Combinational use of mechanical manipulation and programin derivatives to increase Oct4, Sox2, or Nanog expression in fibroblasts

Patent metadata

Field	Value
Publication number	US-10167451-B2
Application number	US-201414579784-A
Country	US
Kind code	B2
Filing date	Dec 22, 2014
Priority date	Dec 22, 2014
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

The present invention provides methods and compositions for inducing pluripotency in differentiated mammalian cells. In particular, the methods include mechanically aggregating the cells into discrete masses or embryoid-like bodies and treated them with a small molecule compound. Provided herein are the compositions of the compounds which are derived from programin (e.g., reversine).

First claim

What is claimed is:

1. A method for increasing Oct4, Sox2, or Nanog expression in fibroblasts, the method comprising:
(a) culturing the fibroblasts in a vessel;
(b) mechanically agitating the fibroblasts that are unattached to the vessel to form a non-adherent cellular aggregate; and
(c) exposing the non-adherent cellular aggregate to an effective amount of a compound of Formula II-IX having any one of the following structures:

thereby increasing Oct4, Sox2, or Nanog expression in the fibroblasts of the non-adherent cellular aggregate.

2. The method of claim 1 , further comprising step (d) detecting the expression level of Oct4, Sox2, or Nanog in the fibroblasts of the non-adherent cellular aggregate.

3. The method of claim 2 , further comprising step (e) isolating the fibroblasts of the non-adherent cellular aggregate.

4. The method of claim 1 , wherein the fibroblasts are human fibroblasts.

5. The method of claim 1 , wherein the fibroblasts are murine fibroblasts.

6. The method of claim 1 , wherein the fibroblasts of step (a) are at a density of about 1×10 5 -1×10 6 cells/cm 2 .

7. The method of claim 1 , wherein mechanically agitating comprises titurating, stirring or rocking the fibroblasts.

8. The method of claim 2 , wherein step (d) comprises an amplification-based assay, a hybridization-based assay, an immunoassay or a flow cytometry assay.

9. The method of claim 2 , wherein the expression level of Oct4, Sox2 or Nanog in the fibroblasts of the non-adherent cellular aggregate is at least about 20-fold to about 200-fold higher compared to the expression level in the fibroblasts prior to step (b).

10. The method of claim 3 , further comprising, subsequent to step (e), expanding the isolated fibroblasts.

Assignees

• Univ Hong Kong Chinese

Inventors

• Lee Kenneth Ka Ho
• Lo Tommy Lok Man
• Cheung Hoi Hung
• Chan Wai Yee

Classifications

• C12N5/0696 Primary

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

• C12N2527/00

  Culture process characterised by the use of mechanical forces, e.g. strain, vibration · CPC title

• C12N2506/1392

  from mesenchymal stem cells from other natural sources · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title