Methods for producing insulin-secreting beta cells from human pluripotent stem cells
First Claim
1. A method of culturing posterior foregut endoderm cells to produce cells of the pancreatic lineage, the method comprising the step of:
- (a) culturing the posterior foregut endoderm cells for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;
i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, andii) Noggin,wherein the cells are cultured on an extracellular matrix coated onto a membrane, andwherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin+ cells.
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Abstract
A method of culturing human pluripotent stem cells to produce pancreatic lineage, the method comprising the steps of (a) culturing the stem cells in the presence of a chemically defined medium comprising an effective amount of FGF, Activin A, and BMP; (b) culturing the cells from step (a) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium (ITS), and FGF; (c) culturing the cells from step (b) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium (ITS), and Noggin-Nicotinamide-Retinoic acid; and (d) culturing the cells from step (c) in the presence of a serum free chemically defined medium (ITSFINE and Noggin) comprising an effective amount of ITS, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and Exendin-4, wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin+ cells.
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Citations
21 Claims
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1. A method of culturing posterior foregut endoderm cells to produce cells of the pancreatic lineage, the method comprising the step of:
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(a) culturing the posterior foregut endoderm cells for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of; i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and ii) Noggin, wherein the cells are cultured on an extracellular matrix coated onto a membrane, and wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin+ cells. - View Dependent Claims (18, 19, 20, 21)
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2. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:
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(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of; i) fibroblast growth factor (FGF), ii) Activin A, and iii) bone morphogenetic protein (BMP); (b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of; i) insulin, transferrin, and selenium (ITS), and ii) fibroblast growth factor (FGF); (c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium under conditions that induce formation of posterior foregut cells, wherein the medium comprises an effective amount of; i) insulin, transferrin, and selenium (ITS), and ii) Noggin-Nicotinamide-Retinoic acid (NNR); and (d) culturing the cells from step (c) for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of; i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and ii) Noggin, wherein the cells are cultured on an extracellular matrix coated onto a membrane, and wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin+ cells. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification