Serum-free in vitro directed differentiation protocol for generating stem cell-derived beta cells and uses thereof

• US 10,443,042 B2
• Filed: 12/18/2015
• Issued: 10/15/2019
• Est. Priority Date: 12/18/2014
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Melton, Douglas A., Gurtler, Mads
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US14/975,158
• Publication Number:
  US 20160177267A1
• Time in Patent Office: 1,397 Days
• CPC Class Codes: C12N 2500/12 Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
• CPC Class Codes: C12N 2500/22 Zinc; Zn chelators insulin-zinc complexes C12N2501/33
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

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.

Claims

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 NaHCO₃, 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 NaHCO₃ 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 NaHCO₃ 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 NaHCO₃ 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.