Use of small molecules to enhance MAFA expression in pancreatic endocrine cells
First Claim
1. An in vitro cell culture comprising (i) a population of human pancreatic endocrine cells, in which at least 10% of the differentiated cells express insulin, PDX1, NKX6.1, and MAFA, and (ii) a differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof, wherein the population is obtained by a step-wise differentiation protocol comprising:
- (a) culturing human pluripotent stem cells in a medium supplemented with either (i) activin A and wnt3A or (ii) GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜
2,6˜
.1-8,12˜
]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to obtain definitive endoderm cells;
(b) differentiating the definitive endoderm cells into pancreatic endocrine cells; and
(c) culturing the pancreatic endocrine cells in the differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof.
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Abstract
The present invention provides methods, cell cultures and differentiation media to promote differentiation of pluripotent stem cells to pancreatic endocrine cells of a mature phenotype. The resulting pancreatic endocrine cells express single hormonal insulin, PDX1, NKX6.1, and MAFA. In one or more differentiation stages, culturing may be carried out in a culture vessel at the air-liquid interface.
168 Citations
20 Claims
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1. An in vitro cell culture comprising (i) a population of human pancreatic endocrine cells, in which at least 10% of the differentiated cells express insulin, PDX1, NKX6.1, and MAFA, and (ii) a differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof, wherein the population is obtained by a step-wise differentiation protocol comprising:
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(a) culturing human pluripotent stem cells in a medium supplemented with either (i) activin A and wnt3A or (ii) GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜
2,6˜
.1-8,12˜
]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to obtain definitive endoderm cells;(b) differentiating the definitive endoderm cells into pancreatic endocrine cells; and (c) culturing the pancreatic endocrine cells in the differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof. - View Dependent Claims (12)
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- 2. A method of inducing MAFA expression in cells comprising treating human pancreatic endocrine cells with a medium supplemented with an inhibitor selected from the group consisting of an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof, wherein the treatment with the inhibitor induces MAFA expression.
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8. An in vitro cell culture comprising (i) a population human pancreatic endocrine cells, in which at least 10% of the differentiated cells express insulin, PDX1, NKX6.1, and MAFA, and (ii) a differentiation medium supplemented with an RSK inhibitor, wherein the population is obtained by a step-wise differentiation protocol comprising:
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(a) culturing human pluripotent stem cells in a medium supplemented with either (i) activin A and wnt3A or (ii) GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜
2,6˜
.1˜
8,12˜
]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to obtain definitive endoderm cells;(b) differentiating the definitive endoderm cells into pancreatic endocrine cells; and (c) culturing the pancreatic endocrine cells in the differentiation medium supplemented with the RSK inhibitor. - View Dependent Claims (9)
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10. An in vitro cell culture comprising (i) a population human pancreatic endocrine cells, in which at least 10% of the differentiated cells express insulin, PDX1, NKX6.1, and MAFA, and (ii) a differentiation medium supplemented with an inhibitor of protein methyltransferase DOT1L, wherein the population is obtained by a step-wise differentiation protocol comprising:
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(a) culturing human pluripotent stem cells in a medium supplemented with either (i) activin A and wnt3A or (ii) GDF-8 and 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜
2,6˜
.1˜
8,12˜
]heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-nonaen-16-one to obtain definitive endoderm cells;(b) differentiating the definitive endoderm cells into pancreatic endocrine cells; and (c) culturing the pancreatic endocrine cells in the differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof. - View Dependent Claims (11)
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- 13. A method for inducing MAFA expression in pancreatic endocrine cells comprising culturing the human foregut endoderm cells with an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof to obtain mature pancreatic endocrine cells which express MAFA.
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14. An in vitro cell culture comprising (i) a population human pancreatic endocrine cells, in which at least 10% of the differentiated cells express insulin, PDX1, NKX6.1, and MAFA, and (ii) a differentiation medium supplemented with L-alanyl-glutamine and an inhibitor selected from the group consisting of an aurora kinase inhibitor, an RSK inhibitor, an inhibitor of protein methyltransferase DOT1L, and combinations thereof, wherein the population is obtained by a step-wise differentiation protocol which comprises culturing human pancreatic endocrine cells in the differentiation medium.
Specification