Mesoderm and definitive endoderm cell populations

Abstract

The present invention provides cell populations that are enriched for mesendoderm and mesoderm, and cell populations that are enriched for endoderm. The cell populations of the invention are useful for generating cells for cell replacement therapy.

1 . A method of making a cell population enriched for endoderm cells comprising culturing embryonic stem cells in the absence of serum and the presence of activin and Wnt for about one to about six days, isolating brach + /HNF3β +  cells, a culturing said isolated cells in the absence of serum and the presence of activin and an inhibitor of Wnt signalling for at least about one day.  
     
     
         2 . The method of  claim 1  wherein the embryonic stem cells are human embryonic stem cells.  
     
     
         3 . The method of  claim 1  wherein Wnt is Wnt 3d.  
     
     
         4 . The method of  claim 1  wherein the inhibitor of Wnt signalling is DKK-1.  
     
     
         5 . The method of  claim 1  wherein activin is present at a concentration of at least about 30 ng/ml.  
     
     
         6 . A method of making a cell population enriched for endoderm cells comprising culturing human embryonic stem cells in the presence of serum for about two to about ten days, isolating brach + /HNF3β +  cells, and culturing said brach + /HNF3β +  cells in the absence of serum and the presence of activin and an inhibitor of Wnt signalling for at least about one day.  
     
     
         7 . The method of  claim 6  wherein the concentration of activin is at least about 30 ng/ml.  
     
     
         8 . The method of  claim 6  wherein the concentration of activin is about 100 ng/ml.  
     
     
         9 . The method of  claim 6  wherein the inhibitor of Wnt signalling is DKK-1.  
     
     
         10 . The method of  claim 9  wherein the concentration of DKK-1 is at least about 30 ng/ml.  
     
     
         11 . An embryonic stem cell in which a nucleic acid encoding a selectable marker is present in the HNF3β locus.  
     
     
         12 . The stem cell of  claim 11  in which the selectable marker is human CD4.  
     
     
         13 . The stem cell of  claim 11  in which a second nucleic acid encoding a second selectable marker is present in the brachury locus.  
     
     
         14 . The stem cell of  claim 13  in which the second selectable marker is green fluorescent protein.