells of the human embryonic stem cell line H1 differentiated according to Example 9. Figure 17A: CDX2, Figure 17B: HHex, Figure 17C: FOXE1, Figure 17D: IPF1 (PDX-1), Figure 17 E: NKX2.1, Figure 17F: NKX2.2, Figure 17G: NKX6.1, Figure 17H: PR OX1, Figure 17I: SOX2, Figure 17J: SOX9.

[0119] As seen in Figures 14 to 17 and Table V, there was a significant increase in NKX 6.1 from day 2 to day 4 of stage 4, while the high expression of PDX-1 was maintained. The expression of SOX2 decreased from stage 3 to stage 4, but 75% of the cells were still SOX2+. Similar to Figure 5, CDX2+ cells, SOX2+ cells, and NKX6.1+ cells were mutually exclusive from the chromogranin population. This indicates that the NKX6.1+ SOX2+ PDX of the cell population on day 4 of stage 4 generated using the protocol outlined in Example 9 was mutually exclusive from the chromogranin population. ​​​-1+ The percentage of CDX2-chromogranin negativity suggests that it is up to 50%. This is, in S4 - S5, PDX-1+ NKX6.1+ SOX2-, CDX2-, chromo The percentage of granin negativity is about 40 - 70%, and PDX-1+ NKX6.1+ SOX2+ is In contrast to the cell population generated in Example 1, which was 2 - 25%. Clearly, the cells generated using the protocol of Example 1 While being PDX-1+ and NXK6.1+, Compared with the cells generated in Example 9, they also had a much higher percentage of pancreatic endoderm, as defined as a low or negative population for SOX2 and CDX2. The data obtained in this example provides evidence that long - term exposure to BMP inhibition in the presence of high glucose and B27 supplement significantly increases the expression of SOX2 in stage 3 and 4 differentiation.

[0120] The data obtained in this example provides evidence that long - term exposure to BMP inhibition in the presence of high glucose and B27 supplement significantly increases the expression of SOX2 in stage 3 and 4 differentiation. The data obtained in this example provides evidence that long - term exposure to BMP inhibition in the presence of high glucose and B27 supplement significantly increases the expression of SOX2 in stage 3 and 4 differentiation. This provides evidence to the contrary.

[0121] (Example 10) Protocols published so far result in the formation of a significant number of SOX2+ populations at stage 3 - 4. Kroon et al. have published a protocol for preparing cells of the pancreatic endoderm lineage from human embryonic stem cells (Nature Biotech 2008, 26:443 - 452 Kroon et al. have published a protocol for preparing cells of the pancreatic endoderm lineage from human embryonic stem cells (Nature Biotech 2008, 26:443 - 452, hereafter referred to as "Kroon"). In the examples provided herein Kroon et al. have published a protocol for preparing cells of the pancreatic endoderm lineage from human embryonic stem cells (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 assayed for the expression of characteristic markers at different stages of differentiation. human embryonic stem cells were differentiated according to the Kroon protocol and assayed for the expression of characteristic markers at different stages of differentiation. human embryonic stem cells were differentiated according to the Kroon protocol and assayed for the expression of characteristic markers at different stages of differentiation.

[0122] Cells of the human embryonic stem cell line H1 were plated on dishes coated with MATRIGEL (trademark) (1:30 dilution) Seed and culture in mTesr (trademark) medium until 70% confluence, and then differentiate as follows using the protocol published by Kroon to date. a) Expose undifferentiated cells to RPMI medium supplemented with 0.2% FBS, 100 ng / mL activin A, and 20 ng / mL WNT-3a for 1 day, and then further treat with RPMI medium supplemented with 0.5% FBS and 1 00 ng / mL activin A for an additional 2 days (stage 1). b) Treat cells at stage 1 with RPMI medium supplemented with 2% FBS and 50 ng / mL FGF7 for 3 days (stage 2). c) Treat cells at stage 2 with DMEM-high glucose medium supplemented with 1% B27, 0.25 μM SANT-1, 2 μM RA , and 50 ng / mL noggin (R & D systems, Minnesota) for 3 days (stage 3). d) Culture cells at stage 3 in DMEM-high glucose medium supplemented with 1% B27 for 3 days (stage 4). e) Scrape cells at stage 4 from the wells and resuspend as clusters in DMEM-high glucose medium supplemented with 1% B27 for 2 days.

