Generation and maintenance of stem cells

• US 9,695,395 B2
• Filed: 09/18/2014
• Issued: 07/04/2017
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Ding, Sheng, Li, Wenlin
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US14/490,433
• Publication Number:
  US 20150079675A1
• Time in Patent Office: 1,020 Days
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• 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
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention provides for the generation and maintenance of pluripotent cells by culturing the cells in the presence of an ALK5 inhibitor.

Claims

1. A method of culturing pluripotent cells through at least one cell division, the method comprising,

culturing pluripotent animal cells in the presence of a sufficient amount of:

(a) a compound selected from one or more of a MEK inhibitor, an Erk inhibitor, a p38 inhibitor, and an FGF receptor inhibitor;

(b) a GSK3β

inhibitor; and

(c) nutrients;

to allow for at least one cell division while maintaining cell pluripotency.

2. The method of claim 1, wherein the culturing step further comprises culturing the cells in the presence of a Leukemia inhibitory factor (LIF).

3. The method of claim 1, wherein the GSK3β

inhibitor is CHIR99021.

4. The method of claim 1, wherein the compound is a MEK inhibitor.

5. The method of claim 4, wherein the MEK inhibitor is PD0325901.

6. The method of claim 1, wherein the pluripotent cells are cultured through at least five cell divisions while maintaining cell pluripotency.

7. The method of claim 1, further comprising introducing a heterologous nucleic acid into the pluripotent cells and culturing the resulting cells to allow for at least one additional cell division while maintaining pluripotency.

8. The method of claim 1, wherein the pluripotent cells are rat, human, non-human primate, ovine, bovine, feline, canine, or porcine cells.

9. A method of producing non-human chimeric animal progeny comprising culturing non-human pluripotent cells by the method of claim 1 and introducing the non-human pluripotent cell into a blastocyst, wherein the blastocyst is from the same species of animal as the non-human pluripotent cells, and introducing the blastocyst into the uterus of an animal of the same species as the non-human pluripotent cells.

10. A culture of pluripotent mammalian cells, comprising a sufficient amount of:

(a) a compound selected from one or more of a MEK inhibitor and an FGF receptor inhibitor; and

(b) a GSK3β

inhibitor;

to allow for at least one cell division while maintaining cell pluripotency.

11. The culture of claim 10, further comprising Leukemia inhibitory factor (LIF).

12. The culture of claim 10, wherein the GSK3β

inhibitor is CHIR99021.

13. The culture of claim 10, wherein the compound is a MEK inhibitor.

14. The culture of claim 13, wherein the MEK inhibitor is PD0325901.

15. The culture of claim 10, wherein the pluripotent mammalian cells are human cells or rat cells.

16. The culture of claim 10, wherein the pluripotent mammalian cells are embryonic stem cells.

17. A cell culture medium, comprising a sufficient amount of:

(a) a compound selected from one or more of a MEK inhibitor and an FGF receptor inhibitor; and

(b) a GSK3β

inhibitor;

to allow for at least one cell division while maintaining cell pluripotency when pluripotent cells are cultured in the medium.

18. The cell culture medium of claim 17, further comprising Leukemia inhibitory factor (LIF).

19. The cell culture medium of claim 17, wherein the medium is in a pre-packaged, sealed container.

20. The cell culture medium of claim 17, wherein the GSK3β

inhibitor is CHIR99021.

21. The cell culture medium of claim 17, wherein the compound is a MEK inhibitor.

22. The cell culture medium of claim 21, wherein the MEK inhibitor is PD0325901.