Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof
Disclosed herein are methods for generating SC-β cells using chemically defined, completely serum free media, and isolated populations of SC-β cells for use in various applications, such as cell therapy.
1. An in vitro method of generating at least one mature β cell that exhibits a glucose-stimulated insulin secretion (GSIS) response in vitro, the method comprising:
culturing glucose-unresponsive PDX1+, NKX6.1+ pancreatic progenitor cells in a medium that is supplemented with at least a transforming growth factor β (TGF-β) signaling pathway inhibitor, a sonic hedgehog pathway inhibitor, and a thyroid hormone signaling pathway activator, thereby generating glucose-unresponsive PDX1+, NKX6.1+, insulin-positive endocrine cells, and
culturing said glucose-unresponsive PDX1+, NKX6.1+, insulin-positive endocrine cells in a serum free medium, thereby generating the at least one mature β cell that exhibits a GSIS response in vitro.
2. The method of claim 1 , further comprising washing the glucose-unresponsive PDX1+, NKX6.1, insulin-positive endocrine cells with CMRLM before treatment with the serum free medium.
3. The method of claim 1 , wherein the serum free medium comprises CMRL 1066 with 2.2 g/L NaHCO 3 , but without HEPES, 1% Pen/Strep, and 2 mM Glutamax.
4. The method of claim 1 , wherein the serum free medium is supplemented with FAF-BSA, human APO-transferrin, trace minerals A+B, ZnSO4, ethanolamine, pyruvate, and defined lipids.
5. The method of claim 1 , comprising culturing the glucose-unresponsive PDX1+, NKX6.1+, insulin-positive endocrine cells in the serum free medium during a 14 day differentiation period, wherein the serum free medium is replenished every second day during the 14 day differentiation period.
6. The method of claim 1 , wherein the medium supplemented with at least a transforming growth factor β (TGF-β) signaling pathway inhibitor, a sonic hedgehog pathway inhibitor, and a thyroid hormone signaling pathway activator is further supplemented with at least an EGF family growth factor, a retinoic signaling pathway activator, a γ-secretase inhibitor, and a protein kinase inhibitor.
7. The method of claim 6 , further comprising washing the glucose-unresponsive PDX1+, NKX6.1+ cells or the glucose-unresponsive PDX1+, NKX6.1+, insulin-positive endocrine cells with BE5.
8. The method of claim 6 , wherein the medium supplemented with at least a transforming growth factor β (TGF-β) signaling pathway inhibitor, a sonic hedgehog pathway inhibitor, and a thyroid hormone signaling pathway activator is not supplemented with Vitamin C.
9. The method of claim 6 , wherein the glucose-unresponsive PDX1+, NKX6.1+ cells are obtained by differentiating a population of PDX1+ pancreatic progenitor cells in a medium that is supplemented with at least a FGF family growth factor, a TGFβ superfamily member, a retinoic signaling pathway activator, a sonic hedgehog pathway inhibitor, and a ROCK inhibitor.
10. The method of claim 9 , wherein at least 50% of cells in the population of PDX1+ pancreatic progenitor cells express PDX1 before the treatment with the medium supplemented with at least a FGF family growth factor, a TGFβ superfamily member, a retinoic signaling pathway activator, a sonic hedgehog pathway inhibitor, and a ROCK inhibitor.
11. The method claim 9 , wherein the population of PDX1+ pancreatic progenitor cells are obtained by differentiating a population of FOXA2+, SOX2+ foregut endoderm cells by treatment with a serum free medium that is supplemented with at least i) a FGF family growth factor, ii) a retinoic acid signaling activator, iii) a sonic hedgehog pathway inhibitor, iv) a BMP pathway signaling inhibitor, v) a protein kinase C (PKC) activator, and vi) a ROCK inhibitor.
