Differentiation of human embryonic stem cells into single hormonal insulin positive cells
First Claim
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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.
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Abstract
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.
14 Citations
6 Claims
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1. An in vitro method for producing a population of pancreatic cells comprising the steps of:
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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.
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2. An in vitro method for differentiating human pluripotent stem cells comprising the steps of:
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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. - View Dependent Claims (4, 5)
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3. An in vitro method for differentiating human pluripotent stem cells comprising the steps of:
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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. - View Dependent Claims (6)
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Specification