Nuclear reprogramming factor and induced pluripotent stem cells

• US 20090047263A1
• Filed: 06/13/2008
• Published: 02/19/2009
• Est. Priority Date: 12/13/2005
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Nakagawa, Masato, Yamanaka, Shinya, Takahashi, Kazutoshi
• Granted Patent: US 8,278,104 B2
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention relates to a nuclear reprogramming factor having an action of reprogramming a differentiated somatic cell to derive an induced pluripotent stem (iPS) cell. The present invention also relates to the aforementioned iPS cells, methods of generating and maintaining iPS cells, and methods of using iPS cells, including screening and testing methods as well as methods of stem cell therapy. The present invention also relates to somatic cells derived by inducing differentiation of the aforementioned iPS cells.

Claims

1. A pluripotent stem cell induced by reprogramming a somatic cell, wherein the reprogramming is performed in the absence of eggs, embryos, or embryonic stem (ES) cells.

2. The pluripotent stem cell of claim 1, wherein the somatic cell is a human cell.

3. The pluripotent stem cell of claim 1, wherein the somatic cell is a mouse cell.

4. The pluripotent stem cell of claim 1, wherein the reprogramming comprises contacting the somatic cell with a nuclear reprogramming factor.

5. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises at least one gene product.

6. The pluripotent stem cell of claim 5, wherein the nuclear reprogramming factor comprises a protein.

7. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor is encoded by a gene contained in a recombinant vector introduced into the cell.

8. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises a gene product of an Oct family gene.

9. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises a gene product of a Klf family gene.

10. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises a gene product of a Myc family gene.

11. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises a gene product of a Sox family gene.

12. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Myc family gene.

13. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Sox family gene.

14. The pluripotent stem cell of claim 13, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, and Sox2.

15. The pluripotent stem cell of claim 14, wherein the nuclear reprogramming factor further comprises one or more gene products of a Sal14 gene.

16. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, a Myc family gene, and a Sox family gene.

17. The pluripotent stem cell of claim 16, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, c-Myc, and Sox2.

18. The pluripotent stem cell of claim 17, wherein the nuclear reprogramming factor further comprises one or more gene products of a Sal14 gene.

19. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28.

20. The pluripotent stem cell of claim 4, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, together with a cytokine.

21. The pluripotent stem cell of claim 20, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

22. The pluripotent stem cell of claim 21, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

23. The pluripotent stem cell of claim 20, wherein the nuclear reprogramming factor further comprises a Myc family gene.

24. The pluripotent stem cell of claim 20, wherein the nuclear reprogramming factor further comprises a gene product of a Sox family gene.

25. The pluripotent stem cell of claim 23, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

26. The pluripotent stem cell of claim 25, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

27. The pluripotent stem cell of claim 24, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

28. The pluripotent stem cell of claim 16, wherein the nuclear reprogramming factor further comprises a gene product of the TERT gene.

29. The pluripotent stem cell of claim 16, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmi1.

30. The pluripotent stem cell of claim 16, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

Fbx15, Nanog, ERas, ECAT15-2, Tc11, and β

-catenin.

31. The pluripotent stem cell of claim 16, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, and Grb2.

74. A somatic cell derived by inducing differentiation of the pluripotent stem cell according to claim 1.

32. A method for preparing an induced pluripotent stem cell by nuclear reprogramming of a somatic cell, which comprises contacting a nuclear reprogramming factor with the somatic cell to obtain an induced pluripotent stem cell.

33. The method of claim 32, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Sox family gene.

34. The method of claim 33 wherein the nuclear reprogramming factor further comprises a cytokine.

35. The method of claim 34, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

36. The method of claim 35, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

37. The method of claim 32, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, a Sox family gene, and a Myc family gene.

38. The method of claim 37, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, c-Myc, and Sox2.

39. The method of claim 38, wherein the nuclear reprogramming factor further includes a gene product of Sal14.

40. The method of claim 32, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28.

41. The method according to claim 32, wherein the somatic cell is a human cell.

42. The method according to claim 32, wherein the somatic cell is a mouse cell.

43. The method of claim 32, wherein the contacting comprises contacting the nuclear reprogramming factor with the somatic cell in the absence of eggs, embryos, or embryonic stem (ES) cells.