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

[0124] Figures 19A to 19G show the following marker FACS histogram expression profiles of cells differentiated according to Example 10 on day 5 of S4. Isotype control (Figure 19A), NKX6.1 (Figure 19B), chromogranin (Figure 19C), SOX2 (Figure 19 D), CDX2 (Figure 19E), KI-67 (Figure 19F), PDX-1 (Figure 19G). The expression percentage of each 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 cell clusters in the suspension were NKX6.1+ PDX-1+ SOX2-, and up to 20% were PDX-1+ NKX6.1+ SOX2+. These results show that a significant proportion of the cell population generated according to Example 10 was NKX6.1+ or SOX2+ at stage 4.

[0126] Table VI below summarizes the percentage of endoderm markers in S3 - S4 of the cells generated in this example.

[0127]

Table 6

[0128] (Example 11) The addition of ascorbic acid significantly decreased the number of multi-hormone cells and simultaneously increased the number of single-hormone insulin-positive cells.

[0129] The effect of ascorbic acid on the expression of markers during the differentiation of pluripotent cells into hormone-producing cells was tested. As follows, glucose was supplemented at all stages of differentiation, and stage 3 ​​​​​Cells were cultured in a medium supplemented with ascorbic acid during the formation of 4 and 5.

[0130] Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were cultured in mTesr (trade mark) 1 medium with 10 μM of Y27632 and 100,000 cells per 2 cm² at a concentration of and seeded as single cells on MATRIGEL (trademark) (1:30 dilution) - coated dishes. Forty - eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - 3 days): Before the start of DE, the cultures were washed and incubated with incomplete PBS (without Mg or Ca) for 30 seconds and then stage 1 medium was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark) - coated dishes were treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 5 mM D - glucose, 100 ng / mL GDF8, and 1 μM of an MCX compound (GSK3B inhibitor) for 1 day. Then, the cells were treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 5 mM glucose, 100 ng / mL GDF8, and 100 nM of the MCX compound on day 2 and subsequently treated with MCDB - 131 medium supplemented with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 5 mM glucose, and 100 ng / mL GDF8 for an additional 1 day. b. Stage 2 (gastrula - 2 days): The cells of stage 1 were treated with 0.1% fatty - acid - free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, and 25 ng / mL FGF7 supplemented MCDB-131 medium for 2 days treatment. c. Stage 3 (foregut - 2 days): Cells at stage 2 were treated with 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 μM SANT-1, 1 μM RA, 200 nM TPB (P KC activator), 100 nM LDN-193189 (BMP receptor inhibitor) supplemented M CDB-131 medium for 1 day. Then, the cells were treated with 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 / m L FGF7, 0.25 μM SANT-1, 1 μM RA, 200 nM TPB (PK C activator), 10 nM LDN-193189 supplemented MCDB-131 medium for an additional 1 day. Some cultures were treated with 0.25 mM ascorbic acid (Catalog number A4544, Sigma, Missouri, USA) during the stage 3 period. d. Stage 4 (pancreatic foregut progenitor cells - 2 days): Cells at stage 3 were treated with or without 0.25 mM a scorbic acid, a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 nM RA, 200 nM TPB, 5 Cells were treated for 2 days in MCDB-131 medium supplemented with 0 nM of LDN-193189. e. Stage 5 (pancreatic endoderm - 2 - 7 days): Cells at stage 4 were treated for 2 - 7 days in MCDB-131 medium supplemented with 1:200 dilution of ITS-X, 2.5 mM glucose, 1 X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 nM RA, with or without 0.25 mM ascorbic acid.

