Use of noggin, an alk5 inhibitor and a protein kinase c activator, to produce endocrine cells

Abstract

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.

What is claimed is: 
     
         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.