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

Patent metadata

Field	Value
Publication number	US-10844356-B2
Application number	US-201715639628-A
Country	US
Kind code	B2
Filing date	Jun 30, 2017
Priority date	Dec 22, 2010
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

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. An isolated induced pluripotent cell, a cell line thereof, or a population thereof, wherein: (a) the cell is in contact with a feeder-free culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor; (b) the induced pluripotent cell is a naïve human induced pluripotent stem cell (iPSC) with ground state pluripotency; and (c) the iPSC (i) exhibits reactivated expression of one or more of GATA6, CDX2, or CGB, or (ii) is a cell in a colony having a domed morphology.

2. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the pluripotent cell is a suspended pluripotent cell.

3. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the pluripotent cells comprise a population of cells enriched for cells expressing one or more markers of pluripotency obtained by selecting and isolating the pluripotent cells expressing the one or more markers.

4. The induced pluripotent cell, cell line thereof, or population thereof of claim 3 , wherein the enriched pluripotent cells maintain pluripotency for at least 5, 10, 15, or 20 passages when cultured in the feeder-free culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor.

5. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the pluripotent cell comprises reactivated expression of one or more of GATA6, CDX2, or CGB.

6. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the pluripotent cell has reduced Xist activity and/or enhanced expression of genes of the X chromosome in comparison to pluripotent cells not cultured in the feeder-free culture medium composition.

7. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the feeder-free culture medium composition is free of additional cytokines and growth factors.

8. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the feeder-free culture medium composition comprises a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor.

9. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the induced pluripotent cell is a single cell dissociated induced pluripotent cell.

10. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the Rock inhibitor is thiazovivin.

11. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the induced pluripotent cell is an isolated cell expressing one or more markers of pluripotency.

12. The induced pluripotent cell, cell line thereof, or population thereof of claim 11 , wherein the induced pluripotent cell has a higher clonality than pluripotent cells not cultured in the feeder-free culture medium composition.

13. The induced pluripotent cell, cell line thereof, or population thereof of claim 11 , wherein the population of induced pluripotent cells is an isolated clonal population of a single induced pluripotent cell.

14. An isolated induced pluripotent cell, a cell line thereof, or a population thereof, wherein the cell:
(a) is in contact with a feeder-free culture medium composition comprising a Rock inhibitor, and one or more of a GSK3 inhibitor, a MEK inhibitor and a TGFβ inhibitor;
(b) comprises one or more exogenous polynucleotides encoding proteins comprising Oct4, and optionally, one or more of Sox2 and Klf4;
(c) is a naïve human induced pluripotent stem cell (iPSC) with ground state pluripotency; and
(d) the iPSC (i) exhibits reactivated expression of one or more of GATA6, CDX2, or CGB, or (ii) is a cell in a colony having a domed morpholgy.

15. The pluripotent cell, cell line thereof, or population thereof of claim 14 , wherein the induced pluripotent cell has a higher clonality than pluripotent cells not cultured in said feeder-free culture medium composition; or wherein the population of induced pluripotent cells is a clonal population.

16. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein (a) the ROCK inhibitor is thiazovivin or Y27632; (b) the GSK3 inhibitor is CHIR99021 or BIO; (c) the MEK inhibitor is PD98059 or PD0325901, and (d) the TGFβ inhibitor is A-83-01 or SB431542.

17. The induced pluripotent cell, cell line thereof, or population thereof of claim 14 , wherein (a) the ROCK inhibitor is thiazovivin or Y27632; (b) the GSK3 inhibitor is CHIR99021 or BIO; (c) the MEK inhibitor is PD98059 or PD0325901, and (d) the TGFβ inhibitor is A-83-01 or SB431542.

18. The induced pluripotent cell, cell line thereof, or population thereof of claim 11 , wherein (a) the ROCK inhibitor is thiazovivin or Y27632; (b) the GSK3 inhibitor is CHIR99021 or BIO; (c) the MEK inhibitor is PD98059 or PD0325901, and (d) the TGFβ inhibitor is A-83-01 or SB431542.

19. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the induced pluripotent cell has improved genomic stability, as indicated by a lower propensity for genomic abnormalities compared to pluripotent cells that have not been contacted with the feeder-free culture medium composition.

20. The induced pluripotent cell, cell line thereof, or population thereof of claim 1 , wherein the iPSC is a cell in a colony having a domed morphology.

Assignees

• Fate Therapeutics Inc

Inventors

• Valamehr Bahram
• Abujarour Ramzey
• Flynn Peter

Classifications

• C12N5/0081

  Purging biological preparations of unwanted cells · CPC title

• C12N2509/10

  Mechanical dissociation · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2501/60

  Transcription factors · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title