Differentiation of pluripotent stem cells to definitive endoderm lineage
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 differentiating pluripotent stem cells into cells expressing markers that are characteristic of the definitive endoderm lineage, comprising the steps of:
a) Plating the pluripotent stem cells on a tissue culture substrate coated with an extracellular matrix, and lacking a feeder cell layer,
b) Culturing the pluripotent stem cells with activin A, a Wnt ligand, and a GSK-3B inhibitor in a first culture medium, and
c) 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 cells expressing markers that are characteristic of the definitive endoderm lineage.