44. The method of claim 43, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Sox family gene.

45. The method of claim 43, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Myc family gene.

46. The method of claim 44 wherein the nuclear reprogramming factor further comprises a cytokine.

47. The method of claim 45, wherein the nuclear reprogramming factor further comprises a cytokine.

48. The method of claim 44, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct3/4 gene, a Klf4 gene, and a Sox2 gene.

49. The method of claim 48, wherein the efficiency of the iPS cell preparation is increased by the addition to the nuclear reprogramming factor of a gene product of Sal14.

50. The method of claim 43, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, a Sox family gene, and a Myc family gene.

51. The method of claim 50, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, c-Myc, and Sox2.

52. The method of claim 51, wherein the nuclear reprogramming factor further includes a gene product of Sal14.

53. The method of claim 43, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28.

54. The method of claim 50, wherein the nuclear reprogramming factor further comprises a gene product of the TERT gene.

55. The method of claim 50, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmi1.

56. The method of claim 50, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

Fbx15, Nanog, ERas, ECAT15-2, Tc11, and β

-catenin.

57. The method of claim 50, wherein the nuclear reprogramming factor further comprises one or more gene products of one or more kinds of the following genes:

ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Sox15, ECAT15-1, Fth117, Sall4, Rex1, UTF1, Stella, Stat3, and Grb2.

58. The method of claim 43, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, together with a cytokine.

59. The method of claim 58, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

60. The method of claim 59, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

61. The method of claim 58, wherein the nuclear reprogramming factor further comprises a Myc family gene.

62. The method of claim 61, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

63. The method of claim 62, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

64. The method of claim 58, wherein the nuclear reprogramming factor further comprises a Sox family gene.

65. The method of claim 64, wherein the cytokine comprises at least one of basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

66. The method of claim 65, wherein the cytokine comprises basic fibroblast growth factor (bFGF) and stem cell factor (SCF).

67. The method according to claim 43, wherein the somatic cell is a human cell.

68. The method according to claim 43, wherein the somatic cell is a mouse cell.

70. An induced pluripotent stem cell obtained by the method of claim 32.

71. An induced pluripotent stem cell obtained by the method of claim 43.

72. A somatic cell derived by inducing differentiation of the pluripotent stem cell according to claim 70.

73. A somatic cell derived by inducing differentiation of the pluripotent stem cell according to claim 71.

76. A method for evaluating a physiological function of a compound comprising treating cells obtained by inducing differentiation of an induced pluripotent stem cell according to claim 70 with the compound.

77. A method for evaluating the toxicity of a compound comprising treating cells obtained by inducing differentiation of an induced pluripotent stem cell according to claim 70 with the compound.

69. A method of inducing a somatic cell to become a pluripotent stem cell comprising contacting the somatic cell with a nuclear reprogramming factor under conditions to obtain a pluripotent stem cell free of rejection.

75. A method for stem cell therapy comprising:

(1) isolating and collecting a somatic cell from a patient;

(2) inducing said somatic cell from the patient into an iPS cell (3) inducing differentiation of said iPS cell, and (4) transplanting the differentiated cell from (3) into the patient.

78. A pluripotent stem cell induced by reprogramming a somatic cell, wherein the reprogramming comprises contacting the somatic cell with a nuclear reprogramming factor.

79. The pluripotent stem cell of claim 78, wherein the somatic cell is a human cell.

80. The pluripotent stem cell of claim 78, wherein the somatic cell is a mouse cell.

81. The pluripotent stem cell of claim 78, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Myc family gene.

82. The pluripotent stem cell of claim 78, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, and a Sox family gene.

83. The pluripotent stem cell of claim 82, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, and Sox2.

84. The pluripotent stem cell of claim 78, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, a Myc family gene, and a Sox family gene.

85. The pluripotent stem cell of claim 84, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

Oct3/4, Klf4, c-Myc, and Sox2.

86. The pluripotent stem cell of claim 78, wherein the nuclear reprogramming factor comprises one or more gene products of each of:

an Oct family gene, a Klf family gene, together with a cytokine.

87. The pluripotent stem cell of claim 86, wherein the nuclear reprogramming factor further comprises a Myc family gene.

88. The pluripotent stem cell of claim 86, wherein the nuclear reprogramming factor further comprises a gene product of a Sox family gene.

89. A somatic cell derived by inducing differentiation of the pluripotent stem cell according to claim 78.