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

1. An in vitro method for producing a population of pancreatic cells comprising the steps of:

a) culturing undifferentiated human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;

b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;

c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;

d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;

e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, ascorbic acid and a retinoid to obtain a population of cells, wherein said population comprises:

(i) greater than 30% of pancreatic endoderm cells that are PDX-1+, NKX6.1+, SOX2− and CDX2−, and

(ii) greater than 10% of single hormonal insulin positive cells.

2. An in vitro method for differentiating human pluripotent stem cells comprising the steps of:

a) culturing human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;

b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;

c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;

d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;

e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells expressing PDX-1, a higher level of NKX6.1, and a lower level of SOX2 as compared to pancreatic foregut cells; and

f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population.

3. An in vitro method for differentiating human pluripotent stem cells comprising the steps of:

a) culturing human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;

b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;

c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;

d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;

e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells expressing PDX-1, a higher level of NKX6.1, and a lower level of SOX2 as compared to pancreatic foregut cells; and

f) differentiating the pancreatic endoderm cells into a pancreatic β-cell population.

4. The method of claim 2 , wherein in at least one step the medium is further supplemented with ascorbic acid.

5. The method of claim 4 , wherein the pancreatic β cells are single hormonal insulin-producing cells which are also NKX6.1+ and PDX-1+.

6. The method of claim 3 , wherein the pancreatic β-cell population is PDX-1+, NKX6.1+, SOX2-, and CDX2-.