Differentiation of Human Embryonic Stem Cells

Abstract

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

What is claimed is: 
     
         1 . A method for differentiating human pluripotent stem cells into pancreatic endocrine cells comprising:
 a. differentiating human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent cells with activin A;   b. differentiating the definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor; and   c. differentiating the pancreatic endoderm cells into cells expressing markers characteristics of the pancreatic endocrine lineage by treating the pancreatic endoderm cells with a gamma secretase inhibitor.   
     
     
         2 . The method of  claim 1 , wherein step a. comprises treating the human pluripotent stem cells with activin A and (i) a GSK-3B inhibitor, (ii) a Wnt ligand, or (iii) both a GSK-3B inhibitor and a Wnt ligand. 
     
     
         3 . The method of  claim 2 , wherein the GSK-3B inhibitor is GSK-3B inhibitor IX or GSK-3B inhibitor XI. 
     
     
         4 . The method of  claim 2 , wherein the Wnt ligand is selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. 
     
     
         5 . The method of  claim 1 , wherein the step of differentiating the human pluripotent stem cells into definitive endoderm cells is achieved by treating the human pluripotent stem cells with activin A for up to about eight days. 
     
     
         6 . The method of  claim 1 , wherein the step a. comprises:
 i) plating the human pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and lacking a feeder cell layer;   ii) culturing the pluripotent stem cells with activin A, a Wnt ligand, and a GSK-3B inhibitor in a first culture medium; and   iii) culturing the pluripotent stem cells with activin A in a second culture medium wherein the second culture medium does not contain a Wnt ligand, for a period of time sufficient to differentiate the pluripotent stem cells into definitive endoderm cells.   
     
     
         7 . The method of  claim 6 , wherein the extracellular matrix is selected from the group consisting of MATRIGEL, growth factor-reduced MATRIGEL, laminin, fibronectin, and human serum. 
     
     
         8 . The method of  claim 1 , wherein the fibroblast growth factor is selected from the selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10. 
     
     
         9 . The method of  claim 1 , wherein the fibroblast growth factor is FGF-2 or FGF-4. 
     
     
         10 . The method of  claim 1 , wherein the step of differentiating the definitive endoderm cells into pancreatic endoderm cells is achieved by treating the definitive endoderm cells with a factor selected from the group consisting of retinoic acid, FGF-2, FGF-4 and FGF-10. 
     
     
         11 . The method of  claim 10 , wherein the cells are treated for about one to about six days. 
     
     
         12 . The method of  claim 1 , wherein the step of differentiating the definitive endoderm cells into pancreatic endoderm cells comprises:
 (i) culturing human definitive endoderm cells in a medium supplemented with retinoic acid or a retinoic acid analog;   (ii) removing the retinoic acid or retinoic acid analog; and   (iii) subsequently culturing the cells in a medium supplemented with at least one fibroblast growth factor.   
     
     
         13 . The method of  claim 12 , wherein the culture medium in step (i), step (iii) or both step (i) and (iii) is supplemented with about 0% to about 2% of serum. 
     
     
         14 . The method of  claim 1 , wherein the gamma secretase inhibitor is L-685,458. 
     
     
         15 . A method for generating pancreatic endocrine cells comprising treating pancreatic endoderm cells with a gamma secretase inhibitor, wherein the pancreatic endocrine cells are derived from human definitive endoderm cells. 
     
     
         16 . The method of  claim 15 , wherein the gamma secretase inhibitor is L-685,458. 
     
     
         17 . The method of  claim 15 , further comprising differentiating human definitive endoderm cells into pancreatic endoderm cells by treating the definitive endoderm cells with at least one fibroblast growth factor, or with retinoic acid and at least one fibroblast growth factor. 
     
     
         18 . The method of  claim 17 , wherein the fibroblast growth factor is selected from the group consisting of FGF-2, FGF-4 and FGF-10. 
     
     
         19 . The method of  claim 15 , further comprising differentiating the human definitive endoderm cells into pancreatic endoderm cells by a method comprising:
 (i) culturing the human definitive endoderm cells in a medium supplemented with retinoic acid or a retinoic acid analog;   (ii) removing the retinoic acid or retinoic acid analog; and   (iii) subsequently culturing the cells in a medium supplemented with at least one fibroblast growth factor.   
     
     
         20 . The method of  claim 19 , wherein the culture medium in step (i), step (iii) or both step (i) and (iii) is supplemented with about 0% to about 2% of serum.