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

• US 9,732,319 B2
• Filed: 12/19/2011
• Issued: 08/15/2017
• Est. Priority Date: 12/22/2010
• Status: Active Grant

• Current Assignee: Fate Therapeutics Inc.
• Original Assignee: Fate Therapeutics Inc.
• Inventors: Valamehr, Bahram, Abujarour, Ramzey, Flynn, Peter
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US13/997,014
• Publication Number:
  US 20140220681A1
• Time in Patent Office: 2,066 Days
• CPC Class Codes: C12N 2501/148 Transforming growth factor alpha [TGF-a]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2502/99 genetically modified cells
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

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.

Claims

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.