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US 12,516,295 B2Granted
Generation of pancreatic progenitor cells and beta cells from human pluripotent stem cells
AssigneeThe Penn State Research Foundation
InventorsXiaojun Lian, Chuanxin Chen
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Abstract
Provided are methods, compositions and kits for generating beta cells from pluripotent stem cells under growth-factor free, defined culture conditions. The beta cells can be generated under conditions that are free of animal products. The generated beta cells secrete insulin, not glucagon, and the amount of insulin secreted is dependent upon the level of glucose stimulus.
Claims4
A method of generating cells of pancreatic beta lineage from pluripotent stem cells comprising:
- a) contacting the cells with a growth-factor free medium comprising a Wnt signaling activator that is CHIR99021 and is present in the growth-factor free medium in a concentration of 2 to 6 μM, and a bone morphogenetic protein (BMP) signaling inhibitor that is Dorsomorphin to obtain a population of definitive endoderm (DE) cells, and wherein more of the DE cells contacted with 2 to 6 μM CHIR99021 are Sox17+ relative to DE cells contacted with CHIR99021 in the same growth-factor free medium but wherein the concentration of CHIR99021 is at least 10 μM, and wherein said DE cells that are contacted with the 2 to 6 μM of the CHIR99021 are obtained in a period of approximately 4 days,
- b) contacting the DE cells with a growth-factor free culture medium comprising Wnt signaling activator that is CHIR99021, the BMP signaling inhibitor that is Dorsomorphin, a retinoic acid signaling activator, and a protein kinase C activator, and a Hedgehog signaling inhibitor to obtain a population of pancreatic progenitor 1 (PP1) cells wherein more than 91% of the PP1 cells are PDX1+,
- c) subjecting the PP1 cells to a growth-factor free medium culture medium comprising a BMP signaling inhibitor that is Dorsomorphin, a ROCK signaling inhibitor, and nicotinamide to obtain a population of pancreatic progenitor 2 (PP2) NKX6.1+ cells,
- d) subjecting the PP2 cells to Hedgehog signaling inhibitor, a retinoic acid signaling activator, a Notch signaling inhibitor, a TGF-beta signaling inhibitor, and 3,3′,5-Triiodo-L-thyronine (T3) to obtain a population of endocrine progenitor (EP) cells,
- e) and culturing the EP cells in growth-factor free, serum-free medium to obtain beta cells, wherein the beta cells produce insulin.
2 more elements
A method of generating beta cells from pluripotent stem cells (PSCs) comprising:
- a) culturing PSCs in a medium comprising a GSK3β inhibitor that is CHIR99021 and a bone morphogenetic protein (BMP) signaling inhibitor that is Dorsomorphin for a period of from 4 to 10 days to obtain definitive endoderm (DE) cells;
- b) culturing DE cells in a medium comprising a GSK3β inhibitor that is CHIR99021, the BMP signaling inhibitor that is Dorsomorphin, and a Hedgehog signaling inhibitor for a period of 2 to 6 days to obtain pancreatic progenitor 1 (PP1) cells;
- c) culturing PP1 cells in a medium comprising the BMP signaling inhibitor that is Dorsomorphin for a period of 2 to 6 days to obtain pancreatic progenitor 2 (PP2) cells;
- d) culturing PP2 cells in a medium comprising 3′,5-Triiodo-L-thyronine (T3), and the Hedgehog signaling inhibitor, a retinoic acid signaling activator, a Notch signaling inhibitor, and a TGF-beta signaling inhibitor for a period of 5-10 days to obtain endocrine progenitor (EP) cells, and
- e) culturing EP cells in growth-factor-free, defined medium for 10-20 days to obtain beta cells,
- wherein steps a), b), c) and d) are carried out under growth-factor free, defined culture conditions.
3 more elements
Dependencies are inferred from the claim text.