Methods for producing mammalian pluripotent stem cell-derived endodermal cells
First Claim
1. A method for producing definitive endodermal cells (DE cells) from mammalian pluripotent stem cells, the method comprising:
- differentiating mammalian pluripotent stem cells under directed differentiation conditions to obtain a cell composition which is characterized in that at least 70% of the cells are DE cells, andexposing the mammalian pluripotent stem cells to a nucleoside-analogue type DNA demethylating agent while undergoing differentiation, wherein the exposing of the mammalian pluripotent stem cells to the nucleoside-analogue type DNA demethylating agent first takes place on day 2 of differentiation, and wherein the cell composition obtained shows increased gene expression of sox17, cxcr4 and hhex compared to a cell composition obtained without exposure to the nucleoside-analogue type DNA demethylating agent.
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Abstract
The present invention relates to the directed differentiation of mammalian pluripotent stem cells, especially human pluripotent stem (hPS) cells, into endodermal cells. In particular, the present invention relates to the treatment of mammalian pluripotent stem cells, especially hPS cells, with a DNA demethylating agent while undergoing differentiation into endodermal. The inventors have, as disclosed herein, found that exposing differentiating mammalian pluripotent stem cells, especially hPS cells, to a DNA demethylating agent leads to an improved morphology and improved yield of endodermal cells. The treatment with a DNA de-methylating agent also leads to a significant down-regulation of expression of the stem cell marker Oct4 and to an improved expression of endoderm specific markers, notably sox17, cxcr4 and hhex.
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Citations
24 Claims
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1. A method for producing definitive endodermal cells (DE cells) from mammalian pluripotent stem cells, the method comprising:
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differentiating mammalian pluripotent stem cells under directed differentiation conditions to obtain a cell composition which is characterized in that at least 70% of the cells are DE cells, and exposing the mammalian pluripotent stem cells to a nucleoside-analogue type DNA demethylating agent while undergoing differentiation, wherein the exposing of the mammalian pluripotent stem cells to the nucleoside-analogue type DNA demethylating agent first takes place on day 2 of differentiation, and wherein the cell composition obtained shows increased gene expression of sox17, cxcr4 and hhex compared to a cell composition obtained without exposure to the nucleoside-analogue type DNA demethylating agent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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Specification