Induction of pluripotent cells

• US 9,005,968 B2
• Filed: 10/15/2010
• Issued: 04/14/2015
• Est. Priority Date: 10/16/2009
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Lin, Tongxiang, Ding, Sheng
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/500,373
• Publication Number:
  US 20120264218A1
• Time in Patent Office: 1,642 Days
• Field of Search: 435/477
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• 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 2501/606 c-Myc
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• 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 slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (>200 fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.

Claims

1. A composition comprising:

an activin receptor-like kinase 5 (ALK5) inhibitor;

a MAP/ERK kinase (MEK) inhibitor; and

a Rho kinase (ROCK) inhibitor, wherein the ROCK inhibitor has the structure;

2. The composition of claim 1, further comprising a glycogen synthase kinase 3 (GSK3) inhibitor.

3. The composition of claim 1, further comprising a histone deacetylase (HDAC) inhibitor.

4. The composition of claim 1, wherein the ALK5 inhibitor is A-83-01 or SB431542.

5. The composition of claim 1, wherein the MEK inhibitor is PD0325901.

6. The composition of claim 1, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen;

or

y and z are 0.

7. The composition of claim 1, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen; and

y and z are 0.

8. The composition of claim 1, wherein the ROCK inhibitor has the structure:

9. The composition of claim 1, comprising one or more mammalian cells.

10. A mixture comprising:

one or more mammalian cells;

an ALK5 inhibitor;

a MEK inhibitor; and

a ROCK inhibitor, wherein the ROCK inhibitor has the structure;

11. The mixture of claim 10, wherein the mammalian cells comprise non-pluripotent cells.

12. The mixture of claim 10, further comprising a glycogen synthase kinase 3 (GSK3) inhibitor.

13. The mixture of claim 10, further comprising an histone deacetylase (HDAC) inhibitor.

19. The mixture of claim 10, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen;

or

y and z are 0.

20. The mixture of claim 10, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen; and

y and z are 0.

21. The mixture of claim 10, wherein the ROCK inhibitor has structure:

14. A method of inducing non-pluripotent mammalian cells into induced pluripotent mammalian stem cells, comprising:

(a) introducing Oct-3/4, Klf4, Sox2, and optionally c-Myc into the non-pluripotent cells; and

(b) contacting the non-pluripotent cells with an ALK5 inhibitor, a MEK inhibitor, and a ROCK inhibitor, wherein the ROCK inhibitor has the structure;

15. The method of claim 14, wherein the introducing step comprises introducing a polynucleotide encoding Oct-3/4, Klf4, Sox2, and optionally c-Myc operably linked to a regulatory sequence into the non-pluripotent cells, wherein the polynucleotide is expressed in the non-pluripotent cells.

16. The method of claim 14, wherein the introducing step comprises introducing Oct-3/4, Klf4, Sox2, and optionally c-Myc polypeptides into the non-pluripotent cells.

17. The method of claim 14, further comprising contacting the non-pluripotent cells with a GSK3 inhibitor.

18. The method of claim 14, further comprising contacting the non-pluripotent cells with an HDAC inhibitor.

22. The method of claim 14, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen;

or

y and z are 0.

23. The method of claim 14, wherein for the ROCK inhibitor:

L2 is methylene;

X is N═

or CH═

;

R1 is hydrogen; and

y and z are 0.

24. The method of claim 14, wherein the ROCK inhibitor has the structure: