Cardiomyocyte production
Methods and composition for the production of cardiomyocytes from differentiation of pluripotent stem cells are provided. For example, in certain aspects methods including differentiating pluripotent stem cells in a large volume of suspension culture in the presence of ROCK inhibitors are described. In further aspects, methods for differentiation of stem cells into cardiomyocytes that overcome variability between different stem cell clones and different batch of culture medium are provided.
1. A method for producing human cardiomyocytes comprising the steps of:
a) culturing a selected human pluripotent stem cell clone having been prepared from a single pluripotent stem cell, the human stem cell clone being either a human embryonic stem cell clone or a human induced pluripotent stem cell clone;
b) individualizing the cultured human pluripotent stem cells of step (a);
c) culturing the individualized human pluripotent stem cells obtained from step (b) under defined conditions in suspension culture in an aggregate promotion media comprising fibroblast growth factor 2 (FGF2), and a Rho-associated protein kinase (ROCK) inhibitor or a myosin II inhibitor, to form cell aggregates;
d) culturing said cell aggregates in cardiac differentiation medium comprising FGF2, Activin, and bone morphogenetic protein 4 (BMP4) to form cardiomyocytes; and
e) obtaining the cardiomyocytes.
2. The method of claim 1 , wherein the selected cell clone is an induced pluripotent stem (iPS) cell clone.
3. The method of claim 1 , wherein the selected cell clone is derived from a single pluripotent stem cell in an adherent culture.
4. The method of claim 3 , wherein the selected cell clone is derived from a single pluripotent stem cell by a process comprising incubating the single pluripotent stem cell in medium comprising a Rho-associated kinase (ROCK) inhibitor or a myosin II inhibitor under conditions to promote cell growth.
5. The method of claim 4 , wherein the myosin II inhibitor is blebbistatin.
6. The method of claim 1 , wherein from about 10 6 to about 10 10 of the pluripotent stem cells are incubated in the suspension culture.
7. The method of claim 1 , wherein the suspension culture for differentiating the stem cells has a volume of from 5 milliliters to 25 liters.
8. The method of claim 1 , wherein the pluripotent stem cells and/or cardiomyocytes differentiated therefrom contain one or more transgenes.
9. The method of claim 8 , wherein the one or more transgenes encode a selectable and/or screenable marker under the control of a cardiomyocyte-specific promoter.
10. The method of claim 1 , further comprising enriching or purifying the differentiated cardiomyocytes.
11. The method of claim 1 , wherein the cardiac differentiation medium comprises externally added fibroblast growth factor 2 (FGF-2) in an amount of from 5 to 200 ng/ml.
12. The method of claim 1 , wherein the suspension culture is rotated or shaken at a speed of about 15 rpm to 100 rpm.