Methods for identifying factors for differentiating definitive endoderm

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 . An in vitro cell culture comprising a medium, human definitive endoderm cells, and human pluripotent stem cells, wherein said definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom, and wherein said human pluripotent stem cells comprise less than 5% of the cells in said cell culture. 
     
     
         2 . The cell culture of  claim 3 , wherein said human pluripotent stem cell comprises an embryonic stem cell. 
     
     
         3 . The cell culture of  claim 4 , wherein said embryonic stem cell is derived from a tissue selected from the group consisting of morula of an embryo and inner cell mass (ICM) of an embryo. 
     
     
         4 . The cell culture of  claim 4 , wherein said embryonic stem cell is derived from a gondal ridge of an embryo. 
     
     
         5 . The cell culture of  claim 1 , wherein said medium lacks B27. 
     
     
         6 . The cell culture of  claim 1 , wherein said medium lacks serum replacement. 
     
     
         7 . The cell culture of  claim 1 , wherein said medium comprises a growth factor of the Nodal/Activin subgroup of the TGFβ superfamily. 
     
     
         8 . The cell culture of  claim 7 , wherein said medium further comprises a Wnt family member. 
     
     
         9 . The cell culture of  claim 7 , wherein said growth factor of the Nodal/Activin subgroup of the TGFβ superfamily is selected from the group consisting of Nodal, Activin A, and Activin B. 
     
     
         10 . The cell culture of  claim 9 , wherein said medium comprises Nodal. 
     
     
         11 . The cell culture of  claim 9 , wherein said medium comprises Activin B. 
     
     
         12 . The cell culture of  claim 1 , wherein said human pluripotent stem cells comprise less than 3% of the cells in said cell culture. 
     
     
         13 . The cell culture of  claim 1 , wherein said human pluripotent stem cells comprise less than 2% of the cells in said cell culture. 
     
     
         14 . An in vitro cell culture comprising a medium, human definitive endoderm cells, and human pluripotent stem cells, wherein at least 15% of said human cells are definitive endoderm cells, said definitive endoderm cells being multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom. 
     
     
         15 . The cell culture of  claim 1  wherein said definitive endoderm cells comprise at least 50% of the cells in said cell culture. 
     
     
         16 . The cell culture of  claim 1  wherein said definitive endoderm cells comprise at least 80% of the cells in said cell culture. 
     
     
         17 . The cell culture of  claim 1  wherein said definitive endoderm cells comprise at least 90% of the cells in said cell culture. 
     
     
         18 . The cell culture of  claim 1  wherein said definitive endoderm cells express a marker selected from the group consisting of SOX17, HNF3β and CXCR4. 
     
     
         19 . The cell culture of  claim 1  wherein said definitive endoderm cells express SOX17. 
     
     
         20 . The cell culture of  claim 1  wherein said definitive endoderm cells express CXCR4.