Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells
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.
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.
2. The method of claim 1 , wherein the pancreatic endocrine cells are positive for NKX6.1, PDX1, and HB9.
3. The method of claim 1 , wherein the pancreatic endocrine cells co-express NKX6.1 and chromogranin-A.
4. The method of claim 3 , wherein at least thirty percent of the pancreatic endocrine cells co-express NKX6.1 and chromogranin-A.
5. The method of claim 1 , wherein the pancreatic endocrine cells co-express NKX6.1 and insulin.
6. The method of claim 5 , wherein at least thirty percent of the pancreatic endocrine cells co-express NKX6.1 and insulin.
7. The method of claim 1 , wherein the method comprises culturing the pancreatic foregut precursor cells at the air-liquid interface in a medium comprising triiodothyronine and an ALK5 inhibitor.
8. The method of claim 1 , wherein culturing pancreatic foregut precursor cells at the air-liquid interface comprises culturing the pancreatic foregut precursor cells on a porous substrate.
9. The method of claim 8 , wherein the porous substrate is uncoated.
10. The method of claim 1 , wherein said ALK5 inhibitor is selected from the group consisting of: SD208, TGF-B inhibitor SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inh V, TGF-β receptor inh I, TGF-β receptor inh I TGF-β receptor inh IV, TGF-β receptor inh VII, TGF-β receptor inh VIII, TGF-β receptor inh II, TGF-β receptor inh VI, and TGF-β receptor inh III.
11. The method of claim 1 , wherein said ALK5 inhibitor is ALK5 inhibitor II.
12. The method of claim 3 , wherein the method increases the number of NXK6.1 positive cells that co-express insulin, chromogranin-A or both chromogranin-A and insulin.
13. The method of claim 1 , wherein the human pluripotent stem cells are of non-embryonic origin.
14. The method of claim 1 , wherein the pancreatic endocrine cells are β cells.
15. An in vitro method of inducing PDX1, NKX6.1, and HB9 expression in cells derived from human pluripotent stem cells, comprising:
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.
16. The method of claim 15 , wherein the human pluripotent stem cells are of non-embryonic origins.
17. The method of claim 15 , wherein the ALK5 inhibitor is ALK5 inhibitor II.
18. The method of claim 15 , wherein the medium further comprises one or more of retinoic acid, ascorbic acid, SANT-1 or LDN-193189.
19. The method of claim 1 , wherein the method comprises culturing pancreatic foregut cells at the air-liquid interface in the medium comprising the thyroid hormone triiodothyronine, thyroxine, an analogue of triiodothyronine, an analogue of thyroxine, or a and mixture thereof.
20. The method of claim 1 , wherein the method comprises culturing pancreatic foregut precursor cells at the air-liquid interface in the medium comprising the ALK5 inhibitor and the thyroid hormone.
21. The method of claim 20 , wherein the thyroid hormone is selected from triiodothyronine, thyroxine, analogues of triiodothyronine, analogues of thyroxine and mixtures thereof.
22. The method of claim 15 , wherein the method comprises differentiating pancreatic foregut precursor cells into cells expressing PDX1, NKX6.1, and HB9 by culturing the pancreatic foregut precursor cells at the air-liquid interface 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.
23. The method of claim 15 , wherein the method comprises differentiating pancreatic precursor foregut cells into cells expressing PDX1, NKX6.1, and HB9 by culturing the pancreatic foregut precursor cells at the air-liquid interface in medium comprising both an ALK5 inhibitor and a thyroid hormone.
24. The method of claim 23 , wherein the thyroid hormone is selected from triiodothyronine, thyroxine, analogues of triiodothyronine, analogues of thyroxine and mixtures thereof.
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.
26. The method of claim 25 , wherein the method comprises culturing the human pancreatic foregut precursor cells at the air-liquid interface in the medium comprising the thyroid hormone selected from triiodothyronine, thyroxine, the analogue of triiodothyronine, the analogue of thyroxine or the mixture thereof.
27. The method of claim 25 , wherein the method comprises culturing the human pancreatic foregut precursor cells at the air-liquid interface in the medium comprising both the ALK5 inhibitor and thyroid hormone.
28. The method of claim 25 , wherein the medium further comprises one or more of a BMP receptor inhibitor, retinoic acid, ascorbic acid, heparin; and zinc sulfate.
29. The method of claim of 25 , wherein the medium further comprising (i) a BMP receptor inhibitor, (ii) retinoic acid, or (iii) a BMP receptor inhibitor and retinoic acid.
30. The method of claim 27 , wherein the medium further comprises (i) a BMP receptor inhibitor, (ii) retinoic acid, or (iii) a BMP receptor inhibitor and retinoic acid.
31. The method of claim 15 , wherein the ALK5 inhibitor, the thyroid hormone, or both the ALK5 inhibitor and the thyroid hormone are present in an amount sufficient to induce expression of PDX1 , NKX6.1 and HB9.
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.
33. The method of claim 32 , wherein the method comprises culturing in i) the medium comprising the ALK5 inhibitor and the thyroid hormone.
34. The method of claim 32 , wherein the method comprises culturing in ii) the medium comprising the thyroid hormone.
35. The method of claim 1 , wherein the medium further comprises one or more of retinoic acid, SANT-1 , and LDN-139189.