Suspension Culturing of Pluripotent Stem Cells
First Claim
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1. A method for differentiation human pluripotent cells, comprising the steps of:
- differentiating foregut endoderm cells to pancreatic endoderm cells by culturing the foregut endoderm cells in a dynamic suspension culture at a pH of about 7.2 to about 7.0 for at least about 24 hours.
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Abstract
The present invention provides methods of differentiating pluripotent cells into beta cell using suspension clustering. The methods of the invention use control of one or more of pH, cell concentration, and retinoid concentration to generate a nearly homogenous population of PDX1/NKX6.1 co-expressing cells by suppressing precocious NGN3 expression and promoting NKX6.1 expression. Also, the nearly homogenous population of PDX1/NKX6.1 co-expressing cells may be further differentiated in vitro to form a population of pancreatic endocrine cells that co-express PDX1, NKX6.1, insulin and MAFA.
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7 Claims
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1. A method for differentiation human pluripotent cells, comprising the steps of:
differentiating foregut endoderm cells to pancreatic endoderm cells by culturing the foregut endoderm cells in a dynamic suspension culture at a pH of about 7.2 to about 7.0 for at least about 24 hours. - View Dependent Claims (2, 3)
- 4. The method of claiml, wherein the pancreatic endoderm cells are substantially negative for the expression of PTF1A and NGN3.
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7. A method for differentiation human pluripotent cells, comprising the steps of:
differentiating foregut endoderm cells to pancreatic endoderm cells by culturing the foregut endoderm cells in a dynamic suspension culture at a pH of about 7.2 to about 7.0 for at least about 24 hours, a cell concentration of equal to or greater than about 1.5 million cells/mL, and a retinoid concentration of about 0.5 to about 1.0 wherein the culturing is carried out in the absence of components to one or more of inhibit, block, activate or agonize TGF-beta signaling and BMP signaling and a sonic hedgehog signaling pathway inhibitor.
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