USE OF NOGGIN, AN ALK5 INHIBITOR AND A PROTEIN KINASE C ACTIVATOR, TO PRODUCE ENDOCRINE CELLS
The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon.
1 . A method of producing a population of NKX6.1 and insulin co-expressing human pancreatic endocrine cells comprising culturing pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5, and a protein kinase C activator, wherein less than 10% of the cells in the population express glucagon.
2 . The method of claim 1 , wherein the method comprises culturing for about 6 to about 12 days.
3 . The method of claim 1 , wherein the inhibitor of ALK5 is ALK5 inhibitor II.
4 . The method of claim 1 , wherein the protein kinase C activator is (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.
5 . The method of claim 1 , wherein at least 30% of the cells express NKX6.1.
6 . The method of claim 1 , wherein at least 40% of the cells express NKX6.1.
7 . The method of claim 1 , wherein at least 50% of the cells express NKX6.1.
8 . The method of claim 1 , wherein at least 60% of the cells express NKX6.1.
9 . The method of claim 1 , wherein at least 5% of the cells express insulin.
10 . A method of producing human pancreatic endocrine cells comprising culturing pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5, and a protein kinase C activator.
11 . The method of claim 10 , wherein less than 10% of the pancreatic endocrine cells in the population express glucagon.
12 . The method of claim 10 , wherein the method comprises culturing for about 6 to about 12 days.
13 . The method of claim 10 , wherein the inhibitor of ALK5 is ALK5 inhibitor II.
14 . The method of claim 10 , wherein the protein kinase C activator is (2S,5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.
15 . The method of claim 10 , wherein at least 30% of the pancreatic endocrine cells express NKX6.1.
16 . The method of claim 10 , wherein at least 40% of the pancreatic endocrine cells express NKX6.1.
17 . The method of claim 10 , wherein at least 50% of the pancreatic endocrine cells express NKX6.1.
18 . The method of claim 10 , wherein at least 60% of the pancreatic endocrine cells express NKX6.1.
19 . The method of claim 10 , wherein at least 5% of the pancreatic endocrine cells express insulin.