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.

1 . A method for producing pancreatic endoderm lineage cells expressing NKX2.2 and PDX1, comprising:
 obtaining definitive endoderm cells; and   differentiating the definitive endoderm cells with a medium comprising a transforming growth factor (TGF)-β receptor agonist, retinoic acid or a retinoic acid analog, and a fibroblast growth factor (FGF),   thereby producing pancreatic endoderm lineage cells expressing NKX2.2 and PDX1.   
     
     
         2 . The method of  claim 1 , wherein the TGF-β receptor agonist is activin A. 
     
     
         3 . The method of  claim 2 , wherein the medium comprises 100 ng/mL activin A. 
     
     
         4 . The method of  claim 1 , wherein the medium comprises 1 μM retinoic acid 
     
     
         5 . The method of  claim 1 , wherein the FGF is FGF-2, FGF4 or FGF-10. 
     
     
         6 . The method of  claim 5 , wherein the FGF is FGF-2. 
     
     
         7 . The method of  claim 6 , wherein the medium comprises 50 ng/mL FGF-2. 
     
     
         8 . The method of  claim 1 , wherein obtaining definitive endoderm cells comprises differentiating pluripotent stem cells into definitive endoderm cells by treating the pluripotent stem cells with a TGF-β receptor agonist. 
     
     
         9 . The method of  claim 8 , wherein the TGF-β receptor agonist is activin A. 
     
     
         10 . A composition comprising an isolated human pancreatic endoderm lineage cell population expressing NKX2.2 and PDX1, a transforming growth factor (TGF)-β receptor agonist, retinoic acid and a fibroblast growth factor (FGF). 
     
     
         11 . The composition of  claim 10 , wherein the TGF-β receptor agonist is activin A. 
     
     
         12 . The composition of  claim 11 , comprising 100 ng/mL activin A. 
     
     
         13 . The composition of  claim 10 , comprising 1 μM retinoic acid 
     
     
         14 . The composition of  claim 10 , wherein the FGF is FGF-2, FGF4 or FGF-10. 
     
     
         15 . The composition of  claim 14 , wherein the FGF is FGF-2. 
     
     
         16 . The composition of  claim 15 , comprising 50 ng/mL FGF-2.