PLURIPOTENT CELLS

• US 20090269845A1
• Filed: 04/24/2008
• Published: 10/29/2009
• Est. Priority Date: 04/24/2008
• Status: Active Grant

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation), Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rezania, Alireza
• Granted Patent: US 7,939,322 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present invention is directed to pluripotent cells that can be readily expanded in culture on tissue culture substrate that is not pre-treated with protein or an extracellular matrix, and do not require a feeder cell line. The present invention also provides methods to derive the pluripotent cell line from human embryonic stem cells.

Claims

1. A method for deriving a population of cells comprising cells expressing pluripotency markers comprising the steps of:

a. Obtaining cells, and

b. Culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix prior to culturing the cells.

2. The method of claim 1, wherein the cells are human embryonic stem cells.

3. The method of claim 2, wherein the human embryonic stem cells are treated with a Rho kinase inhibitor.

4. The method of claim 1, wherein the cells are cells expressing markers characteristic of the definitive endoderm lineage.

5. The method of claim 1, wherein the human embryonic stem cells are cultured in normoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.

6. The method of claim 1, wherein the human embryonic stem cells are cultured in hypoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.

7. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are cultured in normoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.

8. The method of claim 1, wherein the cells expressing markers characteristic of the definitive endoderm lineage are cultured in hypoxic conditions prior to culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.

9. The method of claim 1, wherein the hypoxic conditions are an 02 level from about 1% to about 20%.

10. The method of claim 1, wherein the hypoxic conditions are an 02 level from about 2% to about 10%.

11. The method of claim 1, wherein the hypoxic conditions are an 02 level of about 3%.

12. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration from about 2% to about 5%.

13. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration of about 2%.

14. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration from about 50 ng/ml to about 100 ng/ml.

15. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration of about 100 ng/ml.

16. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration from about 10 ng/ml to about 20 ng/ml.

17. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration of about 200 ng/ml.

18. The method of claim 16, wherein the Wnt ligand is Wnt-3a.

19. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration from about 25 ng/ml to about 50 ng/ml.

20. The method of claim 1, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration of about 50 ng/ml.

21. The method of claim 1, wherein the cells expressing pluripotency markers express at least one of the pluripotency markers selected from the group consisting of ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, and Tral-81.

22. The method of claim 1, wherein the cells expressing pluripotency markers express markers characteristic of pre-primitive streak cells.

23. The method of claim 1, wherein the cells expressing pluripotency markers express markers characteristic of primitive streak cells.

24. The method of claim 1, wherein the cells expressing pluripotency markers express markers characteristic of mesoendoderm cells.

25. The method of claim 1, wherein the cells expressing pluripotency markers express markers characteristic of definite endoderm cells.

26. A method for deriving a population of cells comprising cells expressing pluripotency markers, comprising the steps of:

a. Culturing human embryonic stem cells,

b. Differentiating the human embryonic stem cells into cells expressing markers characteristic of definitive endoderm cells, and

c. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix prior to culturing the cells.

27. The method of claim 26, wherein the human embryonic stem cells are treated with a Rho kinase inhibitor.

28. The method of claim 26, wherein the human embryonic stem cells are cultured on an extracellular matrix, prior to differentiating the cells.

29. The method of claim 26, wherein the human embryonic stem cells are cultured on a feeder cell layer, prior to differentiating the cells.

30. The method of claim 26, wherein the human embryonic stem cells are differentiated on an extracellular matrix, prior to removing the cells.

31. The method of claim 26, wherein the human embryonic stem cells are differentiated on a feeder cell layer, prior to removing the cells.

32. The method of claim 26, wherein the human embryonic stem cells are cultured and differentiated in normoxic conditions, prior to removing the cells.

33. The method of claim 26, wherein the human embryonic stem cells are cultured and differentiated in hypoxic conditions, prior to removing the cells.

34. The method of claim 26, wherein the hypoxic conditions are an 02 level from about 1% to about 20%.

35. The method of claim 26, wherein the hypoxic conditions are an 02 level from about 2% to about 10%.

36. The method of claim 26, wherein the hypoxic conditions are an 02 level of about 3%.

37. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration from about 2% to about 5%.

38. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration of about 2%.

39. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration from about 50 ng/ml to about 100 ng/ml.

40. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration of about 100 ng/ml.

41. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration from about 10 ng/ml to about 20 ng/ml.

42. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration of about 200 ng/ml.

43. The method of claim 42, wherein the Wnt ligand is Wnt-3a.

44. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration from about 25 ng/ml to about 50 ng/ml.

45. The method of claim 26, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration of about 50 ng/ml.

46. The method of claim 26, wherein the cells expressing pluripotency markers express at least one of the pluripotency markers selected from the group consisting of ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, and Tral-81.

47. The method of claim 26, wherein the cells expressing pluripotency markers express markers characteristic of pre-primitive streak cells.

48. The method of claim 26, wherein the cells expressing pluripotency markers express markers characteristic of primitive streak cells.

49. The method of claim 26, wherein the cells expressing pluripotency markers express markers characteristic of mesoendoderm cells.

50. The method of claim 26, wherein the cells expressing pluripotency markers express markers characteristic of definite endoderm cells.

51. A method for deriving a population of cells comprising cells expressing pluripotency markers comprising the steps of:

a. Culturing human embryonic stem cells, and

b. Removing the cells, and subsequently culturing them under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix prior to culturing the cells.

52. The method of claim 51, wherein the human embryonic stem cells are treated with a Rho kinase inhibitor.

53. The method of claim 51, wherein the human embryonic stem cells are cultured on an extracellular matrix, prior to removing the cells.

54. The method of claim 51, wherein the human embryonic stem cells are cultured on a feeder cell layer, prior to removing the cells.

55. The method of claim 51, wherein the human embryonic stem cells are cultured in normoxic conditions, prior to removing the cells.

56. The method of claim 51, wherein the human embryonic stem cells are cultured a in hypoxic conditions, prior to removing the cells.

57. The method of claim 51, wherein the hypoxic conditions are an 02 level from about 1% to about 20%.

58. The method of claim 51, wherein the hypoxic conditions are an 02 level from about 2% to about 10%.

59. The method of claim 51, wherein the hypoxic conditions are an 02 level of about 3%.

60. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration from about 2% to about 5%.

61. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration of about 2%.

62. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration from about 50 ng/ml to about 100 ng/ml.

63. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration of about 100 ng/ml.

64. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration from about 10 ng/ml to about 20 ng/ml.

65. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration of about 200 ng/ml.

66. The method of claim 65, wherein the Wnt ligand is Wnt-3a.

67. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration from about 25 ng/ml to about 50 ng/ml.

68. The method of claim 51, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing IGF-1 at a concentration of about 50 ng/ml.

69. The method of claim 51, wherein the cells expressing pluripotency markers express at least one of the pluripotency markers selected from the group consisting of ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tral-60, and Tral-81.

70. The method of claim 51, wherein the cells expressing pluripotency markers express markers characteristic of pre-primitive streak cells.

71. The method of claim 51, wherein the cells expressing pluripotency markers express markers characteristic of primitive streak cells.

72. The method of claim 51, wherein the cells expressing pluripotency markers express markers characteristic of mesoendoderm cells.

73. The method of claim 51, wherein the cells expressing pluripotency markers express markers characteristic of definite endoderm cells.

74. A method to expand cells expressing markers characteristic of the definitive endoderm lineage, comprising the steps of:

culturing the cells under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix.

75. The method of claim 74, wherein the cells expressing markers characteristic of the definitive endoderm lineage are derived from pluripotent cells formed by the methods of the present invention.

76. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration from about 0.2% to about 5%.

77. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing serum at a concentration of about 0.5%.

78. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration from about 50 ng/ml to about 100 ng/ml.

79. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing activin-A at a concentration of about 100 ng/ml.

80. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration from about 10 ng/ml to about 20 ng/ml.

81. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a Wnt ligand at a concentration of about 200 ng/ml.

82. The method of claim 80, wherein the Wnt ligand is Wnt-3a.

83. The method of claim 74, wherein the cells are cultured under hypoxic conditions, on a tissue culture substrate that is not pre-treated with a protein or an extracellular matrix, in medium containing a GSK-3B inhibitor.