IP Library › Granted Patent US 8,017,393

Granted Patent B2

US 8,017,393 · App. 11/787,262 · Granted Sep 13, 2011

Hemangio-colony forming cells

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Quick Facts

Patent No.: US 8,017,393
App. No.: 11/787,262
Granted: Sep 13, 2011
Kind: B2

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Abstract

Methods of generating and expanding human hemangio-colony forming cells in vitro and methods of expanding and using such cells are disclosed. The methods permit the production of large numbers of hemangio-colony forming cells as well as derivative cells, such as hematopoietic and endothelial cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications.

Claims (74)

1. A method for generating and expanding CD34 − CD31 − human hemangio-colony forming cells in vitro, said method comprising the steps of:

(a) culturing a cell culture comprising human embryo-derived cells in serum-free media in the presence of vascular endothelial growth factor (VEGF) and bone morphogenic protein 4 (BMP-4) in an amount sufficient to induce the differentiation of said embryo-derived cells into embryoid bodies;

(b) adding basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), stem cell factor (SCF), Flt-3L (FL), thrombopoietin (TPO), and tPTD-HOXB4 to said culture comprising embryoid bodies and continuing to culture said culture in serum-free media;

(d) culturing a cell culture comprising said single cells in serum-free media in the presence of insulin, transferrin, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), bone morphogenic protein 4 (BMP-4), and tPTD-HOXB4 in an amount sufficient to expand CD34 − CD31 − human hemangio-colony forming cells in said culture comprising said single cells,

wherein said embryo-derived cells, embryoid bodies and CD34 − CD31 − hemangio-colony forming cells are grown in serum-free media throughout steps (a)-(d) of said method.

2. The method according to claim 1 , wherein the VEGF and BMP-4 are added to the cell culture of step (a) within 0-48 hours of initiation of said cell culture.

3. The method according to claim 1 , wherein the VEGF is added to the cell culture of step (a) within 0-48 hours of initiation of said cell culture.

4. The method according to claim 1 , wherein the BMP-4 is added to the cell culture of step (a) within 0-48 hours of initiation of said cell culture.

5. The method according to claim 1 , wherein the concentrations in step (a) of VEGF is 20 or 25-100 nm/ml, and BMP-4 is 15 or 25-100 ng/ml.

6. The method according to claim 1 , wherein the concentrations in step (a) of VEGF is 20 ng/ml, and BMP-4 is 15 ng/ml.

7. The method according to claim 1 , wherein the concentrations in step (a) of VEGF is 25-100 ng/ml, and BMP-4 is 25-100 ng/ml.

8. The method according to claim 1 , wherein the concentration in step (a) of VEGF is 20 ng/ml.

9. The method according to claim 1 , wherein the concentration in step (a) of VEGF is 50 ng/ml.

10. The method according to claim 1 , wherein the concentration in step (a) of VEGF is 25-100 ng/ml.

11. The method according to claim 1 , wherein the concentration in step (a) of BMP-4 is 15 ng/ml.

12. The method according to claim 1 , wherein the concentration in step (a) of BMP-4 is 50 ng/ml.

13. The method according to claim 1 , wherein the concentration in step (a) of BMP-4 is 25-100 ng/ml.

14. The method according to claim 1 , wherein in step (b) the bFGF, VEGF, BMP-4, SCF, FL, TPO and tPTD-HOXB4 are added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

15. The method according to claim 1 , wherein in step (b) the bFGF is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

16. The method according to claim 1 , wherein in step (b) the VEGF is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

17. The method according to claim 1 , wherein in step (b) the BMP-4 is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

18. The method according to claim 1 , wherein in step (b) the SCF is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

19. The method according to claim 1 , wherein in step (b) the FL is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

20. The method according to claim 1 , wherein in step (b) the TPO is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

21. The method according to claim 1 , wherein in step (b) the tPTD-HOXB4 is added to said culture comprising embryoid bodies within 48-72 hours from the start of step (a).

