Optimized Methods for Differentiation of Cells into Cells With Hepatocyte and Hepatocyte Progenitor Phenotypes, Cells Produced by the Methods, and Methods of Using the Cells
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Abstract
The invention is directed to methods for culturing cells so that the cells are induced to differentiate into cells that express hepatocyte phenotypes and hepatocyte progenitor phenotypes. More particularly, the invention relates to methods for culturing cells so that the cells are induced to differentiate into cells that express a definitive endodermal phenotype, a liver-committed endodermal phenotype, a hepatoblast phenotype, and hepatocyte phenotype. The invention is also directed to cells produced by the methods of the invention. The cells are useful, among other things, for treatment of liver deficiency, liver metabolism studies, and liver toxicity studies.
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Citations
82 Claims
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1-62. -62. (canceled)
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63. A method for inducing cells to differentiate into cells with a hepatocyte phenotype, comprising:
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(a) culturing cells with about 5 ng/ml to about 500 ng/ml Wnt3a and about 10 ng/ml to about 1,000 ng/ml ActivinA; (b) then culturing the cells of step (a) with about 1 ng/ml to about 100 ng/ml bFGF and about 5 ng/ml to about 500 ng/ml BMP4; (c) then culturing the cells of step (b) with about 5 ng/ml to about 500 ng/ml aFGF, about 1 ng/ml to about 100 ng/ml FGF4 and about 2.5 ng/ml to about 250 ng/ml FGF8b; and (d) then culturing the cells of step (c) with about 2 ng/ml to about 200 ng/ml HGF and about 10 ng/ml to about 1,000 ng/ml Follistatin. - View Dependent Claims (64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 81, 82)
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- 76. A composition, comprising cells in a culture medium comprising about 5 ng/ml to about 500 ng/ml Wnt3a and about 10 ng/ml to about 1,000 ng/ml ActivinA, wherein the cells are embryonic stem cells or cells that are not embryonic stem cells, embryonic germ cells or germ cells, and can differentiate into at least one cell type of each of the endodermal, ectodermal and mesodermal embryonic lineages.
Specification