| 主权项 |
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. |
