Method for inducing differentiation of pluripotent stem cells into cardiomyocytes

• US 8,293,529 B2
• Filed: 04/27/2007
• Issued: 10/23/2012
• Est. Priority Date: 04/28/2006
• Status: Expired due to Fees

• Current Assignee: Daiichi Sankyo Company Limited
• Original Assignee: Daiichi Sankyo Company Limited
• Inventors: Koshimizu, Uichi, Tanaka, Tomofumi, Kawashima, Kayoko, Kadokura, Michinori
• Primary Examiner(s): Vogel, Nancy T
• Application Number:
  US12/298,565
• Publication Number:
  US 20090325288A1
• Time in Patent Office: 2,006 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The present invention provides a method for inducing differentiation of cardiomyocytes efficiently and selectively from stem cells.

A method for inducing differentiation of cardiomyocytes from pluripotent stem cells, which comprises: (i) culturing the pluripotent stem cells in a culture medium containing no substance that promotes activation of the canonical Wnt signaling pathway during the time period between initiation of differentiation induction and 24 hours before the period of elevated canonical Wnt gene expression; and then (ii) culturing the pluripotent stem cells in a culture medium containing a substance that promotes activation of the canonical Wnt signaling pathway during a time period of 24 to 96 hours, starting from 24 to 0 hours before the period of elevated canonical Wnt gene expression.

Claims

1. A method for inducing differentiation of cardiomyocytes from mammalian pluripotent stem cells, which comprises:

i) culturing the mammalian pluripotent stem cells in a culture medium containing no substance that promotes activation of the canonical Wnt signaling pathway during the time period between initiation of differentiation induction and 24 hours before the period of elevated canonical Wnt gene expression; and

then

ii) culturing the mammalian pluripotent stem cells in a culture medium containing a substance that promotes activation of the canonical Wnt signaling pathway during a time period of 24 to 96 hours, starting from 24 to 0 hours before the period of elevated canonical Wnt gene expression, wherein the substance that promotes activation of the canonical Wnt signaling pathway is a substance selected from the group consisting of a canonical Wnt protein, a GSK3β

inhibitor, and a Wnt agonist.

2. The method according to claim 1, wherein the mammalian pluripotent stern cells are cultured in a culture medium containing a substance that promotes activation of the canonical Wnt signaling pathway, starting from 24 hours before the period of elevated canonical Wnt gene expression.

3. The method according to claim 1, wherein the mammalian pluripotent stem cells are cultured in a culture medium containing a substance that promotes activation of the canonical Wnt signaling pathway during a time period of 48 to 72 hours.

4. The method according to claim 1, wherein the substance that promotes activation of the canonical Wnt signaling pathway is a canonical Wnt protein.

5. The method according to claim 4, wherein the canonical Wnt protein is at least one Wnt protein selected from the group consisting of Wnt-1, Wnt-3a and Wnt-5a.

6. The method according to claim 4, wherein the concentration of the canonical Wnt protein in the culture medium is 0.1 ng/ml, to 500 ng/mL.

7. The method according to claim 1, wherein the substance that promotes activation of the canonical Wnt signaling pathway is a GSK3β

inhibitor.

8. The method according to claim 7, wherein the GSK3β

inhibitor is at least one inhibitor selected from the group consisting of GSK3β

inhibitor VII, L803-mts, SB216763 and GSK3β

inhibitor IX (BIO).

9. The method according to claim 7, wherein the concentration of the GSK3β

inhibitor in the culture medium is 2 μ

mol/L to 100 μ

mol/L for GSK3β

inhibitor VII, 5 μ

mol/L to 500 μ

mol/L for L803-mts, 10 nmol/L to 1 μ

mol/L for SB216763, or 10 nmol/L to 1 μ

mol/L for GSK3β

inhibitor IX (BIO).

10. The method according to claim 1, wherein the substance that promotes activation of the canonical Wnt signaling pathway is a Wnt agonist.

11. The method according to claim 10, wherein the Wnt agonist is 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)-primidine.

12. The method according to claim 10, wherein the concentration of the Wnt agonist in the culture medium is 1 nmol/L to 1000 nmol/L.

13. The method according to claim 1, wherein the mammalian pluripotent stem cells are mammalian embryonic stem cells, mammalian embryonic germ cells or mammalian germline stem cells.

14. The method according to claim 13, wherein the mammalian pluripotent stem cells are mammalian embryonic stem cells.

15. The method according to claim 13, wherein the mammalian pluripotent stem cells are of human origin.