Method of making induced pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9499797-B2
Application number	US-201213572593-A
Country	US
Kind code	B2
Filing date	Aug 10, 2012
Priority date	May 2, 2008
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

A method of producing an induced pluripotent stem cell includes introducing into a somatic cell one or more non-viral expression vectors. The vectors include one or more of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and Nanog gene. The somatic cell is then cultured in a medium that supports pluripotent stem cells. At least a portion of the one or more introduced non-viral expression vectors is not substantially integrated in the chromosome.

First claim

What is claimed is:

1. A method of producing an induced pluripotent stem cell, comprising the steps of:
concurrently introducing two or more plasmids into a mammalian somatic cell in vitro, wherein the two or more plasmids encode the following reprogramming factors:
Oct 3/4, Sox 2, Klf4, and a Myc family member selected from the group consisting of c-Myc, N-Myc, L-Myc and T58A mutant of c-Myc,

wherein at least one plasmid vector encodes two or more of the reprogramming factors via an intervening sequence enabling polycistronic expression, and
culturing the mammalian somatic cell in a medium that supports pluripotent stem cells such that iPS cells are obtained, wherein at least a portion of the one or more plasmids are not integrated into a chromosome of the somatic cell.

2. The method of claim 1 , wherein the reprogramming factors further comprise one or more factors selected from the group consisting of Lin28, Lin28B, Nanog, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmi1.

3. The method of claim 1 , wherein the genes that encode Klf4 and Sox 2 are incorporated in one plasmid with an intervening sequence enabling polycistronic expression.

4. The method of claim 3 , wherein the intervening sequence is a 2A sequence derived from foot-and-mouth disease virus.

5. The method of claim 3 , wherein the plasmid comprising the genes that encode Klf4 and Sox 2 further comprise the Oct 3/4 gene.

6. The method of claim 1 , wherein the genes that encode Sox 2 and Klf4 are incorporated in a first plasmid vector with an intervening sequence enabling polycistronic expression, and the Myc family gene is incorporated in a second plasmid vector.

7. The method of claim 1 , wherein the mammalian somatic cell is a human somatic cell.

8. The method of claim 1 , wherein the intervening sequence is a 2A sequence derived from foot-and-mouth disease virus.

9. The method of claim 6 , wherein the intervening sequence is a 2A sequence derived from foot-and-mouth disease virus.

Assignees

• Yamanaka Shinya
• Okita Keisuke
• Univ Kyoto

Inventors

• Yamanaka Shinya
• Okita Keisuke

Classifications

• C12N2506/1361

  from dental pulp or dental follicle stem cells · CPC title

• C12N2506/13

  from connective tissue cells, from mesenchymal cells · CPC title

• C12N2501/603

  Oct-3/4 · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N2501/605

  Nanog · CPC title