Methods and compositions for culturing endoderm progenitor cells in suspension

1. A method of producing human self-renewing endoderm progenitor cells comprising:

a) culturing undifferentiated human induced pluripotent stem cells (iPS cells) in a defined, serum-free, feeder cell and feeder cell conditioned media free, medium comprising Activin A or Nodal; and

b) culturing the iPS cells of step a) in defined, serum-free, feeder-cell free medium, the media comprising Activin A, BMP4, VEGF and FGF-2 to produce definitive endoderm (DE) cells, said cells characterized as express both CXCR4 and CD117; and

c) washing the DE cells of step b); and

d) culturing the washed DE cells of step a) with BMP4, VEGF, FGF2, and EGF in defined, serum-free, feeder-cell free medium to selectively promote the growth of self-renewing endoderm progenitor cells (EPC cells), wherein the EPC cells are characterized as express CD34 and HNF4α.

2. The method of claim 1 , further comprising e) culturing the endoderm progenitor cells with islet beta cell promoting growth factors to effect the formation of mono-hormonal pancreatic islet beta cells.

3. The method of claim 1 , wherein the serum-free medium in step a) comprises Activin A.

4. The method of claim 1 , wherein the culturing in step a) is carried out as an adherent culture.

5. The method of claim 1 , wherein the cells from step a) comprise a population of definitive endoderm cells and endoderm progenitor cells.

6. The method of claim 1 , wherein the culture of step b) further comprises TGFβ.

7. The method of claim 1 , wherein the endoderm progenitor cells express CXCR4, CD34, and CD117.

8. The method of claim 7 , wherein the endoderm progenitor cells further express at least one of Sox17, FoxA1, FoxA2, and CD31.

9. The method of claim 1 , wherein the culturing occurs under hypoxic conditions.

10. The method of claim 9 , wherein the pluripotent stem cells are maintained under hypoxic conditions prior to step a).

11. The method of claim 2 , wherein the islet beta cell promoting growth factors comprise an inhibitor of AMP-activated protein kinase (AMPK), a BMP antagonist, a hedgehog inhibitor, a gamma-secretase inhibitor, an ALK5 inhibitor, retinoic acid, FGF10, B27, and Wnt3A or a GSK3 inhibitor.

12. The method of claim 2 , further comprising:

i) culturing the endoderm progenitor cells with a BMP antagonist, Wnt3A, and FGF10 to effect the formation of foregut endoderm cells;

ii) culturing the foregut endoderm cells with B27, a BMP antagonist, a Hedgehog inhibitor, retinoic acid, and FGF10 to effect the formation of pancreatic endoderm cells;

iii) culturing the pancreatic endoderm cells with B27, a BMP antagonist, a gamma-secretase inhibitor, and an ALK5 inhibitor to effect the formation of endocrine precursor cells; and

iv) culturing said endocrine precursor cells with a BMP antagonist, an ALK5 inhibitor, insulin, glucose, and nicotinamide to effect the formation of mono-hormonal pancreatic islet beta cells.

13. The method of claim 12 , wherein the cells comprise an inducible expression cassette encoding Erg1 and wherein step iv) further comprises inducing the cells to express Erg1.

14. The method of claim 12 , wherein the cells comprise an inducible expression cassette encoding Gfi1 and wherein step iv) further comprises inducing the cells to express Gfi1.

15. The method of claim 12 , wherein the mono-hormonal pancreatic islet beta cells are cultured for one to four weeks.

16. The method of claim 15 , wherein the mono-hormonal pancreatic islet beta cells are cultured in a suspension culture.

17. The method of claim 2 , wherein the culturing occurs under hypoxic conditions.

18. The method of claim 2 , wherein the culture is free of MEF feeder cells and MEF-conditioned medium.

19. The method of claim 2 , wherein the mono-hormonal pancreatic islet beta cells express PDX-1, insulin, and C-peptide, but not glucagon.

20. The method of claim 19 , wherein the mono-hormonal pancreatic islet beta cells do not express somatostatin.

21. A method of producing human self-renewing endoderm progenitor cell aggregates comprising:

a) culturing human pluripotent stem cells in defined, feeder-free, serum-free medium comprising Activin A or Nodal, and BMP4 and one or both of VEGF and FGF-2, to effect the induction of endoderm cells; and

b) culturing the endoderm cells in a culture with BMP4, VEGF, FGF2, and EGF or homologs thereof in serum-free medium to effect the formation of self-renewing endoderm progenitor cell (EPC) aggregates.

22. The method of claim 21 , wherein the culture of step b) further comprises Activin A.

23. The method of claim 21 , wherein the culture of step b) further comprises TGFβ.

24. A method of producing human self-renewing endoderm progenitor cells comprising:

a) culturing human induced pluripotent stem (iPS) cells in a defined, feeder-free, serum-free medium comprising Activin A, BMP4 and one or both of VEGF and FGF-2; and

b) culturing the resultant cells from step a) in a culture with Activin A, BMP4, VEGF, FGF2, and EGF in defined, feeder-free, serum-free medium to selectively promote the growth of self-renewing endoderm progenitor cells (EPCs).

25. The method of claim 24 , wherein the culture of step b) further comprises TGFβ.

26. The method of claim 1 , wherein the human pluripotent stem cells cultured in step a) are mouse pluripotent stem cells.

27. The method of claim 1 , wherein the human pluripotent stem cells cultured in step a) are human pluripotent stem cells.

28. The method of claim 21 , wherein the human pluripotent stem cells cultured in step a) are mouse pluripotent stem cells.

29. The method of claim 21 , wherein the human pluripotent stem cells cultured in step a) are human pluripotent stem cells.

30. The method of claim 1 , wherein the culturing step b) is carried out in a suspension culture.

31. The method of claim 21 , wherein the culturing step b) is carried out in a suspension culture.

32. The method of claim 24 , wherein the culturing step b) is carried out in a suspension culture.