[0131] Figures 20A - 20J show real-time PCR analysis of the expression of the following genes in cells of the human embryonic stem cell line H1 differentiated according to Example 11. Figure 20A: Somatostatin, 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 that the addition of ascorbic acid at stage 3, or at stages 3 and 4, significantly reduces the expression of somatostatin and glucagon at stages 4 - 5, while increasing the expression of insulin (see Figures 20A, Figure 20G, and Figure 20I). Furthermore, at stages 4 - 5, the expression of pancreatic endoderm markers such as PDX-1 and NKX6. 1 was not significantly changed by the addition of 0.25 mM ascorbic acid (see Figures 20B and 20D). At stages 4 - 5, the expression of Pax6 was downregulated and the expression of Pax4 was maintained (see Figures 20C and 20D). Cultures treated with + / - ascorbic acid at S3 - S5 were immunostained for insulin, glucagon, and somatostatin hormones at the end of stage 5. Table VII shows the in ​​​​​​​​The average percentages of glucagon- and somatostatin-positive cells, as well as multi-hormone cells (expressing two or more hormones within a single cell), are summarized. This is the summary of the average percentage of glucagon- and somatostatin-positive cells, as well as multi-hormone cells (expressing two or more hormones within a single cell).

[0132] [Table 7]

[0133] (Example 12) Optimal dosage of ascorbic acid at stage 3 This example was performed to determine the optimal dosage of ascorbic acid used to generate single hormone, PDX-1 positive, and NKX6.1 positive insulin-positive cells.

[0134] Cells of the human embryonic stem cell line H1 of various passages (passage 40 to passage 52) were seeded as single cells on MATRIGEL (trademark) (1:30 dilution)-coated dishes in mTesr (trademark) 1 medium at a concentration of 10 μM of Y27632 and 100,000 cells per cm ². 2 100,000 cells per cm² ². After 48 hours from seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - 3 days): Before the start of DE, the cultures were washed and incubated for 30 seconds with incomplete PBS (without Mg or Ca), and then the medium of stage 1 was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark)-coated dishes were treated for 1 day with MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 5 mM D-glucose, and 100 ng / mL GDF 8 and 1 μM of MCX compound (GSK3B inhibitor). Then, the cells were treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax (trademark), 5 mM D-glucose, and 100 ng / mL GDF 8 and 1 μM of MCX compound (GSK3B inhibitor) supplemented MCDB-131 medium. Then, the cells were treated with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, Sodium, 1X GlutaMax™, 5 mM glucose, 100 ng / mL of GDF8, and 100 nM of the MCX compound were added to MCDB-131 medium on day 2 and treated, followed by an additional 1 day with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM glucose, and 100 ng / mL of GDF8 in MCDB-131 medium supplemented with these components. b. Stage 2 (gastrula - 2 days): The cells from stage 1 were treated for 2 days with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose in MCDB-131 medium supplemented with 0.25 mM ascorbic acid and with or without 25 ng / mL of FGF7. c. Stage 3 (foregut - 2 days): The cells from stage 2 were treated with 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 / m L of FGF7, 0.25 μM SANT-1, + / - 0.25 mM ascorbic acid, 1 μM RA, and 200 nM TPB, and on day 1 with 100 nM LDN-1 93189 in MCDB-131 medium supplemented with these components, followed by a 1:20 0 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 of FGF7, 0.25 μM SANT-1, + / - 0.25 mM ascorbic acid, 1 μM RA, and 200 nM TPB, and for an additional 1 day with 10 nM LDN-1 Treated with MCDB-131 medium supplemented with 93189. d. Stage 4 (pancreatic foregut progenitor cells - 2 days): Cells from stage 3 were cultured in ITS-X at a 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 0 nM RA, 200 nM TPB, and 50 nM LDN-193189 supplemented M CDB-131 medium, with or without the addition of 0.25 mM to 1 mM ascorbic acid, for 2 days. e. Stage 5 (pancreatic endoderm - 2 - 9 days): Cells from stage 4 were cultured in 0.25 mM as corbic acid, with or without the addition of ITS-X at a 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, and 50 nM RA supplemented MCD B-131 medium for 2 - 9 days.

