COMPOSITION FOR REPROGRAMMING SOMATIC CELLS TO GENERATE INDUCED PLURIPOTENT STEM CELLS, COMPRISING Bmi1 AND LOW MOLECULAR WEIGHT SUBSTANCE, AND METHOD FOR GENERATING INDUCED PLURIPOTENT STEM CELLS USING THE SAME

• US 20110275157A1
• Filed: 05/09/2011
• Published: 11/10/2011
• Est. Priority Date: 05/10/2010
• Status: Active Grant

• Current Assignee: STEM Lab, Inc.
• Original Assignee: Korea University Research and Business Foundation
• Inventors: LEE, Jung Han, HWANG, Ji Hye, GWON, Su Hyun, MOON, Jai-Hee, YOON, Byung Sun, HEO, June Seok, YOU, Seungkwon, JUN, Eun Kyoung, KIM, Jun Sung, KIM, Ji Hyun
• Granted Patent: US 8,518,700 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Provided is a composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising: a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1) protein or a nucleic acid molecule encoding the Bmi1 protein; and b) at least one low molecular weight substance selected from the group consisting of a set of a MEK/ERK (mitogen-activated protein kinase/extracellular regulated kinase) inhibitor and a GSK (glycogen synthase kinase) inhibitor, a set of a G9a HMTase (G9a histone methyltransferase) inhibitor and a DMNT (DNA methyltransferase) inhibitor, and a histone deacetylase inhibitor. Also, a method is provided for reprogramming somatic cells to generate embryonic stem cell-like cells using the composition. In addition to reducing the number of the reprogramming factors conventionally needed, the composition and method allow the generation of pluripotent embryonic stem cell-like cells which have high potential in the cell therapy of various diseases.

Claims

1. A composition for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising:

a) a Bmi1 (B cell-specific Moloney murine leukemia virus integration site

     1) protein or a nucleic acid molecule encoding the Bmi1 protein; and

b) at least one low molecular weight substance selected from the group consisting of a set of a MEK/ERK (mitogen-activated protein kinase/extracellular regulated kinase) inhibitor and a GSK (glycogen synthase kinase) inhibitor, a set of a G9a HMTase (G9a histone methyltransferase) inhibitor and a DMNT (DNA methyltransferase) inhibitor, and a histone deacetylase inhibitor.

2. A method for reprogramming somatic cells to generate embryonic stem cell-like cells, comprising:

i) introducing a Bmi1 (B cell-specific Moloney murine leukemia virus integration site

     1) protein or a nucleic acid molecule encoding the Bmi1 protein into the somatic cells;

ii) culturing the Bmi1-transduced somatic cells under a condition used to culture neural stem cells; and

iii) treating the cells with at least one low molecular weight substance selected from the group consisting of a set of a MEK/ERK (mitogen-activated protein kinase/extracellular regulated kinase) inhibitor and a GSK (glycogen synthase kinase) inhibitor, a set of a G9a HMTase (G9a histone methyltransferase) inhibitor and a DMNT (DNA methyltransferase) inhibitor, and a histone deacetylase inhibitor.

3. The method of claim 2, wherein the condition used to culture neural stem cells in step ii) is a medium which contains bFGF (basic Fibroblast Growth Factor), but is free of EGF (Epidermal Growth Factor).

4. The method of claim 3, wherein the method further comprises selecting Oct4-expressing cells after step ii) and the cells are treated with a set of a MEK/ERK (mitogen-activated protein kinase/extracellular regulated kinase) inhibitor and a GSK (glycogen synthase kinase) inhibitor in step iii).

5. The method of claim 4, wherein the MEK inhibitor is PD0325901 and the GSK inhibitor is CHIR99021 inhibitory of GSK3β

.

6. The method of claim 3, wherein the cells are treated with a set of a G9a HMTase (G9a histone methyltransferase) inhibitor and a DMNT (DNA methyltransferase) inhibitor.

7. The method of claim 6, wherein the G9a HMTase inhibitor is BIX01294 and the DMNT inhibitor is RG108.

8. The method of claim 2, wherein the cells are treated with a histone deacetylase inhibitor in step iii).

9. The method of claim 8, wherein the histone deacetylase inhibitor is VPA (valproic acid).

10. Embryonic stem cell-like cells, established by the method of any one of claims 2 to 9.