22. The method according to claim 1 , wherein the concentration in step (b) of VEGF is 20 or 25-100 ng/ml, BMP-4 is 15 or 25-100 ng/ml, SCF is 20 or 50 ng/ml, FL is 10-50 ng/ml, TPO is 20 or 50 ng/ml, and tPTD-HOXB4 is 1.5-5 U/ml.

23. The method according to claim 1 , wherein the concentration in step (b) of VEGF is 50 ng/ml, BMP-4 is 50 ng/ml, SCF is 20 ng/ml, FL is 50 ng/ml, TPO is 50 ng/ml, and tPTD-HOXB4 is 1.5-5 U/ml.

24. The method according to claim 1 , wherein the concentration in step (b) of VEGF is 20 ng/ml.

25. The method according to claim 1 , wherein the concentration in step (b) of VEGF is 50 ng/ml.

26. The method according to claim 1 , wherein the concentration in step (b) of BMP-4 is 25-100 ng/ml.

27. The method according to claim 1 , wherein the concentration in step (b) of BMP-4 is 15 ng/ml.

28. The method according to claim 1 , wherein the concentration in step (b) of BMP-4 is 50 ng/ml.

29. The method according to claim 1 , wherein the concentration in step (b) of BMP-4 is 25-100 ng/ml.

30. The method according to claim 1 , wherein the concentration in step (b) of SCF is 20 ng/ml.

31. The method according to claim 1 , wherein the concentration in step (b) of SCF is 50 ng/ml.

32. The method according to claim 1 , wherein the concentration in step (b) of FL is 20 ng/ml.

33. The method according to claim 1 , wherein the concentration in step (b) of FL is 50 ng/ml.

34. The method according to claim 1 , wherein the concentration in step (b) of FL is 10-50 ng/ml.

35. The method according to claim 1 , wherein the concentration in step (b) of TPO is 20 ng/ml.

36. The method according to claim 1 , wherein the concentration in step (b) of TPO is 50 ng/ml.

37. The method according to claim 1 , wherein the concentration in step (b) of tPTD-HOXB4 is 1.5-5 U/ml.

38. The method according to claim 1 , wherein erythropoietin (EPO) is further added during step (b).

39. The method according to claim 38 , wherein the concentration in step (b) of EPO is 3-6 U/ml.

40. The method according to claim 1 , wherein in step (b) the bFGF, VEGF, BMP-4, SCF, FL, TPO, and tPTD-HOXB4 are added to said culture multiple times throughout step (b).

41. The method according to claim 1 , wherein the bFGF, VEGF, BMP-4, SCF, FL, TPO, and tPTD-HOXB4 are added to said culture in step (b) multiple times throughout step (b), and wherein the VEGF, BMP-4, SCF, and tPTD-HOXB4 are added to said culture in step (d) multiple times throughout step (d).

42. The method according to claim 1 , wherein in step (b) the bFGF, VEGF, BMP-4, SCF, FL, TPO, and tPTD-HOXB4 are added once a day.

43. The method according to claim 1 , wherein in step (b) the bFGF, VEGF, BMP-4, SCF, FL, TPO, and tPTD-HOXB4 are added once every other day.

44. The method according to claim 1 , wherein the concentrations in step (d) of insulin is 10 μg/ml, transferrin is 200 μg/ml, GM-CSF is 20 μg/ml, IL-3 is 20 ng/ml, IL-6 is 10 or 20 ng/ml, G-CSF is 20 ng/ml, EPO is 3-6 U/ml, SCF is 20 or 50 ng/ml, VEGF is 20 or 25-100 ng/ml, BMP-4 is 15 or 25-100 ng/ml, and tPTD-HOXB4 is 1.5-5 U/ml.

45. The method according to claim 1 , wherein the concentrations in step (d) of insulin is 10 μg/ml, transferrin is 200 μg/ml, GM-CSF is 20 μg/ml, IL-3 is 20 ng/ml, IL-6 is 10 ng/ml, G-CSF is 20 ng/ml, EPO is 3 U/ml, SCF is 20 ng/ml, VEGF is 20 ng/ml, BMP-4 is 15 ng/ml, and tPTD-HOXB4 is 1.5-5 U/ml.