12. The method of claim 11 , wherein the serum-free medium supplemented with at least a FGF family growth factor, a retinoic acid signaling activator, a sonic hedgehog pathways inhibitor, a BMP pathway signaling inhibitor, a protein kinase C (PKC) activator, and a ROCK inhibitor comprises MCDB131, 1% Pen/Strep, 1.23 g/L NaHCO 3 and 2 mM Glutamax.
13. The method of claim 11 , wherein the serum-free medium supplemented with at least a FGF family growth factor, a retinoic acid signaling activator, a sonic hedgehog pathway inhibitor, a BMP pathway signaling inhibitor, a protein kinase C (PKC) activator, and a ROCK inhibitor is further supplemented with FAF-BSA, ITS-X, Glucose, and Vitamin C.
14. The method of claim 11 , comprising differentiating the population of FOXA2+, SOX2+ foregut endoderm cells during a 2 day differentiation period, wherein the serum-free medium supplemented with at least a FGF family growth factor, a retinoic acid signaling activator, a sonic hedgehog pathway inhibitor, a BMP pathway signaling inhibitor, a protein kinase C (PKC) activator, and a ROCK inhibitor is replenished without the PKC activator on the second day of the 2 day differentiation period.
15. The method of claim 11 , wherein at least 50% of cells in the population of FOXA2+, SOX2+ foregut endoderm cells express FOXA2 and/or SOX2 before treatment with the serum-free medium supplemented with at least a FGF family growth factor, a retinoic acid signaling activator, a sonic hedgehog pathway inhibitor, a BMP pathway signaling inhibitor, a protein kinase C (PKC) activator, and a ROCK inhibitor.
16. The method of claim 11 , wherein the population of FOXA2+, SOX2+ foregut endoderm cells are obtained by differentiating a population of SOX17+ definitive endoderm cells by treatment with a serum free medium that is supplemented with at least a FGF family growth factor.
17. The method of claim 16 , wherein the medium supplemented with at least a FGF family growth factor comprises MCDB131, 1% Pen/Strep, 1.23 g/L NaHCO 3 and 2 mM Glutamax.
18. The method of claim 16 , wherein the medium supplemented with at least a FGF family growth factor is further supplemented with FAF-BSA, ITS-X, Glucose, and Vitamin C.
19. The method of claim 16 , comprising differentiating the population of SOX17+ definitive endoderm cells in a 3 day differentiation period, wherein the population of SOX17+ definitive endoderm cells is treated with the medium supplemented with at least a FGF family growth factor on day 1 and day 3 of the 3 day differentiation period.
20. The method of claim 16 , wherein at least 50% of cells in the population of SOX17+ definitive endoderm cells express SOX17 before treatment with the medium supplemented with at least a FGF family growth factor.
21. The method of claim 16 , wherein between at least 25% and 75% of the SOX17+ definitive endoderm cells in the population of SOX17+ definitive endoderm cells differentiate into FOXA2+, SOX2+ foregut endoderm cells.
22. The method of claim 16 , wherein the population of FOXA2+, SOX2+ foregut endoderm cells are obtained by differentiating a population of pluripotent stem cells by treatment with a serum free medium that is supplemented with at least a TGFβ superfamily member and a WNT signaling pathway activator.
23. The method of claim 22 , wherein the medium supplemented with at least a TGFβ superfamily member and a WNT signaling pathway activator comprises MCDB131, 1% Pen/Strep, 2.5 g/L NaHCO 3 and 2 mM Glutamax.
24. The method of claim 22 , wherein the medium supplemented with at least a TGFβ superfamily member and a WNT signaling pathway activator is further supplemented with FAF-BSA, ITS-X, Glucose, and Vitamin C.
25. The method of claim 22 , comprising differentiating the population of pluripotent stem cells in a 3 day differentiation period, wherein the population of pluripotent stem cells is treated on day 1 with the WNT signaling pathway activator and on day 2 without the WNT signaling pathway activator during the 3 day differentiation period.
26. The method of claim 16 , wherein between at least 25% and 75% of the pluripotent stem cells in the population of pluripotent stem cells differentiate into SOX17+ definitive endoderm cells.