Differentiation of human embryonic stem cells into single hormonal insulin positive cells
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12N-005/00
C12N-005/02
C12N-005/071
출원번호
US-0708369
(2012-12-07)
등록번호
US-9388386
(2016-07-12)
발명자
/ 주소
Rezania, Alireza
출원인 / 주소
Janssen Biotech, Inc.
대리인 / 주소
Gianneschi, Lois A.
인용정보
피인용 횟수 :
0인용 특허 :
7
초록▼
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of cells, wherein greater than 10% of the cells in the population express markers characteristic of single hormonal pancreati
The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides methods to produce a population of cells, wherein greater than 10% of the cells in the population express markers characteristic of single hormonal pancreatic beta cells.
대표청구항▼
1. An in vitro method for producing a population of pancreatic cells comprising the steps of: a) culturing undifferentiated human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;
1. An in vitro method for producing a population of pancreatic cells comprising the steps of: a) culturing undifferentiated human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, ascorbic acid and a retinoid to obtain a population of cells, wherein said population comprises:(i) greater than 30% of pancreatic endoderm cells that are PDX-1+, NKX6.1+, SOX2− and CDX2−, and(ii) greater than 10% of single hormonal insulin positive cells. 2. An in vitro method for differentiating human pluripotent stem cells comprising the steps of: a) culturing human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells expressing PDX-1, a higher level of NKX6.1, and a lower level of SOX2 as compared to pancreatic foregut cells; andf) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. 3. An in vitro method for differentiating human pluripotent stem cells comprising the steps of: a) culturing human pluripotent stem cells in a medium supplemented with 5 mM to 20 mM glucose, a TGF-β ligand, and a WNT activator, to generate a population of definite endoderm (DE) cells;b) culturing the DE cells in a medium supplemented with 5 mM to 20 mM glucose, and a FGF ligand to generate a population of gut tube cells;c) culturing the gut tube cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a FGF ligand, a PKC activator, a TGF-β ligand, a retinoid, and a gradient of a BMP inhibitor to generate a population of posterior foregut endoderm cells expressing PDX-1 and SOX2;d) culturing the posterior foregut cells in medium supplemented with 5 mM to 20 mM glucose, a PKC activator, a shh inhibitor, a retinoid, and a BMP inhibitor to generate a population of pancreatic foregut cells expressing PDX-1 and NKX6.1, and expressing lower level of SOX2 as compared to the posterior foregut cells;e) culturing the pancreatic foregut cells in medium supplemented with 5 mM to 20 mM glucose, a shh inhibitor, a TGF-β inhibitor, and a retinoid to obtain a population of pancreatic endoderm cells expressing PDX-1, a higher level of NKX6.1, and a lower level of SOX2 as compared to pancreatic foregut cells; andf) differentiating the pancreatic endoderm cells into a pancreatic β-cell population. 4. The method of claim 2, wherein in at least one step the medium is further supplemented with ascorbic acid. 5. The method of claim 4, wherein the pancreatic β cells are single hormonal insulin-producing cells which are also NKX6.1+ and PDX-1+. 6. The method of claim 3, wherein the pancreatic β-cell population is PDX-1+, NKX6.1+, SOX2-, and CDX2-.
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이 특허에 인용된 특허 (7)
D'Amour, Kevin Allen; Agulnick, Alan D.; Baetge, Emmanuel E., Definitive endoderm.
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