Cell culture platform for single cell sorting and enhanced reprogramming of iPSCs

Patent metadata

Field	Value
Publication number	US-9732319-B2
Application number	US-201113997014-A
Country	US
Kind code	B2
Filing date	Dec 19, 2011
Priority date	Dec 22, 2010
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

The invention provides cell culture conditions for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). More particularly, the invention provides a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated status; improved efficiency of reprogramming; and generation of a naïve pluripotent cell.

First claim

The invention claimed is:

1. A method of obtaining induced pluripotent cells having improved genomic stability comprising the steps of:
(a) introducing into cells without ground state pluripotency one or more polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4, wherein the cells without ground state pluripotency are somatic cells, progenitor cells, or multipotent cells; and
(b) culturing the cells resulting from step (a) in a culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor, and a TGFβ inhibitor for a sufficient amount of time to reprogram the cells without ground state pluripotency into pluripotent cells, wherein the pluripotent cells have improved genomic stability, as indicated by a lower propensity for genomic abnormalities than pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b).

2. The method of claim 1 , further comprising:
contacting the cells resulting from step (b) with one or more dissociating enzyme to obtain single cell dissociated pluripotent cells.

3. The method of claim 2 , further comprising:
suspending the single cell dissociated pluripotent cells.

4. The method of claim 2 , further comprising: sorting the single cell dissociated pluripotent cells by selecting and isolating the single cell dissociated pluripotent cell expressing one or more markers of pluripotency to enrich for pluripotent cells expressing the one or more markers.

5. The method of claim 4 , further comprising:
culturing the enriched pluripotent cells in the culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor, wherein the pluripotent cells maintain pluripotency for at least 5, 10, 15, or 20 passages.

6. The method of claim 1 , wherein the pluripotent cells have ground state pluripotency.

7. The method of claim 6 , wherein the pluripotent cells comprise reactivated genes associated with extraembryonic cells.

8. The method of claim 1 , wherein the pluripotent cells resulting from step (b) have reduced Xist activity and/or enhanced expression of genes of the X chromosome in comparison to pluripotent cells obtained by step (a) but not cultured in the culture medium composition of (b).

9. The method of claim 1 , wherein the culture medium composition is free of additional cytokines and growth factors.

10. The method of claim 1 , wherein the pluripotent cells resulting from step (b) are obtained with a higher efficiency in comparison to pluripotent cells obtained from step (a) but not cultured in the culture medium composition of step (b).

11. The method of claim 10 , wherein the efficiency is increased by at least 10%, at least 50%, at least 100%, at least 300%, or at least 500%.

12. The method of claim 1 , wherein the Rock inhibitor is thiazovivin.

13. The method of claim 1 , wherein the culture medium composition in step (b) is feeder-free.

Assignees

• Valamehr Bahram
• Abujarour Ramzey
• Flynn Peter
• Fate Therapeutics Inc

Inventors

• Valamehr Bahram
• Abujarour Ramzey
• Flynn Peter

Classifications

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title

• C12N2502/99

  genetically modified cells · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title