[0135] Figures 21A - 21J show the data of real-time PCR analysis of the expression of the following genes in cells of human embryonic stem cell line H1 differentiated according to Example 12. Figure 21A: Somatostatin, Figure 21B: PDX1, Figure 21C: Pax6, Figure 21D: Pax4, Figure 21E: N KX6.1, Figure 21F: NGN3, Figure 21G: NeuroD, Figure 21H: Insulin, Figure 21I: Glucagon, Figure 21J: Chromogranin. Consistent with the data from Example 10, the addition of ascorbic acid at stages 2 - 4 significantly reduces the expression of somatostatin, glucagon, and Pax6, while maintaining the expression of insulin and Pax4 at stage 5. Furthermore, compared with 0.25 mM ascorbic acid, there was no significant benefit in using 0.5 - 1 mM ascorbic acid in S4. Finally, the addition of ascorbic acid at stage 2 was also proven effective in maintaining insulin expression while reducing the expression of glucagon and somatostatin at stages S3 - 5. Therefore, ascorbic acid acts to regulate the expression of single - hormone cells in a stage - specific manner. The addition of ascorbic acid is important at the early stage of the differentiation protocol, but was not proven effective in reducing the number of multi - hormone cells at the later stage.

[0136] (Example 13) The combination of retinoic acid and ascorbic acid is necessary to generate single - hormone insulin - positive cells. This example was conducted to clarify the requirements for generating single - hormone insulin - positive cells during the differentiation of pluripotent cells.

[0137] Cells of the human embryonic stem cell line H1 of various passages (passage 40 - passage 52) were seeded as single cells on MATRIGEL (trademark) (1:30 dilution) - coated dishes at a concentration of 100,000 cells per cm² in mTesr (trademark) 1 medium with 10 μM Y27632. 2 Forty - eight hours after seeding, the cultures were differentiated into cells of the pancreatic endocrine system as follows. a. Stage 1 (definitive endoderm (DE) - 3 days): Before the start of DE, the cultures were washed and incubated in incomplete PBS (without Mg or Ca) for 30 seconds, and then stage 1 medium was added. Human embryonic stem cells cultured as single cells on MATRIGEL (trademark) - coated dishes The stem cells were treated for 1 day in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 100 ng / mL GDF8, and 1 μM of the MCX compound (GSK3B inhibitor). Subsequently, the cells were treated on day 2 in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, sodium bicarbonate, GlutaMa x™, 5 mM glucose, 100 ng / mL GDF8, and 100 nM of the MCX compound, and then treated for an additional 1 day in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X Glut aMax™, 5 mM glucose, and 100 ng / mL GDF8. b. Stage 2 (gastrula - 2 days): The cells were treated for 2 days in MCDB-131 medium supplemented with 0.1% fatty acid-free BSA, 0.0012 g / mL sodium bicarbonate, 1X GlutaMax™, 5 mM D-glucose, 0.25 mM ascorbic acid, and 25 ng / mL FGF7. c. Stage 3 (foregut - 2 days): The cells were treated in MCDB-131 medium supplemented with 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, and then treated in MCDB-131 medium supplemented with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 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 on day 2, 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. 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. 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, and 200 nM TPB, and additionally 10 nM LDN-193189 was supplemented for one more day and treated with MCDB-131 medium. d. Stage 4 (pancreatic foregut progenitor cells - 2 days): The cells were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, 2% fatty acid-free BSA, 0.25 μM SANT-1, 50 nM RA , 200 nM TPB, 50 nM LDN-193189, and 0.1 mM ascorbic acid supplemented in MCDB-131 medium for 2 days. e. Stage 5 (pancreatic endoderm - 3 days): The cells were treated with a 1:200 dilution of ITS-X, 2.5 mM glucose, 1X GlutaMax™, 0.0015 g / mL sodium bicarbonate, and 2% fatty acid-free BSA supplemented in MCDB-131 medium under the following culture conditions for 3 days. · +0.1 mM 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 0 nM 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 inh + 50 nM LDN-193189

