Method of efficiently establishing induced pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-10077429-B2
Application number	US-201314438106-A
Country	US
Kind code	B2
Filing date	Oct 23, 2013
Priority date	Oct 23, 2012
Publication date	Sep 18, 2018
Grant date	Sep 18, 2018

Abstract

The present invention provides a method of improving iPS cell establishment efficiency, comprising contacting a protein involved in primitive streak (PrS) formation, preferably Foxh1, or a nucleic acid that encodes the same with a somatic cell in a nuclear reprogramming step. Also provided is a method of producing an iPS cell, comprising contacting the protein involved in PrS formation or a nucleic acid that encodes the same, and nuclear reprogramming substance(s) with a somatic cell.

First claim

The invention claimed is:

1. A method of improving induced pluripotent stem (iPS) cell establishment efficiency, comprising contacting a protein involved in primitive streak (PrS) formation or a nucleic acid that encodes the same with a somatic cell in vitro in a nuclear reprogramming step,
wherein the protein is selected from the group consisting of forkhead box protein h1 (Foxh1), brachyury, Lin-11/Isl-1/Mec-3 homeobox protein 1 (LHX1), forkhead box protein b1 (Foxb1), forkhead box protein f1 (Foxf1), forkhead box protein f2 (Foxf2), and forkhead box protein g1 (Foxg1), and
wherein the nuclear reprogramming step is performed by introducing nuclear reprogramming substances into the somatic cell, wherein the nuclear reprogramming substances comprise any of the following (a) to (d):
(a) Oct3/4, Sox2, and Klf4,
(b) (i) Oct3/4,
(ii) a member of the Sox family selected from the group consisting of Sox1, Sox2, Sox3, Sox15, Sox17, and Sox18,
(iii) a member of the Klf family selected from the group consisting of Klf1, Klf2, Klf4, and Klf5, and
(iv) a member of the Myc family selected from the group consisting of c-Myc, L-Myc, and n-Myc,

(c) Oct3/4, Sox2, Nanog, and Lin28, and
(d) nucleic acids that encode any of (a) to (c), wherein (a) to (c) are proteins,
thereby improving iPS cell establishment efficiency compared to that obtained by introducing the nuclear reprogramming substances alone into the somatic cell.

2. The method according to claim 1 , wherein the protein is Foxh1.

3. The method according to claim 1 , wherein the somatic cell is further contacted with Glioma-associated oncogene-similar 1 (Glis1) or a nucleic acid that encodes the same and/or a p53 inhibitor in vitro in the nuclear reprogramming step.

4. The method according to claim 1 , wherein the protein is brachyury.

5. The method according to claim 1 , wherein the protein is LHX1.

6. The method according to claim 1 , wherein the protein is Foxb1.

7. The method according to claim 1 , wherein the protein is Foxf1.

8. The method according to claim 1 , wherein the protein is Foxf2.

9. The method according to claim 1 , wherein the protein is Foxg1.

10. The method according to claim 1 , wherein the nuclear reprogramming substances are Oct3/4, Sox2, and Klf4 proteins, or nucleic acids that encode Oct3/4, Sox2, and Klf4 proteins.

11. The method according to claim 1 , wherein the nuclear reprogramming substances are proteins (i) Oct3/4, (ii) a member of the Sox family selected from the group consisting of Sox1, Sox2, Sox3, Sox15, Sox17 and Sox18, (iii) a member of the Klf family selected from the group consisting of Klf1, Klf2, Klf4, and Klf5, and (iv) a member of the Myc family selected from the group consisting of c-Myc, L-Myc, and n-Myc, or nucleic acids that encode (i)-(iv).

12. The method according to claim 1 , wherein the nuclear reprogramming substances are Oct3/4, Sox2, Nanog, and Lin28 proteins, or nucleic acids that encode Oct3/4, Sox2, Nanog, and Lin28 proteins.

Assignees

• Univ Kyoto

Inventors

• Takahashi Kazutoshi
• Tanabe Koji
• Yamanaka Shinya

Classifications

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N2501/603

  Oct-3/4 · CPC title

• C12N2510/00

  Genetically modified cells · CPC title