46. The method according to claim 1 , wherein the concentrations in step (d) of insulin is 10 μg/ml, transferrin is 200 μg/ml, GM-CSF is 20 μg/ml, IL-3 is 20 ng/ml, IL-6 is 20 ng/ml, G-CSF is 20 ng/ml, EPO is 3-6 U/ml, SCF is 50 ng/ml, VEGF is 50 ng/ml, BMP-4 is 50 ng/ml, and tPTD-HOXB4 is 1.5-5 U/ml.

47. The method according to claim 1 , wherein the concentrations in step (d) of insulin is 10 μg/ml.

48. The method according to claim 1 , wherein the concentrations in step (d) of transferrin is 200 μg/ml.

49. The method according to claim 1 , wherein the concentrations in step (d) GM-CSF is 20 μg/ml.

50. The method according to claim 1 , wherein the concentrations in step (d) of IL-3 is 20 ng/ml.

51. The method according to claim 1 , wherein the concentrations in step (d) of IL-6 is 10 ng/ml.

52. The method according to claim 1 , wherein the concentrations in step (d) of IL-6 is 20 ng/ml.

53. The method according to claim 1 , wherein the concentrations in step (d) of G-CSF is 20 ng/ml.

54. The method according to claim 1 , wherein the concentrations in step (d) of EPO is 3-6 U/m.

55. The method according to claim 1 , wherein the concentrations in step (d) of SCF is 20 ng/ml.

56. The method according to claim 1 , wherein the concentrations in step (d) of SCF is 50 ng/ml.

57. The method according to claim 1 , wherein the concentrations in step (d) of VEGF is 20 ng/ml.

58. The method according to claim 1 , wherein the concentrations in step (d) of VEGF is 50 ng/ml.

59. The method according to claim 1 , wherein the concentrations in step (d) of VEGF is 25-100 ng/ml.

60. The method according to claim 1 , wherein the concentrations in step (d) of BMP-4 is 15 ng/ml.

61. The method according to claim 1 , wherein the concentrations in step (d) of BMP-4 is 50 ng/ml.

62. The method according to claim 1 , wherein the concentrations in step (d) of BMP-4 is 25-100 ng/ml.

63. The method according to claim 1 , wherein the concentrations in step (d) of tPTD-HOXB4 is 1.5-5 U/ml.

64. The method according to claim 1 , wherein the insulin, transferrin, GM-CSF, IL-3, IL-6, G-CSF, EPO, SCF, VEGF, BMP-4, and tPTD-HOXB4 are added to said culture in step (d) multiple times throughout step (d).

65. The method according to claim 1 , wherein the insulin, transferrin, GM-CSF, IL-3, IL-6, G-CSF, EPO, SCF, VEGF, BMP-4, and tPTD-HOXB4 are added once a day in step (d).

66. The method according to claim 1 , wherein the insulin, transferrin, GM-CSF, IL-3, IL-6, G-CSF, EPO, SCF, VEGF, BMP-4, and tPTD-HOXB4 are added once every other day in step (d).

67. The method according to claim 1 , further comprising, following step (d), purifying said CD34 − CD31 − human hemangio-colony forming cells from said culture.

68. The method according to claim 67 , wherein said purifying said hemangio-colony forming cells comprises using immunoaffinity column chromatography with an anti-CD71 antibody.

69. The method according to any one of claims 1 - 66 , further comprising, following step (d), isolating said human hemangio-colony forming cells from said culture.

Assignments (2)

CHANGE OF NAME Recorded May 31, 2017

From: OCATA THERAPEUTICS, INC.

To: ASTELLAS INSTITUTE FOR REGENERATIVE MEDICINE

Reel/Frame 042634/0795 →

CHANGE OF NAME Recorded Feb 10, 2015

From: ADVANCED CELL TECHNOLOGY, INC.

To: OCATA THERAPEUTICS, INC.

Reel/Frame 034944/0418 →