[0138] ​​​ Figures 22A to 22L show data from real-time PCR analysis of the expression of Pax4 (Figure 22A), Pax6 (Figure 22B), PDX1 (Figure 22C), PTF1a (Figure 22D), glucagon (Figure 22E), insulin (Figure 22F), NeuroD (Figure 22G), ngn3 (Figure 22H), Zic1 (Figure 22I), CDX2 , albumin (Figure 22K), and NKX6.1 (Figure 22L) in cells of the embryonic stem cell line H1 differentiated according to Example 13 and harvested on the 3rd day of S5. (Figure 22J), albumin (Figure 22K), NKX6.1 (Figure 22L) from real-time PCR analysis of the expression. The cultures treated with the above combinations were immunostained for insulin, glucagon, and somatostatin hormones at the end of stage 5. Table VIII summarizes the average percentages of insulin-positive cells, glucagon- and somatostatin-positive cells, and multi-hormone cells (expressing two or more hormones within one cell).

[0139] As shown in Figure 22 and Table VIII below, the addition of low-dose retinoic acid and ascorbic acid at stage 5 significantly decreased the total number of hormone-positive cells compared to cultures treated with vitamin C alone at S5, while increasing the percentage of single-hormone insulin-positive cells. Furthermore, the combination of retinoic acid, ascorbic acid, sonic hedgehog inhibitor, and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. The cultures treated with the above combinations were immunostained for insulin, glucagon, and somatostatin hormones at the end of stage 5. Table VIII summarizes the average percentages of insulin-positive cells, glucagon- and somatostatin-positive cells, and multi-hormone cells (expressing two or more hormones within one cell). positive cells, glucagon and somatostatin positive cells, and multi-hormone cells (expressing two or more hormones within one cell). is a summary of the average percentages of insulin-positive cells, glucagon- and somatostatin-positive cells, and multi-hormone cells (expressing two or more hormones within one cell).

[0140] As shown in Figure 22 and Table VIII below, the addition of low-dose retinoic acid and ascorbic acid at stage 5 significantly decreased the total number of hormone-positive cells compared to cultures treated with vitamin C alone at S5, while increasing the percentage of single-hormone insulin-positive cells. Furthermore, the combination of retinoic acid, ascorbic acid, sonic hedgehog inhibitor, and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. and ascorbic acid at stage 5 significantly decreased the total number of hormone-positive cells compared to cultures treated with vitamin C alone at S5, while increasing the percentage of single-hormone insulin-positive cells. Furthermore, the combination of retinoic acid, ascorbic acid, sonic hedgehog inhibitor, and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. while increasing the percentage of single-hormone insulin-positive cells. Furthermore, the combination of retinoic acid, ascorbic acid, sonic hedgehog inhibitor, and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. inhibitor, and ALK5 inhibitor further increased the number of single-hormone insulin-positive cells compared to cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. cultures treated with ascorbic acid (vitamin C) alone. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. This data indicates that a unique combination of factors is required to generate single-hormone insulin-positive cells. is required.

