Chemically defined production of cardiomyocytes from pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9234176-B2
Application number	US-201314079312-A
Country	US
Kind code	B2
Filing date	Nov 13, 2013
Priority date	Nov 13, 2012
Publication date	Jan 12, 2016
Grant date	Jan 12, 2016

Abstract

Methods are provided for producing a cardiomyocyte population from a mammalian pluripotent stem cell population. Aspects of the methods include using a Wnt signaling agonist and antagonist, each in minimal media, to modulate Wnt signaling. Also provided are kits for practicing the methods described herein.

First claim

That which is claimed is:

1. A method of producing a cardiomyocyte population from a human pluripotent stem cell population, the method comprising:
(a) contacting the human pluripotent stem cell population with an effective amount of a Wnt signaling agonist in a minimal media for a period of about 12-60 hours, to produce an agonist-contacted cell population; and
(b) contacting the agonist-contacted cell population with an effective amount of a Wnt signaling antagonist in a minimal media for a period of at least 12 hours
wherein the minimal media is selected from the group consisting of: CDM3, D3 media, D4 media, and D11 media.

2. The method according to claim 1 , wherein the Wnt signaling agonist is an inhibitor of GSK-3β.

3. The method according to claim 2 , wherein the inhibitor of GSK-3β is BIO, CHIR-99021, or a combination thereof.

4. The method according to claim 1 , wherein the Wnt signaling antagonist is a compound selected from the group consisting of: C59, IWR-1, IWP-2, IWP-4, XAV-939, and combinations thereof.

5. The method according to claim 4 , wherein the Wnt signaling antagonist is C59.

6. The method according to claim 1 , wherein the human pluripotent stem cell population is a population of embryonic stem cells or a population of induced pluripotent stem cells.

7. The method according to claim 1 , wherein the minimal media is a chemically defined minimal media.

8. The method according to claim 7 , wherein the chemically defined minimal media is CDM3 media.

9. The method according to claim 1 , wherein prior to contact with the Wnt signaling agonist, the mammalian pluripotent stem cell population is cultured in maintenance media.

10. The method according to claim 9 , wherein the maintenance media is supplemented with a ROCK inhibitor for a period of about 24 hours each time the human pluripotent stem cell population is passaged.

11. The method according to claim 1 , wherein the human pluripotent stem cell population is cultured as a monolayer on a matrix.

12. The method according to claim 1 , further comprising the step of verifying the presence of cardiomyocytes in the antagonist-contacted cell population.

13. The method according to claim 12 , wherein verifying comprises one or more of: determining a cardiomyocyte electrophysiological profile; determining responsiveness of the antagonist-contacted cell population to known cardioactive drugs; or contacting the antagonist-contacted cell population with an antibody specific for a cardiomyocyte marker protein, and determining the percentage of cells positive for expression, wherein cells positive for expression are cardiomyocytes.

14. The method according to claim 12 , wherein at least 80% of the cells of the antagonist-contacted cell population are determined to be cardiomyocytes.

15. The method according to claim 1 , wherein cells are not contacted with the compounds SB431542, LY364947, dorsomorphin, LDN 193189, or SB203580.

16. The method according to claim 1 , further comprising a step of contacting the antagonist-contacted cell population with a minimal media lacking glucose for a period of time sufficient to enrich the cell population for cardiomyocytes.

Assignees

• Univ Leland Stanford Junior

Inventors

• Wu Joseph
• Robbins Robert C
• Burridge Paul W

Classifications

• C12N2501/415

  Wnt; Frizzeled · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title

• C12N5/0657 Primary

  Cardiomyocytes; Heart cells · CPC title

• C12N2501/727

  Kinases (EC 2.7.) · CPC title

• C12N2500/38

  Vitamins · CPC title