Chemically defined production of cardiomyocytes from pluripotent stem cells
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.
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.