CHEMICAL APPROACHES FOR GENERATION OF INDUCED PLURIPOTENT STEM CELLS

• US 20100267141A1
• Filed: 04/02/2010
• Published: 10/21/2010
• Est. Priority Date: 03/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Shi, Yan, Ding, Sheng, Li, Wenlin, Lin, Tongxiang, Zhu, Saiyong, Desponts, Caroline
• Granted Patent: US 9,394,524 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• 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 an induced pluripotent stem cell from a mammalian non-pluripotent cell, the method comprising contacting the cell with a TGFβ

receptor/ALK5 inhibitor under conditions to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell.

2. The method of claim 1, further comprising contacting the cell with a second agent selected from the group consisting of a MEK inhibitor and an Erk inhibitor.

3. The method of claim 2, wherein the second agent is a MEK inhibitor.

4. The method of claim 1, further comprising contacting the cell with a GSK-3 inhibitor

5. The method of claim 2, further comprising contacting the cell with a GSK-3 inhibitor.

6. The method of claim 1, wherein the conditions without the TGFβ

receptor/ALK5 inhibitor are sufficient to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell, wherein the presence of the TGFβ

receptor/ALK5 inhibitor improves efficiency of the method.

7. The method of claim 1, wherein the conditions without the TGFβ

receptor/ALK5 inhibitor are not sufficient to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell.

8. A method of producing an induced pluripotent stem cell from a mammalian non-pluripotent cell, the method comprising, contacting the cell with a GSK-3 inhibitor under conditions to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell.

9. The method of claim 8, further comprising contacting the cell with an agent that inhibits H3K9 methylation or promotes H3K9 demethylation.

10. The method of claim 8, wherein the conditions without the GSK-3 inhibitor are sufficient to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell, wherein the presence of the GSK-3 inhibitor improves efficiency of the method.

11. The method of claim 8, wherein the conditions without the GSK-3 inhibitor are not sufficient to produce the induced pluripotent stem cell from the mammalian non-pluripotent cell.

12. A eukaryotic cell medium comprising a TGFβ

receptor/ALK5 inhibitor.

13. The medium of claim 12, wherein the medium further comprises a second agent selected from the group consisting of a MEK inhibitor and an Erk inhibitor.

14. The medium of claim 13, wherein the second agent is a MEK inhibitor.

15. The medium of claim 12, further comprising a GSK-3 inhibitor.

16. The medium of claim 13, further comprising a GSK-3 inhibitor.

17. The medium of claim 12, wherein the medium does not comprise cells.

18. The medium of claim 12, wherein the medium comprises cells.

19. A kit for inducing pluripotency in non-pluripotent cells, the kit comprising:

a TGFβ

receptor/ALK5 inhibitor; and

(i) one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide and/or polynucleotide encoding one or more of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Sox polypeptide;

or

(ii) a MEK inhibitor and/or an Erk inhibitor;

or

(iii) both (i) and (ii).

20. The kit of claim 19, further comprising a histone deacetylase (HDAC) inhibitor.