Stem cell culture medium and method

Patent metadata

Field	Value
Publication number	US-10626366-B2
Application number	US-201615250848-A
Country	US
Kind code	B2
Filing date	Aug 29, 2016
Priority date	Sep 22, 2006
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

Stem cells such as embryonic stem cells (ES cells), including human ES cells, are cultured in a medium comprising a ROCK inhibitor, and a stem cell culture medium, optionally serum free, comprises a ROCK inhibitor.

First claim

The invention claimed is:

1. A method of differentiating human pluripotent stem cells to neural cells, which comprises
(a) pre-treating human pluripotent stem cells with a Rho-kinase (ROCK) inhibitor for about 1 hour before dissociating the human pluripotent stem cells into single cells,
(b) dissociating the human pluripotent stem cells into single cells, and
(c) replating the single cells in (i) an adherent culture containing a neural cell differentiation medium with a ROCK inhibitor for at least 12 hours after dissociation and replating or (ii) a suspension culture containing a neural cell differentiation medium with a ROCK inhibitor for at least 2 days after the dissociating and replating,
thereby differentiating human pluripotent stem cells to neural cells.

2. The method of claim 1 , wherein the ROCK inhibitor is present in the medium during a portion of the period of inducing differentiation of the human pluripotent stem cells to neural cells.

3. The method of claim 1 or 2 , wherein the method comprises inducing differentiation of the human pluripotent stem cells to neural cells and their precursors according to a serum-free floating culture of embryoid body-like aggregates (SFEB) method.

4. The method of claim 3 , wherein the method comprises inducing differentiation of the human pluripotent stem cells to neural cells and their precursors in the presence of a factor selected from the group consisting of Nodal inhibitors, Wnt inhibitors, and BMP inhibitors.

5. The method of claim 1 or 2 , wherein the neural cells are neural precursor cells.

6. The method of claim 5 , wherein the neural precursor cells are Nestin and Pax6 positive cells.

7. The method of claim 1 or 2 ,
wherein (b) dissociating the human pluripotent stem cells into single cells occurs in the presence of a ROCK inhibitor.

8. The method of claim 1 or 2 , wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

9. The method of claim 1 or 2 , wherein the human pluripotent stem cells are human embryonic stem cells.

10. The method of claim 2 , wherein the ROCK inhibitor is present in the medium for between 12 hours and six days.

11. The method of claim 2 , wherein the ROCK inhibitor is present in the medium for between one and five passages.

12. The method of claim 2 , wherein after the portion of the period during which differentiation to neural cells is induced, the ROCK inhibitor is withdrawn from the medium.

13. The method of claim 1 , wherein (c) the single cells are replated in (i) an adherent culture containing a neural cell differentiation medium with a ROCK inhibitor for at least 12 hours after dissociation and replating.

14. The method of claim 13 , wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

15. The method of claim 13 , wherein the human pluripotent stem cells are human embryonic stem cells.

16. The method of claim 1 , wherein (c) the single cells are replated in (ii) a suspension culture containing a neural cell differentiation medium with a ROCK inhibitor for at least 2 days after the dissociating and replating.

17. The method of claim 16 , wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

18. The method of claim 16 , wherein the human pluripotent stem cells are human embryonic stem cells.

19. The method according to claim 16 , wherein the ROCK inhibitor is present in the suspension culture containing a neural cell differentiation medium for at least 6 days after dissociating and replating.

20. The method of claim 19 , wherein the ROCK inhibitor is Y-27632, Fasudil, or H-1152.

21. The method of claim 19 , wherein the human pluripotent stem cells are human embryonic stem cells.

Assignees

• Riken

Inventors

• Sasai Yoshiki
• Watanabe Kiichi

Classifications

• C12N5/0606 Primary

  Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696) · CPC title

• C12N2501/999

  Small molecules not provided for elsewhere · CPC title

• C12N2506/00

  Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells · CPC title

• C12N2501/70

  Enzymes · CPC title

• C12N5/0603

  Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies · CPC title