Differentiation of human embryonic stem cells
The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce a population of cells, wherein greater than 80% of the cells in the population express markers characteristic of the definitive endoderm lineage.
1. An in vitro culture comprising an isolated population of in vitro differentiated cells and a serum-free cell culture medium supplemented with L-alanyl-L-glutamine in which the concentration of glucose does not exceed 10.5 mM,
wherein greater than 80% of the cells in the population are definitive endoderm cells and express CXCR4 and CD99, and
wherein said population of cells is obtained by a step-wise differentiation process comprising:
(i) seeding clusters of pluripotent stem cells in a serum-free medium supplemented with L-alanyl-L-glutamine, and glucose at a concentration that does not exceed 10.5 mM;
(ii) culturing the pluripotent stem cells for about one day in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, about 20 ng/ml of Wnt-3a, and glucose at a concentration that does not exceed 10.5 mM; followed by
(iii) culturing the cells for about three days in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, and glucose at a concentration that does not exceed 10.5 mM, whereby the culture does not require further purification or selection of the definitive endoderm cells.
2. The isolated population of cells of claim 1 , wherein the concentration of glucose does not exceed 5.5 mM.
3. An in vitro culture comprising an isolated population of in vitro differentiated cells and a serum-free cell culture medium supplemented with L-alanyl-L-glutamine and glucose at a concentration that does not exceed 10.5 mM,
wherein greater than 80% of the cells in the population are definitive endoderm cells and express CXCR4 and, CD99, and
wherein said population of cells is obtained by a step-wise differentiation process comprising the steps of:
(i) seeding pluripotent stem cells in a serum-free medium supplemented with L-alanyl-L-glutamine and glucose at a concentration that does not exceed 10.5 mM,
(ii) culturing the pluripotent stem cells for about one day in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, about 20 ng/ml Wnt-3a and glucose at a concentration that does not exceed 10.5 mM; followed by
(iii) culturing the cells for about three days in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, and glucose at concentration that does not exceed 10.5 mM, whereby the culture does not require further purification or selection of the definitive endoderm cells.
4. The in vitro culture of claim 3 , wherein the concentration of glucose does not exceed 5.5 mM.
5. The in vitro culture of claim 3 , wherein the concentration of glucose in said step-wise differentiation process does not exceed 5.5 mM.
6. A method for generating a population of cells wherein 80% of the cells in the population are definitive endoderm cells and express CXCR4 and CD99, wherein said population of cells is obtained by a step-wise differentiation process comprising the steps of:
(i) seeding pluripotent stem cells in a serum-free medium supplemented with L-alanyl-L-glutamine and glucose at a concentration that does not exceed 10.5 mM,
(ii) culturing the pluripotent stern cells for about one day in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, about 20 ng/ml Wnt-3a and glucose at a concentration that does not exceed 10.5 mM; followed by
(iii) culturing the cells for about three days in a serum-free medium supplemented with L-alanyl-L-glutamine, about 100 ng/ml activin A, and glucose at concentration that does not exceed 10.5 mM, whereby the culture does not require further purification or selection of the definitive endoderm cells.