Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
First Claim
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1. An in vitro method for producing pancreatic endocrine cells from human pluripotent stem cells, comprising the steps of:
- a) differentiating human pluripotent stem cells into pancreatic foregut precursor cells; and
b) differentiating the pancreatic foregut precursor cells into pancreatic endocrine cells by culturing the pancreatic foregut precursor cells at an air-liquid interface in i) a medium comprising an ALK5 inhibitor and a thyroid hormone or ii) a medium comprising a thyroid hormone, wherein the thyroid hormone is triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine or a mixture 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 expressing PDX1, NKX6.1, and HB9 by culturing in a culture vessel at the air-liquid interface. The invention also provides for in vivo maturation of cells cultured at the air-liquid interface.
179 Citations
35 Claims
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1. An in vitro method for producing pancreatic endocrine cells from human pluripotent stem cells, comprising the steps of:
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a) differentiating human pluripotent stem cells into pancreatic foregut precursor cells; and b) differentiating the pancreatic foregut precursor cells into pancreatic endocrine cells by culturing the pancreatic foregut precursor cells at an air-liquid interface in i) a medium comprising an ALK5 inhibitor and a thyroid hormone or ii) a medium comprising a thyroid hormone, wherein the thyroid hormone is triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine or a mixture thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 19, 20, 21, 35)
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15. An in vitro method of inducing PDX1, NKX6.1, and HB9 expression in cells derived from human pluripotent stem cells, comprising:
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a) differentiating human pluripotent stem cells into pancreatic foregut precursor cells; and b) differentiating the pancreatic foregut precursor cells into cells expressing PDX1, NKX6.1, and HB9 by culturing the pancreatic foregut precursor cells at an air-liquid interface in a medium comprising i) an ALK5 inhibitor and a thyroid hormone, or ii) in a medium comprising a thyroid hormone, wherein the thyroid hormone is triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine or a mixture thereof. - View Dependent Claims (16, 17, 18, 22, 23, 24, 31)
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- 25. An in vitro method for differentiating human pancreatic foregut precursor cells into pancreatic endocrine cells comprising culturing the human pancreatic foregut precursor cells at an air-liquid interface in i) a medium comprising with an ALK5 inhibitor and a thyroid hormone, or ii) a medium comprising a thyroid hormone, wherein the thyroid hormone is triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine or a mixture thereof.
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32. An in vitro method for producing pancreatic endocrine cells comprising differentiating pancreatic endoderm cells into pancreatic endocrine cells by culturing the pancreatic endoderm cells at an air-liquid interface in i) a medium comprising an ALK5 inhibitor and a thyroid hormone;
- or ii) a medium comprising, the thyroid hormone, wherein the thyroid hormone is triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine or a mixtures thereof.
- View Dependent Claims (33, 34)
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