Efficient method for nuclear reprogramming

• US 20100075421A1
• Filed: 11/25/2008
• Published: 03/25/2010
• Est. Priority Date: 12/10/2007
• Status: Abandoned Application

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Koyanagi, Michiyo, Yamanaka, Shinya
• Application Number:
  US12/292,717
• Publication Number:
  US 20100075421A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2330/10 naturally occurring
• 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 2501/65 MicroRNA
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

A method of preparing induced pluripotent stem cells, comprising a nuclear reprogramming step with a nuclear reprogramming factor in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof.

Claims

1. A method of preparing induced pluripotent stem cells, comprising nuclear reprogramming at least one somatic cell with nuclear reprogramming factor and at least one miRNA, wherein the at least one miRNA increases efficiency of the nuclear reprogramming of the at least one somatic cell compared to nuclear reprogramming of the at least one somatic cell with the nuclear reprogramming factor in the absence of the at least one miRNA.

2. The method according to claim 1, wherein the at least one miRNA is expressed in embryonic stem cells at a higher level than in somatic cells.

3. The method according to claim 1, wherein a gene encoding the nuclear reprogramming factor and/or the at least one miRNA is introduced into the at least one somatic cell.

4. The method according to claim 3, wherein a vector comprising the gene and/or a vector encoding the at least one miRNA is introduced into the at least one somatic cell.

5. The method according to claim 4, wherein the vector comprising the gene or encoding the at least one miRNA is a retroviral vector.

6. The method according to claim 3, wherein the gene is selected from an Oct family gene, a Klf family gene, and a Sox family gene.

7. The method according to claim 3, wherein the gene is selected from Oct3/4, Klf4, and Sox2.

8. The method according to claim 3, wherein the nuclear reprogramming factor comprises Oct3/4, Klf4, and Sox2.

9. The method according to claim 3, wherein the at least one miRNA is introduced into the at least one somatic cell as primary miRNA.

10. The method according to claim 3, wherein the at least one miRNA is introduced into the at least one somatic cell as pre-miRNA.

11. The method according to claim 1, wherein the at least one miRNA comprises at least one miRNA represented by SEQ ID NOs:

1 to 14.

12. The method according to claim 1, wherein the at least one miRNA comprises at least one miRNA contained in miRNA cluster hsa-miR-302-367 cluster, hsa-miR-371-373 cluster and hsa-miR-520c.

13. The method according to claim 1, wherein the at least one miRNA regulates DNA methylation.

14. The method according to claim 11, wherein the at least one miRNA regulates de novo DNA methylation.

15. The method according to claim 11, wherein the at least one miRNA down-regulates DNA methylation.

16. The method according to claim 1, wherein the at least one miRNA comprises at least 10 contiguous nucleotides of at least one miRNA represented by SEQ ID NOs:

1 to 14.

17. The method according to claim 1, wherein the at least one miRNA comprises at least 30 contiguous nucleotides of at least one miRNA represented by SEQ ID NOs:

1 to 14.

18. The method according to claim 1, wherein the at least one miRNA comprises at least 60 contiguous nucleotides of at least one miRNA represented by SEQ ID NOs:

1 to 14.

19. The method according to claim 1, wherein the nuclear reprogramming factor does not include c-Myc and/or Sox2.

20. The method according to claim 1, wherein the at least one miRNA comprises hsa-miR-302-367 cluster, hsa-miR-371-373 cluster and hsa-miR-520C miRNA.

21. The method according to claim 20, wherein the nuclear reprogramming factor comprises an Oct family gene member, a Sox family gene member, and a Klf family gene member.

22. The method according to claim 1, wherein the at least one miRNA comprises mmu-miR-295/295* and 294/294*.

23. The method according to claim 1, wherein the at least one miRNA comprises hsa-miR-302-367 cluster and hsa-miR-371-373 cluster miRNA.

24. The method according to claim 1, wherein the nuclear reprogramming factor comprises a Klf family gene, and an Oct family gene.

25. The method according to claim 24, wherein the nuclear reprogramming factor further comprises a Myc family gene.

26. The method according to claim 24, wherein the nuclear reprogramming factor further comprises a Sox family gene.

27. The method according to claim 25, wherein the nuclear reprogramming factor further comprises a Sox family gene.

28. The method according to claim 24, wherein the nuclear reprogramming factor comprises KLF4 and OCT3/4.

32. An induced pluripotent stem cell obtained by the method of claim 1.

34. A somatic cell derived by inducing differentiation of the pluripotent stem cell according to claim 32.

36. The method according to claim 25, wherein the nuclear reprogramming factor excludes a Sox family gene.

37. The method according to claim 26, wherein the nuclear reprogramming factor excludes a Myc family gene.

38. The method according to claim 1, where the at least one somatic cell comprises a plurality of somatic cells.

29. A method of increasing the efficiency of nuclear reprogramming comprising:

adding a nuclear reprogramming factor and at least one miRNA to at least one somatic cell so that the number of induced pluripotent stem cells produced is greater than in the absence of the added miRNA.

33. An induced pluripotent stem cell obtained by the method of claim 29.

35. A somatic cell derived by inducing differentiation of the pluripotent stem cell of claim 33.

30. An induced pluripotent stem cell induced by reprogramming a somatic cell, wherein the reprogramming is performed by adding at least one miRNA and in the absence of eggs, embryos, or embryonic stem (ES) cells.

31. The induced pluripotent stem cell according to claim 30, wherein the induced pluripotent stem cell is a human cell.