SOMATIC CELL REPROGRAMMING

• US 20080233610A1
• Filed: 03/21/2008
• Published: 09/25/2008
• Est. Priority Date: 03/23/2007
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Yu, Junying, Thomson, James A.
• Granted Patent: US 8,440,461 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/60 Transcription factors
• 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 2502/99 genetically modified cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• 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 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention relates to methods for reprogramming a somatic cell to pluripotency by administering into the somatic cell at least one or a plurality of potency-determining factors. The invention also relates to pluripotent cell populations obtained using a reprogramming method.

Claims

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

exposing a plurality of potency-determining factors to the primate somatic cells under conditions sufficient to reprogram the cells, wherein the potency-determining factors do not comprise c-Myc and Klf4; and

culturing the exposed cells to obtain reprogrammed cells having a higher potency level than the primate somatic cells.

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

3. The method of claim 2, wherein the reprogrammed cells are substantially genetically identical to the post-natal individual.

4. The method of claim 1, wherein the primate somatic cells are obtained by in vitro differentiation of a stem cell.

5. The method of claim 1, wherein the exposing step includes the step of introducing a vector encoding one or more potency-determining factors into the primate somatic cells.

6. The method of claim 5, wherein the vector is a viral-based vector.

7. The method of claim 6, wherein the viral-based vector is a retroviral vector.

8. The method of claim 7, wherein the retroviral vector is a lentiviral vector.

9. The method of claim 1, wherein the potency-determining factors are introduced to the somatic cells as a reprogramming sequence in which a nucleic acid sequence encoding the potency-determining factor is operably linked to a heterologous promoter.

10. The method of claim 1, wherein the plurality of potency-determining factors is selected from the group consisting of Oct-4, Sox2, Nanog and Lin28.

11. The method of claim 1, wherein the potency-determining factors are Oct-4, Sox2, and at least one of Nanog and Lin28.

12. The method of claim 1, wherein the potency-determining factors are Oct-4 and Sox2.

13. The method of claim 1, wherein the reprogrammed cells are pluripotent.

14. The method of claim 1, wherein the reprogrammed cells (i) express a cell marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

15. An enriched population of primate pluripotent cells produced according to a method comprising the step of:

introducing a plurality of potency-determining factors into primate somatic cells under conditions sufficient to express the potency-determining factors, thereby reprogramming the somatic cells to produce euploid primate pluripotent cells, wherein the potency-determining factors do not comprise c-Myc or Klf4.

16. The enriched population of cells as claimed in claim 15, wherein the primate pluripotent cells (i) express a cell surface marker selected from the group consisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81;

(ii) exhibit morphology characteristic of pluripotent cells; and

(iii) form teratomas when introduced into an immunocompromised animal.

17. The enriched population of cells as claimed in claim 15, wherein the potency-determining factors are Oct-4, Sox2 and at least one of Nanog and Lin28.

18. The enriched population of cells as claimed in claim 15, wherein the potency-determining factors are Oct-4 and Sox2.

19. The enriched population of cells as claimed in claim 15, wherein the primate pluripotent cells account for at least 60% of the population.

20. The enriched population of cells as claimed in claim 15, wherein the primate pluripotent cells account for at least 80% of the population.

21. The enriched population of cells as claimed in claim 15, wherein the primate pluripotent cells account for at least 95% of the population.

22. A cell culture comprising euploid pluripotent cells having a genome of a pre-existing differentiated cell of an individual primate.

23. The cell culture of claim 22, wherein the primate is a human.

24. The cell culture of claim 22, wherein the cells further comprise in the genome a plurality of introduced polynucleotides encoding potency-determining factors, wherein the potency-determining factors do not comprise c-Myc and Klf4.

25. A method for assessing suitability of at least one putative potency-determining factor to convert primate somatic cells to pluripotent cells, the method comprising the steps of:

exposing primate somatic cells to the at least one putative potency-determining factor, the primate somatic cells being receptive to uptake of the factor and comprising a marker gene under control of a regulated promoter active in a pluripotent cell; and

evaluating whether the marker gene is expressed in the cells after exposure to the at least one factor, expression indicating suitability of the at least one factor to convert the primate somatic cells to pluripotent cells.

26. The method of claim 25, wherein the regulated promoter is an Oct4 promoter.

27. The method of claim 25, wherein the at least one putative potency-determining factor is Oct-4, Sox2 and at least one of Nanog and Lin28.