COMBINATIONAL USE OF MECHANICAL MANIPULATION AND PROGRAMIN TO GENERATE PLURIPOTENT STEM CELLS FROM SOMATIC CELLS

• US 20160177271A1
• Filed: 12/22/2014
• Published: 06/23/2016
• 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
• Granted Patent: US 10,167,451 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 producing a pluripotent stem cell from differentiated cells, the method comprising:

(a) culturing the differentiated cells in a vessel;

(b) mechanically agitating the differentiated cells to form a cellular aggregate;

(c) exposing the 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, wherein the differentiated cells of step (a) is obtained from a subject or a cell line.

3. The method of claim 2, wherein the subject is a mammalian subject.

4. The method of claim 2, wherein the cell line is a mammalian cell line.

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

6. The method of claim 1, wherein the differentiated cells are progenitor cells.

7. The method of claim 1, wherein the differentiated cells of step (a) are at a density of about 1×

10⁵-1×

10⁶ cells/cm².

8. The method of claim 1, wherein the cellular aggregate does not adhere to an inner surface of the vessel.

9. The method of claim 8, wherein the inner surface of the vessel is coated with a non-adherent substrate.

10. The method of claim 1, wherein mechanically agitating comprises titurating, stirring or rocking the differentiated cells.

11. The method of claim 1, wherein step (d) comprises an amplification-based assay, a hybridization-based assay, an immunoassay or a flow cytometry assay.

12. The method of claim 1, wherein the higher expression level of Oct4, Sox2 or Nanog is at least about 20-fold to about 200-fold higher compared to the differentiated cell.

13. The method of claim 1, further comprising, subsequent to step (e), expanding the pluripotent stem cell.

14. The method of claim 1, further comprising subsequent to step (e), inducing the pluripotent stem cell to undergo differentiation.

15. (canceled)

16. A method for producing a cellular aggregate from differentiated cells, wherein the cellular aggregate comprise a cell that expresses at least one pluripotent stem cell marker, the method comprising:

(a) culturing the differentiated cells at a density of about 1×

10⁵ to about 1×

10⁶ cells/ml in a vessel;

(b) mechanically agitating the differentiated cells to form the cellular aggregate; and

(c) culturing the cellular aggregate under conditions to produce a cell that expresses at least one pluripotent stem cell marker.

17. The method of claim 16, further comprising (d) exposing the cellular aggregate to an effective amount of a compound of Formula II-IX having any one of the following structures:

18. The method of claim 16, wherein the at least one pluripotent stem cell marker is Oct4, Sox2 or Nanog.

19. The method of claim 16, further comprising subsequent to step (c) expanding (c) the cell that expresses the at least one pluripotent stem cell marker.

20. The method of claim 16, further comprising subsequent to step (c) isolating the cell that expresses the at least one pluripotent stem cell marker.

21. A compound of Formula II-IX having the any one of the following structures:

22-24. -24. (canceled)