[0141] [Table 8] The present invention may include the following aspects. [1] An in vitro differentiated population of pancreatic endoderm cells obtained from the stepwise differentiation of pluripotent cells, wherein the cells at each stage of differentiation are cultured in a medium containing 5 mM to 20 mM glucose, a differentiated population of pancreatic endoderm cells. [2] More than 30% of the differentiated pancreatic endoderm cells are PDX-1+NKX6.1+, SOX2 -, and CDX2-, the differentiated population of pancreatic endoderm cells according to [1] above. [3] More than 10% of the cells of the differentiated population are single hormone insulin-positive cells, the differentiated population of pancreatic endoderm cells according to [1 or [2] above. [4] The stepwise differentiation includes culturing undifferentiated human embryonic stem cells in a medium supplemented with a TGF-β ligand, a differentiated population of pancreatic endoderm cells according to any one of [1] to [3] above. [5] The stepwise differentiation includes culturing undifferentiated human embryonic stem cells in a medium supplemented with a WNT activator, a differentiated population of pancreatic endoderm cells according to any one of [1] to [4] above. [6] The stepwise differentiation includes culturing definitive endoderm cells in a medium supplemented with an FGF ligand, a differentiated population of pancreatic endoderm cells according to [1] above. [7] The stepwise differentiation includes culturing intestinal cells in a medium supplemented with a gradient of a shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a BMP inhibitor, a differentiated population of pancreatic endoderm cells according to any one of [1] to [4] above. ​​​ [8] wherein said stepwise differentiation comprises culturing posterior foregut cells in a medium supplemented with a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor A population of differentiated pancreatic endoderm cells according to [5] above, comprising the step of culturing posterior foregut cells in a further supplemented medium . [9] wherein said stepwise differentiation comprises culturing cells in a medium supplemented with ascorbic acid A population of differentiated pancreatic endoderm cells according to any one of [1] to [6] above

[10] An in vitro method for the stepwise differentiation of pluripotent cells into a population of cells of the pancreatic endoderm lineage comprising culturing the cells at each stage of differentiation in a medium containing 5 mM to 20 mM glucose .

[11] culturing said pluripotent cells in a medium supplemented with a TGF-β ligand and a WNT activator thereby further comprising the step of differentiating said pluripotent cells into definitive endoderm (DE) cells, the in vitro method according to

[10] above .

[12] culturing said DE cells in a medium supplemented with an FGF ligand, thereby further comprising the step of differentiating said DE cells into gut tube cells, the in vitro method according to

[11] above .

[13] culturing gut tube cells in a medium supplemented with an shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a BMP inhibitor, thereby further comprising the step of differentiating said gut tube cells into posterior foregut endoderm cells, the in vitro method according to

[12] above .

[14] culturing posterior foregut endoderm cells in a medium supplemented with a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor thereby differentiating said posterior foregut endoderm cells into pancreatic foregut cells ​An in vitro method according to

[13] further comprising a step of differentiation.

[15] A step of differentiating the pancreatic foregut cells into pancreatic endoderm cells by culturing the pancreatic foregut cells in a medium supplemented with an shh inhibitor, a TGF-β inhibitor, and a retinoid. An in vitro method according to

[14] further comprising a step of differentiating the pancreatic foregut cells into pancreatic endoderm cells by culturing the pancreatic foregut cells in a medium supplemented with an shh inhibitor, a TGF-β inhibitor, and a retinoid. An in vitro method according to

[14] further comprising a step of differentiating the pancreatic foregut cells into pancreatic endoderm cells by culturing the pancreatic foregut cells in a medium supplemented with an shh inhibitor, a TGF-β inhibitor, and a retinoid.

[16] An in vitro method according to

[15] further comprising a step of differentiating the pancreatic endoderm cells into a pancreatic β cell population. An in vitro method according to

[15] further comprising a step of differentiating the pancreatic endoderm cells into a pancreatic β cell population.

[17] An in vitro method according to any one of

[10] to

[16] , further supplementing ascorbic acid to the medium in at least one step. An in vitro method according to any one of

[10] to

[16] , further supplementing ascorbic acid to the medium in at least one step.

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

[0010] to

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

[19] An in vitro method according to

[18] , wherein more than 30% of the pancreatic endoderm cells in the culture are PDX-1+, NKX6.1+, SOX2-, and CDX2-. An in vitro method according to

[18] , wherein more than 30% of the pancreatic endoderm cells in the culture are PDX-1+, NKX6.1+, SOX2-, and CDX2-.

