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

Patent metadata

Field	Value
Publication number	US-12415989-B2
Application number	US-202017039006-A
Country	US
Kind code	B2
Filing date	Sep 30, 2020
Priority date	Dec 22, 2010
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

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 sorting a population of human cells to obtain a population of cells enriched for pluripotent cells comprising:
a) suspending a mixed population of dissociated cells to thereby produce a suspension comprising the mixed population of cells, wherein the mixed population of cells comprises human induced pluripotent stem cells (hiPSCs) having ground state pluripotency and human non-pluripotent cells; and
b) sorting the cells in the suspension for cells expressing one or both of SSEA4 and TRA181, thereby obtaining an enriched population of cells enriched for the hiPSCs, wherein the hiPSCs of the enriched population of cells comprise the hiPSCs having ground state pluripotency; and
wherein the hiPSCs having ground state pluripotency in the mixed population of cells and the enriched population of cells have the ability to reactivate GATA6, CDX2, and CGB upon differentiation.

2. The method of claim 1 , wherein the suspension comprises at least one of i) a TGFβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor, or iv) a Rock inhibitor.

3. The method of claim 1 , wherein sorting is by magnetic beads or flow cytometry.

4. The method of claim 1 , further comprising culturing the enriched population of cells in a culture medium comprising at least one of i) a TGFβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor, or iv) a Rock inhibitor, optionally in combination with soluble fibronectin.

5. The method of claim 1 , wherein the sorting comprises sorting the cells in the suspension for cells expressing both of SSEA4 and TRA181.

6. The method of claim 1 , wherein the hiPSCs are produced by contacting non-pluripotent cells with one or more pluripotency factors to induce reprogramming.

7. The method of claim 4 , wherein hiPSCs cultured in the culture medium comprise hiPSCs with ground state pluripotency.

8. The method of claim 4 , wherein the human induced pluripotent stem cells cultured in the culture medium have a higher clonality than human induced pluripotent stem cells not cultured in the culture medium.

9. The method of claim 7 , further comprising culturing the hiPSCs with ground state pluripotency to form colonies having a domed morphology.

10. The method of claim 1 , further comprising preparing the suspension of dissociated cells by culturing an initial cell population comprising the human induced pluripotent stem cells (hiPSCs) in a culture medium, wherein the culturing comprises inhibiting each of TGFβ, GSK3, MEK, and ROCK.

11. The method of claim 1 , wherein the suspension of dissociated cells is feeder-free.

Assignees

• Fate Therapeutics Inc

Inventors

• Valamehr Bahram
• Abujarour Ramzey
• Flynn Peter

Classifications

• C12N5/0607

  Non-embryonic pluripotent stem cells, e.g. MASC (induced pluripotent stem cells C12N5/0696) · CPC title

• C12N5/0081

  Purging biological preparations of unwanted cells · CPC title

• C12N2533/54

  Collagen; Gelatin · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C12N2506/1307

  from adult fibroblasts · CPC title