Differentiation of human embryonic stem 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 a population of cells, wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage.

What is claimed is: 
     
         1 . A method for generating a population of cells wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage, comprising the steps of:
 a) culturing a population of pluripotent stem cells,   b) differentiating the population of pluripotent stem cells in a medium with a pH of 7.6 or higher and supplemented with:
 (i.) activin A; 
 (ii.) wnt3a; and 
 (iii.) glucose at a concentration that does not exceed 10.5 mM, thereby generating a population of cells, wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage. 
   
     
     
         2 . The method of  claim 2 , wherein the concentration of glucose does not exceed 5.5 mM. 
     
     
         3 . The method of  claim 1 , wherein the cells express CXCR4 and CD99. 
     
     
         4 . The method of  claim 1 , wherein the cells expressing markers characteristic of the definitive endoderm lineage are definitive endoderm cells. 
     
     
         5 . The method of  claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells. 
     
     
         6 . The method of  claim 1 , wherein the pluripotent stem cells are human embryonic stem cells. 
     
     
         7 . The method of  claim 1 , wherein the medium comprises about 20 ng/ml of wtn-3A. 
     
     
         8 . The method of  claim 1 , wherein the medium comprises about 100 ng/ml of activin A. 
     
     
         9 . The method of  claim 1 , wherein the medium is serum free. 
     
     
         10 . The method of  claim 1 , wherein the step of differentiation comprises
 culturing the pluripotent stem cells for about one day in serum-free medium supplemented with about 100 ng/ml of activin A, about 20 ng/ml of wnt-3a and glucose at a concentration that does not exceed 10.5 mM followed by   culturing the cells for about three days in serum-free medium supplemented with about 100 ng/ml of activin A and glucose at a concentration that does not exceed 10.5 mM.   
     
     
         11 . A method of differentiating pluripotent stem cells into definitive endoderm cells comprising culturing pluripotent stem cells in a medium with a pH of 7.6 or higher and supplemented with:
 a) activin A;   b) wnt3a; and   c) glucose at a concentration that does not exceed 10.5 mM.   
     
     
         12 . The method of  claim 11 , wherein the method further comprises culturing the pluripotent stem cells prior to differentiating. 
     
     
         13 . The method of  claim 11 , wherein the concentration of glucose does not exceed 5.5 mM. 
     
     
         14 . The method of  claim 11 , wherein the pluripotent stem cells are human pluripotent stem cells. 
     
     
         15 . The method of  claim 11 , wherein the pluripotent stem cells are human embryonic stem cells. 
     
     
         16 . The method of  claim 11 , wherein the medium comprises about 100 ng/ml of activin A. 
     
     
         17 . The method of  claim 11 , wherein the medium comprises about 20 ng/ml of wnt-3a. 
     
     
         18 . The method of  claim 11 , wherein the medium is serum free.