Differentiation of human embryonic stem cells
First Claim
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1. A method to generate a population of pancreatic endocrine cells that co-express NKX6.1 and insulin, the method comprising the steps of:
- a) culturing human pluripotent stem cells;
b) differentiating the human pluripotent stem cells of step a) into definitive endoderm cells by culturing the human pluripotent stem cells in medium comprising a TGF-β
receptor agonist;
c) differentiating the definitive endoderm cells of step b) into pancreatic endoderm cells by culturing the definitive endoderm cells in the presence of an FGF family member and retinoic acid; and
d) differentiating the pancreatic endoderm cells of step c) into pancreatic endocrine cells by culturing the pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5 and a protein kinase C activator, wherein the pancreatic endocrine cells co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon.
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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 cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon.
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14 Claims
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1. A method to generate a population of pancreatic endocrine cells that co-express NKX6.1 and insulin, the method comprising the steps of:
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a) culturing human pluripotent stem cells; b) differentiating the human pluripotent stem cells of step a) into definitive endoderm cells by culturing the human pluripotent stem cells in medium comprising a TGF-β
receptor agonist;c) differentiating the definitive endoderm cells of step b) into pancreatic endoderm cells by culturing the definitive endoderm cells in the presence of an FGF family member and retinoic acid; and d) differentiating the pancreatic endoderm cells of step c) into pancreatic endocrine cells by culturing the pancreatic endoderm cells in a medium supplemented with noggin, an inhibitor of ALK5 and a protein kinase C activator, wherein the pancreatic endocrine cells co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method to generate a population of pancreatic endocrine cells that co-express NKX6.1 and insulin, the method comprising the steps of:
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a) culturing human pluripotent stem cells; b) differentiating the human pluripotent stem cells of step a) into definitive endoderm cells by culturing the human pluripotent stem cells in medium comprising a TGF-β
receptor agonist;c) differentiating the definitive endoderm cells of step b) into pancreatic endoderm cells by culturing the definitive endoderm cells in the presence of an FGF family member and retinoic acid; and d) differentiating the pancreatic endoderm cells of step c) into pancreatic endocrine cells by culturing the pancreatic endoderm cells in a medium supplemented with noggin, a TGF-β
receptor signaling inhibitor and (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam, wherein the pancreatic endocrine cells co-express NKX6.1 and insulin and wherein less than 10% of the cells in the population express glucagon. - View Dependent Claims (10, 11, 12, 13, 14)
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Specification