Methods and compositions for culturing endoderm progenitor cells in suspension

• US 10,266,807 B2
• Filed: 04/03/2014
• Issued: 04/23/2019
• Est. Priority Date: 04/03/2013
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Inventors: Rajesh, Deepika, Burton, Sarah Alice
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US14/244,396
• Publication Number:
  US 20140329321A1
• Time in Patent Office: 1,846 Days
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Provided herein are methods for the in vitro differentiation of induced pluripotent stem cells, which have been expanded and/or maintained under defined conditions, into endodermal precursor cells (EPCs) that are capable of producing mono-hormonal beta cells.

Claims

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.

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.

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

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

.

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.

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

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

.

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