METHODS AND COMPOSITIONS FOR CULTURING ENDODERM PROGENITOR CELLS IN SUSPENSION

• US 20140329321A1
• Filed: 04/03/2014
• Published: 11/06/2014
• Est. Priority Date: 04/03/2013
• Status: Active Application

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: RAJESH, Deepika, BURTON, Sarah Alice
• Granted Patent: US 10,266,807 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• 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
• 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 self-renewing endoderm progenitor cells comprising:

a) culturing pluripotent stem cells in serum-free medium comprising Activin A or Nodal; and

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

2. The method of claim 1, further comprising c) 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.

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

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

11. The method of claim 1, wherein the suspension culture of step b) further comprises Activin A.

12. The method of claim 1, wherein the suspension culture of step b) further comprises TGFI3.

22. The method of claim 1, wherein the suspension cultures in step b) are maintained in spinner flasks.

27. The method of claim 1, wherein the endoderm progenitor cells express CXCR4, CD34, and CD117 (c-KIT).

28. The method of claim 27, wherein the endoderm progenitor cells further express at least one of Sox17, FoxA1, FoxA2, and CD31.

29. The method of claim 1, wherein the formation of self-renewing endoderm progenitor cells does not require the presence of MEF feeder layers or MEF-conditioned medium.

30. The method of claims 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

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

33. The method of claim 32, wherein the pluripotent stem cells are maintained under hypoxic conditions prior to step a).

34. The method of claim 2, wherein the endoderm progenitor cells are purified prior to the culturing of step c).

35. The method of claim 2, wherein the islet beta cell promoting growth factors comprise a potent 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.

36. 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.

37. The method of claim 36, wherein the cells comprise an inducible expression cassette encoding Erg1 and wherein step iv) further comprises inducing the cells to express Erg1.

38. The method of claim 36, wherein the cells comprise an inducible expression cassette encoding Gfi1 and wherein step iv) further comprises inducing the cells to express Gfi1.

46. The method of claim 36, wherein the mono-hormonal pancreatic islet beta cells are cultured for one to four weeks.

47. The method of claim 46, wherein the mono-hormonal pancreatic islet beta cells are cultured in a suspension culture.

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

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

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

53. The method of claim 52, wherein the mono-hormonal pancreatic islet beta cells do not express somatostatin.

4-7. -7. (canceled)

10. (canceled)

13-21. -21. (canceled)

23-26. -26. (canceled)

31. (canceled)

39-45. -45. (canceled)

48-49. -49. (canceled)

54-57. -57. (canceled)

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

a) culturing pluripotent stem cells in serum-free medium comprising Activin A or Nodal to effect the induction of endoderm cells; and

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

60. The method of claim 58, wherein the suspension culture of step b) further comprises Activin A.

61. The method of claim 58, wherein the suspension culture of step b) further comprises TGFI3.

59. (canceled)

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

a) culturing pluripotent stem cells in serum-free medium comprising Activin A; and

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

63. The method of claim 62, wherein the suspension culture of step b) further comprises TGFI3.