[20] An in vitro method for differentiating human embryonic stem cells into pancreatic β cells, comprising: a) culturing undifferentiated human embryonic stem cells in a medium supplemented with glucose, a TGF-β ligand, and a WNT activator to generate a population of definitive endoderm (DE) cells; b) culturing the DE cells in a medium supplemented with glucose and an FGF ligand to generate a population of gut tube cells; and c) culturing the DE cells in a medium supplemented with glucose, an shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, and a retinoid to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2. b) culturing the DE cells in a medium supplemented with glucose and an FGF ligand to generate a population of gut tube cells; c) culturing the DE cells in a medium supplemented with glucose, an shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, and a retinoid to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2. c) culturing the DE cells in a medium supplemented with glucose, an shh inhibitor, an FGF ligand, a PKC activator, a TGF-β ligand, and a retinoid to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2. culturing the intestinal cells in a medium supplemented with a retinoid and a BMP inhibitor; d) culturing the posterior foregut cells in a medium supplemented with glucose, a PKC activator, an shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells that express PDX-1 and NKX6.1 and express SOX2 at a lower level compared to the posterior foregut cells; e) culturing the pancreatic foregut cells in a medium supplemented with glucose, an shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells that express a higher level of NKX6.1 and a lower level of SOX2 compared to the pancreatic foregut cells and PDX-1; f) differentiating the pancreatic endoderm cells into a pancreatic β cell population.

[21] The method according to

[20] , wherein the pancreatic β cell population is PDX-1+, NKX6.1+, SOX2−, and CDX2−.

[22] The method according to

[20] or

[21] , wherein ascorbic acid is further supplemented to the medium in at least one step.

[23] The method according to

[22] , wherein the pancreatic β cells are single hormone insulin-producing cells that are also NKX6.1+ and PDX-1+. ​​​​​​​​​

Claims

1. An in vitro method for the stepwise differentiation of pluripotent cells into a population of cells comprising pancreatic endocrine cells, comprising: i) differentiating said pluripotent cells into foregut endoderm cells; ii) differentiating said foregut endoderm cells in a first medium supplemented with retinoic acid, an shh inhibitor, a bone morphogenetic protein (BMP) inhibitor, and a protein kinase C (PKC) activator to generate foregut progenitor cells; iii) differentiating said foregut progenitor cells in a second medium comprising an shh inhibitor, a BMP inhibitor, and retinoic acid to generate pancreatic endoderm cells; and iv) differentiating said pancreatic endoderm cells in a third medium comprising an shh inhibitor and retinoic acid, thereby generating a population of cells comprising said pancreatic endocrine cells.

2. The in vitro method according to claim 1, wherein the shh inhibitor in the first medium is SANT-1.

3. The in vitro method according to claim 1 or 2, wherein the BMP inhibitor in the first medium is LDN-193189 or noggin.

4. The in vitro method according to any one of claims 1 to 3, wherein the BMP inhibitor in the second medium is LDN-193189 or noggin.

5. The in vitro method according to any one of claims 1 to 4, wherein an ALK5 inhibitor is further supplemented in the second medium.

6. The in vitro method according to any one of claims 1 to 5, wherein the shh inhibitor in the second medium is SANT-1.

7. The in vitro method according to any one of claims 1 to 6, wherein ascorbic acid is further supplemented in the second medium.

8. The in vitro method according to any one of claims 1 to 7, wherein the shh inhibitor in the third medium is SANT-1.

9. The in vitro method according to any one of claims 1 to 8, wherein ascorbic acid is further supplemented in the third medium.

10. The in vitro method according to any one of claims 1 to 9, wherein an ALK5 inhibitor is further supplemented in the third medium.

11. The in vitro method according to any one of claims 1 to 10, wherein the pancreatic endocrine cells are single hormone insulin-producing cells that are also NKX6.1+ and PDX-1+.

12. The in vitro method according to any one of claims 1 to 11, wherein the first medium contains 5 mM to 20 mM glucose.

13. The in vitro method according to any one of claims 1 to 12, wherein the second medium further contains 5 mM to 20 mM glucose.

14. The in vitro method according to any one of claims 1 to 13, wherein the third medium further contains 5 mM to 20 mM glucose.

15. The in vitro method according to any one of claims 1 to 14, wherein the PKC activator is TPB.

Images