Methods for production of functional beta cells

1. A method of maturing an immature pancreatic beta cell into a mature pancreatic beta cell expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1 comprising:

culturing the immature pancreatic beta cell in a medium supplemented with one or more of UNC0638, UNC0642, UNC0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568, thereby maturing the immature pancreatic beta cell into the mature beta cell.

2. The method of claim 1 , wherein the medium is further supplemented with i) heparin, ii) N-Acetyl Cysteine, and iii) one or more of T3, T4 and an analogue thereof.

3. The method of claim 1 , wherein the medium lacks an ALK5 inhibitor.

4. The method of claim 1 , wherein the medium is further supplemented with an ALK5 inhibitor.

5. The method of claim 1 , wherein the medium is supplemented with

i) ZM447439;

ii) heparin;

iii) N-Acetyl Cysteine; and

iv) one or more of T3, T4 and an analogue thereof;

and wherein the medium lacks an ALK5 inhibitor.

6. The method of claim 5 , wherein the medium is supplemented with T3.

7. The method of claim 6 , wherein the medium is supplemented with AZT.

8. The method of claim 7 , wherein the medium is supplemented with DEZA.

9. The method of claim 1 , wherein the immature beta cell is obtained by step-wise differentiation of definitive endoderm cells, primitive gut tube cells, foregut endoderm cells, pancreatic endoderm cells, or pancreatic endocrine precursor cells.

10. The method of claim 1 , wherein the method comprises culturing the immature pancreatic beta cell at the air-liquid interface.

11. The method of claim 1 , wherein the method comprises culturing the immature pancreatic beta cell in suspension clusters.

12. A method of differentiating pluripotent stem cells into mature beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A:

a) differentiating pluripotent stem cells into definitive endoderm cells;

b) differentiating the definitive endoderm cells of step a) into primitive gut tube cells;

c) differentiating the primitive gut tube cells of step b) into foregut endoderm cells;

d) differentiating the foregut endoderm cells of step c) into pancreatic endoderm cells;

e) differentiating the pancreatic endoderm cells of step d) into pancreatic endocrine precursor cells;

f) differentiating the pancreatic endocrine precursor cells of step e) into immature beta cells; and

g) culturing the immature beta-cells of step f) in a medium supplemented with one or more of UNC0638, UNC0642, UNC0646, TC-E5003, A366, PF03814735, ZM447439, SB747651A, PFI1, LY303511, MS436, AZT, DEZA, pyroxamide, CI9994 or MC1568 to mature the immature beta cells into the mature beta cells expressing PDX1, NKX6.1, MAFA, UCN3 and SLC2A1.

13. The method of claim 12 , wherein the mature beta cells exhibit glucose-dependent mitochondrial respiration.

14. The method of claim 12 , wherein the mature beta cells exhibit glucose-stimulated insulin secretion.

15. The method of claim 12 , wherein the mature beta cells secrete insulin in multiple phases.

16. The method of claim 12 , wherein the medium is further supplemented with:

i) heparin;

ii) N-Acetyl Cysteine; and

iii) one or more of T3, T4 or an analogue thereof.

17. The method of claim 12 , wherein the medium lacks an ALK5 inhibitor.

18. The method of claim 12 , wherein the medium is further supplemented with T3.

19. The method of claim 12 , wherein the medium is supplemented with

i) ZM447439;

ii) heparin;

iii) N-Acetyl Cysteine; and

iv) one or more of T3, T4 or and an analogue thereof, and

wherein the medium lacks an ALK5 inhibitor.

20. The method of claim 19 , wherein the medium is supplemented with T3.

21. The method of claim 20 , wherein the medium is further supplemented with AZT.

22. The method of claim 21 , wherein the medium is further supplemented with DEZA.

23. The method of claim 12 , wherein the method comprises culturing the immature beta cells at the air-liquid interface.

24. The method of claim 12 , wherein the method comprises culturing the immature beta cells in suspension clusters.

25. The method of claim 12 , wherein step a) comprises differentiating the pluripotent stem cells into the definitive endoderm cells by culturing the pluripotent stem cells in a medium supplemented with MCX compound and GDF-8.

26. The method of claim 12 , wherein step b) comprises differentiating the definitive endoderm cells into the primitive gut tube cells by culturing the definitive endoderm cells in a medium supplemented with FGF7 and ascorbic acid.

27. The method of claim 12 , wherein step c) comprises differentiating the primitive gut tube cells into the foregut endoderm cells by culturing the primitive gut tube cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid.

28. The method of claim 12 , wherein the step d) comprises differentiating the foregut endoderm cells into the pancreatic endoderm cells by culturing the foregut endoderm cells in a medium supplemented with FGF7, retinoic acid, SANT-1, a PKC activator, a BMP inhibitor and ascorbic acid.

29. The method of claim 12 , wherein step e) comprises differentiating the pancreatic endoderm cells into pancreatic endocrine precursor cells by culturing the pancreatic endoderm cells in a medium supplemented with SANT-1, a PKC activator, a BMP inhibitor, and ascorbic acid.

30. The method of claim 29 , wherein the medium is further supplemented one or more of T3, T4 or an analogue thereof.

31. The method of claim 12 , wherein step f) comprises culturing the pancreatic endocrine precursor cells into immature beta-cells by culturing the pancreatic endocrine precursor cells in a medium supplemented with a i) a BMP inhibitor, ii) ascorbic acid, and iii) one or more of T3, T4 and an analogue thereof.

32. The method of claim 31 , wherein the medium is further supplemented with an ALK 5 inhibitor or a gamma secretase inhibitor.

33. The method of claim 12 , wherein step g) comprises culturing immature beta-cells in a medium lacking an ALK5 inhibitor and supplemented with ZM447439, AZT, N-acetyl cysteine, DEZA, Formulation I and one or more of T3, T4 and an analogue thereof.

34. The method of claim 33 , wherein the mature beta cells exhibit glucose-dependent mitochondrial respiration.

35. The method of claim 33 , wherein the mature beta cells exhibit glucose-stimulated insulin secretion.

36. The method of claim 34 , wherein the glucose-dependent mitochondrial respiration has an oxygen consumption rate response following glucose stimulation in the range of about 20% to about 80% over basal oxygen consumption rate.

37. The method of claim 36 , wherein the oxygen consumption rate response occurred at least 15 minutes following glucose stimulation.

38. The method of claim 35 , wherein the glucose-stimulated insulin secretion comprises a bi-phasic insulin secretion in response to glucose stimulation.

39. The method of claim 38 , wherein a first phase of the bi-phasic insulin secretion has at least a four-fold increase to at least an eight-fold increase over baseline secretion, and a second phase has at least a two-fold increase to at least a four-fold increase over the baseline secretion.

40. The method of claim 38 , wherein the insulin secretion occurs at least five minutes to at least ten minutes following the glucose stimulation.

41. The method of claim 6 , wherein the medium is supplemented with 1 nM to 100 nM T3.

42. The method of claim 30 , wherein the medium is supplemented 1 nM to 1 μM T3.

43. The method of claim 12 , wherein the pluripotent stem cells are human H1 or H9 cells.