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

• US 10,167,451 B2
• Filed: 12/22/2014
• Issued: 01/01/2019
• Est. Priority Date: 12/22/2014
• Status: Active Grant

• Current Assignee: Chinese University of Hong Kong
• Original Assignee: Chinese University of Hong Kong
• Inventors: Lee, Kenneth Ka Ho, Lo, Tommy Lok Man, Cheung, Hoi Hung, Chan, Wai Yee
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/579,784
• Publication Number:
  US 20160177271A1
• Time in Patent Office: 1,471 Days
• CPC Class Codes: C12N 2500/40 Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/1392 from mesenchymal stem cells from other natural sources
• CPC Class Codes: C12N 2527/00 Culture process characterised by the use of mechanical forces, e.g. strain, vibration
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

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).

Claims

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;

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×

10⁵-1×

10⁶ cells/cm².

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.