OCT4 and SOX2 with SV40 T antigen produce pluripotent stem cells from primate somatic cells

• US 8,268,620 B2
• Filed: 10/23/2009
• Issued: 09/18/2012
• Est. Priority Date: 10/24/2008
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James, Yu, Junying
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/605,220
• Publication Number:
  US 20100184227A1
• Time in Patent Office: 1,061 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: C12N 15/85 for animal cells
• 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 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Methods for reprogramming primate somatic cells to pluripotency using an episomal vector that does not encode an infectious virus are disclosed. Pluripotent cells produced in the methods are also disclosed.

Claims

1. A method of reprogramming primate somatic cells, the method comprising the steps of:

introducing into a primate somatic cell at least one non-viral episomal vector that encodes at least one potency-determining factor under conditions sufficient to reprogram the cells, wherein the at least one vector encodes at least potency-determining factors OCT4 and SOX2, wherein the cells express the introduced OCT4 and SOX2 and further comprise SV40 T Antigen; and

culturing the cells obtained from the introducing step in a medium that supports pluripotent cell growth to obtain pluripotent reprogrammed cells substantially free of any vector component associated with introducing the potency-determining factors to the somatic cells.

2. The method of claim 1, wherein the primate somatic cells are obtained from a fetal or post-natal primate individual.

3. The method of claim 1, wherein the primate is a human.

4. The method of claim 1, wherein the reprogrammed cells are genetically identical to a fetal or post-natal individual.

5. The method of claim 1, wherein at least one vector further encodes at least one potency-determining factor selected from the group consisting of LIN28, NANOG, c-Myc, and KLF4.

6. The method of claim 1, wherein a single vector encodes at least OCT4 and SOX2 and comprises at least one operably positioned internal ribosomal entry site.

7. The method of claim 6, wherein the single vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4.

8. The method of claim 6, wherein the vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, c-Myc, IRES 2, KLF4, a third promoter, NANOG, IRES2, and LIN28.

9. The method of claim 1, wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, SV40 T antigen, IRES2, and KLF4, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, NANOG, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, c-Myc, IRES 2, and LIN28, wherein the promoters need not be identical.

10. The method of claim 9, wherein each of the first, second, third, and fourth promoters is an elongation factor 1α

(EF1α

) gene promoter.

11. The method of claim 1, wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, KLF4, IRES2, c-Myc, a third promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a fourth promoter, OCT4, IRES2, SOX2, a fifth promoter, SV40 T antigen, IRES2, and KLF4, wherein the promoters need not be identical.

12. The method of claim 11, wherein each of the first, third, fourth, and fifth promoters is an EF1α

gene promoter and wherein the second promoter is a cytomegalovirus immediate early gene (CMV) promoter.

13. The method of claim 1, wherein a first vector comprises, in order, a first promoter, OCT4, IRES2, SOX2, a second promoter, NANOG, IRES2, and LIN28, wherein a second vector comprises, in order, a third promoter, OCT4, IRES2, SOX2, a fourth promoter, SV40 T antigen, IRES2, and KLF4, and wherein a third vector comprises, in order, a fifth promoter, OCT4, IRES2, SOX2, a sixth promoter, c-Myc, IRES 2, and KLF4, wherein the promoters need not be identical.

14. The method of claim 1, wherein at least one vector encodes a plurality of factors and at least one vector comprises at least one internal ribosomal entry site.

15. The method of claim 1, wherein OCT4 and SOX2 are human.

16. The method of claim 1, wherein LIN28, NANOG, c-Myc, and KLF4 are human.