Differentiation of human embryonic stem cells
First Claim
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, andwherein 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.
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Abstract
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.
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Citations
6 Claims
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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. - View Dependent Claims (2)
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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. - View Dependent Claims (4, 5)
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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:
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(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.
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Specification