Combined Chemical and Genetic Approaches for Generation of Induced Pluripotent Stem Cells

• US 20110110899A1
• Filed: 03/17/2009
• Published: 05/12/2011
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Shi, Yan, Desponts, Caroline, Ding, Sheng, Li, Wenlin, zhou, Hongyan, Lin, Tongxiang, Zhu, Saiyong
• Granted Patent: US 9,534,205 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 11/08 Bronchodilators
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• 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 2501/605 : Nanog
• CPC Class Codes: C12N 2501/608 : Lin28
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/08 : from cells of the nervous system
• CPC Class Codes: C12N 2506/094 : from keratinocytes
• CPC Class Codes: C12N 2506/1307 : from adult fibroblasts
• CPC Class Codes: C12N 2506/28 : from vascular endothelial cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention provides for identification and use of small molecules to induce pluripotency in mammalian cells as well as other methods of inducing pluripotency.

Claims

1. A method of producing induced pluripotent stem cells from mammalian non-pluripotent cells, the method comprising, contacting the cells with at least one of:

an agent that inhibits H3K9 methylation or promotes H3K9 demethylation;

an L-type Ca channel agonist;

an activator of the cAMP pathway;

DNA methyltransferase (DNMT) inhibitor;

a nuclear receptor ligand;

a GSK3 inhibitor;

a MEK inhibitor;

a TGFβ

receptor/ALK5 inhibitor;

a HDAC inhibitor; and

an erk inhibitor, thereby producing induced pluripotent stem cells.

3. The method of claim 1 further comprising introducing one or more exogenous polypeptides selected from the group consisting of a KIf polypeptide, an Oct polypeptide, a Myc polypeptide, and/or a Sox polypeptide into the non-pluripotent cells.

2. (canceled)

4. (canceled)

5. A method of producing induced pluripotent stem cells from mammalian non-pluripotent cells, the method comprising, a) introducing one or more exogenous polypeptides selected from the group consisting of:

an Oct polypeptide, a KIf polypeptide, a Myc polypeptide, and a Sox polypeptide into the non-pluripotent cells;

b) contacting the non-pluripotent cells with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, thereby producing induced pluripotent stem cells.

6. The method of claim 5, wherein step a) comprises:

(i) at least two cycles of contacting the non-pluripotent cells with one or more exogenous polypeptides selected from the group consisting of;

a KIf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide;

followed by culturing the cells in the absence of the exogenous polypeptides;

(ii) contacting the non-pluripotent cells with an exogenous Oct polypeptide and an exogenous Sox polypeptide;

(iii) introducing the one or more exogenous polypeptides into the non-pluripotent cells in vivo;

(iv) introducing the one or more exogenous polypeptides into the non-pluripotent cells ex vivo;

or

(v) introducing the one or more exogenous polypeptides into the non-pluripotent cells in vitro.

12. The method of claim 5, wherein the non-pluripotent cells are somatic cells.

13. The method of claim 5, wherein the non-pluripotent cells are progenitor cells.

17. The method of claim 5, further comprising, c) selecting cells that exhibit pluripotent stem cell characteristics.

18. The method of claim 5, wherein the induced pluripotent stem cells are differentiated into a desired cell type.

19. The method of claim 18, wherein the desired cell type is introduced into the animal.

20. The method of claim 19, wherein the animal is a human.

23. The method of claim 5, wherein the agent that inhibits H3K9 methylation is BIX-01294.

42. The method of claim 13, wherein step (b) comprises contacting cells with a MAPK/ERK kinase (MEK) inhibitor such that growth of non-pluripotent cells is inhibited and growth of pluripotent stem cells is promoted.

46. The method of claim 42, wherein the MEK inhibitor is PD0325901.

7-11. -11. (canceled)

14-16. -16. (canceled)

21-22. -22. (canceled)

24-27. -27. (canceled)

29-30. -30. (canceled)

31. A method for screening for agents that induce reprogramming or dedifferentiation of non-pluripotent mammalian cells into pluripotent stem cells, the method comprising,

a) introducing at least one of, but not all of, an exogenous Oct polypeptide, an exogenous KIf polypeptide, an exogenous Myc polypeptide, and an exogenous Sox polypeptide into the non-pluripotent cells;

b) contacting the non-pluripotent cells to a library of different agents;

c) screening the contacted cells for pluripotent stem cell characteristics; and

;

d) correlating the development of stem cell characteristics with a particular agent from the library, thereby identifying an agent that stimulates dedifferentiation of cells into pluripotent stem cells.

41. The method of claim 31, wherein the Oct polypeptide is Oct4, the KIf polypeptide is KIf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

32-40. -40. (canceled)

43-45. -45. (canceled)

47. A mixture of non-pluripotent mammalian cells and an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, wherein the cells are in contact with at least one or more of an exogenous Oct polypeptide, an exogenous KIf polypeptide, an exogenous Sox polypeptide, and an exogenous Myc polypeptide.

49. The mixture of claim 47, wherein the agent that inhibits H3K9 methylation is BIX-01294.

53. The mixture of claim 47, wherein the cells are human cells.

55. The mixture of claim 47, wherein the cells comprise progenitor cells.

57. The mixture of claim 47, wherein the Oct polypeptide is Oct4, the KIf polypeptide is KIf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

48. (canceled)

50-52. -52. (canceled)

54. (canceled)

56. (canceled)

58-68. -68. (canceled)

69. A mixture of non-pluripotent mammalian cells and at least one of an agent that inhibits H3K9 methylation or promotes H3K9 demethylation, an L-type Ca channel agonist;

an activator of the cAMP pathway;

DNA methyltransferase (DNMT) inhibitor;

a nuclear receptor ligand;

a GSK3 inhibitor;

a MEK inhibitor;

a TGFβ

receptor/ALK5 inhibitor;

a HDAC inhibitor; and

an erk inhibitor.

71. The mixture of claim 69, wherein one or more exogenous polypeptides selected from the group consisting of:

a KIf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide have been introduced into the non-pluripotent cells.

70. (canceled)

72-74. -74. (canceled)

75. A composition comprising:

(a) at least one of;

an agent that inhibits H3K9 methylation;

an L-type Ca channel agonist;

an activator of the cAMP pathway;

DNA methyltransferase (DNMT) inhibitor;

a nuclear receptor ligand;

a GSK3 inhibitor;

a MEK inhibitor;

a TGFβ

receptor/ALK5 inhibitor;

a HDAC inhibitor; and

an erk inhibitor; and

(b) one or more polypeptides selected from the group consisting of;

a KIf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.

76. (canceled)

77. (canceled)

78. A kit comprising (i) an agent that inhibits H3K9 methylation and (ii) an agent selected from the group consisting of an L-type Ca channel agonist;

an activator of the cAMP pathway;

DNA methyltransferase (DNMT) inhibitor;

a nuclear receptor ligand;

a GSK3 inhibitor;

a MEK inhibitor;

a TGFβ

receptor/ALK5 inhibitor;

a HDAC inhibitor; and

an erk inhibitor.

79. The kit of claim 78, comprising mammalian cells.

80. The kit of claim 78, comprising one or more polypeptides selected from the group consisting of:

a KIf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Sox polypeptide.