Definitive endoderm cells and human pluripotent stem cells

Abstract

Disclosed herein are methods of identifying one or more differentiation factors that are useful for differentiating cells in a cell population comprising definitive endoderm cells into cells which are capable of forming tissues and/or organs that are derived from the gut tube.

1 .- 20 . (canceled) 
     
     
         21 . An in vitro method of producing human definitive endoderm cells, comprising:
 obtaining a cell population comprising pluripotent human stem cells; and   providing said cell population with a TGFβ superfamily growth factor selected from the group consisting of activn A, activin B, BMP4, nodal, and combinations thereof, thereby producing human definitive endoderm cells.   
     
     
         22 . The method of  claim 21 , further comprising removing TGFβ superfamily growth factor from the cell population. 
     
     
         23 . The method of  claim 21 , wherein at least 15% of the pluripotent human stem cells differentiate into definitive endoderm cells. 
     
     
         24 . The method of  claim 21 , wherein the TGFβ superfamily growth factor is activin A. 
     
     
         25 . The method of  claim 21 , further comprising providing a Wnt family member to the cell population. 
     
     
         26 . The method of  claim 25 , wherein the Wnt family member is Wnt3a. 
     
     
         27 . The method of  claim 21 , wherein at least 10 ng/ml activin A is provided to the cell population. 
     
     
         28 . The method of  claim 21 , wherein at least 100 ng/ml activin A is provided to the cell population. 
     
     
         29 . The method of  claim 21 , further comprising providing serum to the cell population. 
     
     
         30 . The method of  claim 21 , wherein the pluripotent human stem cells comprise embryonic stem cells. 
     
     
         31 . The method of  claim 29 , wherein the embryonic stem cells are derived from tissue selected from the group consisting of the morula and the inner cell mass (ICM) of the embryo. 
     
     
         32 . The method of  claim 29 , wherein providing serum to the cell population comprises providing increasing concentrations of serum to the cell population. 
     
     
         33 . An in vitro cell culture, comprising:
 human pluripotent cells;   human definitive endoderm cells; and   a medium comprising an effective amount of a TGFβ superfamily member and an effective amount of a TGFβ superfamily growth factor selected from the group consisting of activn A, activin B, BMP4, nodal and combinations thereof, wherein the effective amount of a TGFβ superfamily growth factor selected from the group consisting of activn A, activin B, BMP4, nodal and combinations thereof promote differentiation of pluripotent cells to definitive endoderm cells.   
     
     
         34 . The in vitro cell culture of  claim 33 , wherein at least 15% of said pluripotent cells differentiate into definitive endoderm cells. 
     
     
         35 . The in vitro cell culture of  claim 33 , further comprising a Wnt family member. 
     
     
         36 . The in vitro cell culture of  claim 35 , wherein said Wnt family member is Wnt3a. 
     
     
         37 . The in vitro cell culture of  claim 33 , further comprising serum in the medium. 
     
     
         38 . The in vitro cell culture of  claim 37 , wherein the medium comprises less than 2% serum. 
     
     
         39 . An in vitro method of producing human definitive endoderm cells, comprising:
 obtaining a cell population comprising pluripotent human stem cells; and   providing said cell population with activn A, activin B and BMP4, thereby generating human definitive endoderm cells.