Highly efficient method for establishing induced pluripotent stem cell

Patent metadata

Field	Value
Publication number	US-10519425-B2
Application number	US-201314402310-A
Country	US
Kind code	B2
Filing date	May 23, 2013
Priority date	May 23, 2012
Publication date	Dec 31, 2019
Grant date	Dec 31, 2019

Abstract

The present invention provides a production method of iPS cell, including a step of introducing the following (1) and (2):
(1) an episomal vector containing a nuclear reprogramming factor; and (2) an episomal vector containing EBNA-1, which is different from (1), into a somatic cell, as well as a method for improving iPS cell establishment efficiency. The present invention also provides an agent for improving iPS cell establishment efficiency, which contains an episomal vector containing a nucleic acid encoding EBNA-1, and a kit for producing an iPS cell further containing an episomal vector containing a nucleic acid encoding a nuclear reprogramming factor.

First claim

The invention claimed is:

1. A method of producing a human iPS cell, comprising a step of introducing the following (1) and (2):
(1) one or more episomal vectors containing a nucleic acid encoding a nuclear reprogramming factor, oriP, and a nucleic acid encoding EBNA-1; and
(2) a plasmid vector containing a nucleic acid encoding EBNA-1 and not containing oriP

into a human somatic cell, thereby producing a human iPS cell.

2. The method according to claim 1 , further comprising introducing a nucleic acid, encoding an inhibitor of p53 function, in the form of an episomal vector.

3. The method according to claim 2 , wherein the inhibitor of p53 function is p53 shRNA or a dominant negative mutant of p53.

4. The method according to claim 3 , wherein the dominant negative mutant of p53 is p53DD.

5. The method according to claim 1 , wherein the nuclear reprogramming factor is one or more selected from the group consisting of the members of Oct family, Klf family, Sox family, Myc family, Lin family and Glis family.

6. The method according to claim 5 , wherein the nuclear reprogramming factors are Oct3/4, Klf4, Sox2, L-Myc and Lin28.

7. The method according to claim 6 , wherein the nuclear reprogramming factors are Oct3/4, Klf4, Sox2, L-Myc, Lin28 and Glis1.

8. The method according to claim 1 , wherein the nucleic acid encoding a nuclear reprogramming factor is divided and contained in 2 or 3 episomal vectors.

9. The method according to claim 1 , wherein the episomal vectors containing the nucleic acid encoding a nuclear reprogramming factor are the following (1) or (2):
(1) pCEB-hSK-O and pCEB-hUL-G or
(2) pCXLE-hOCT3/4, pCXLE-hSK and pCXLE-hUL.

10. The method according to claim 2 , wherein the episomal vectors containing the nucleic acid encoding a nuclear reprogramming factor are pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL.

11. The method according to claim 2 , wherein the episomal vectors containing the nucleic acid encoding a nuclear reprogramming factor are pCE-hOCT3/4-shp53, pCE-hSK and pCE-hUL.

12. The method according to claim 2 , wherein the episomal vectors containing the nucleic acid encoding a nuclear reprogramming factor are pCE-hOCT3/4, pCE-hSK and pCE-hUL, and the episomal vector containing the nucleic acid encoding an inhibitor of p53 function is pCE-mp53DD.

13. The method according to claim 9 , wherein the plasmid vector containing the nucleic acid encoding EBNA-1 is pCXWB-EBNA1.

14. The method according to claim 10 , wherein the plasmid vector containing the nucleic acid encoding EBNA-1 is pCXB-EBNA1.

15. The method according to claim 1 , wherein the somatic cell is selected from human fibroblast (HDF) and human blood cell.

16. The method according to claim 15 , wherein the blood cell is a peripheral blood mononuclear cell (PMNC).

17. The method according to claim 16 , wherein the peripheral blood mononuclear cell (PMNC) is a T cell.

18. A method of producing a human iPS cell, comprising a step of introducing the following (1) and (2):
(1) one or more episomal vectors containing a nucleic acid encoding a nuclear reprogramming factor, oriP, and a nucleic acid encoding EBNA-1; and
(2) a plasmid vector containing a nucleic acid encoding EBNA-1 and not containing a replication origin functional in a mammalian cell

into a human somatic cell, thereby producing a human iPS cell.

19. The method of claim 18 , wherein the plasmid vector of (2) does not contain a nucleic acid encoding a nuclear reprogramming factor.

20. The method of claim 1 , wherein the plasmid vector of (2) does not contain a nucleic acid encoding a nuclear reprogramming factor.

Assignees

• Univ Kyoto

Inventors

• Yamanaka Shinya
• Okita Keisuke

Classifications

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N2501/606

  c-Myc · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N15/113

  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 title

• C12N2510/00

  Genetically modified cells · CPC title