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 increased production of cardiomyocytes, wherein the method comprises the steps of:
a) incubating pluripotent stem cells from a selected cell clone in a suspension culture under conditions to promote aggregate formation; and
b) differentiating the stem cells into cardiomyocytes in a cardiac differentiation medium prepared from a selected batch of culture medium, the differentiation medium having been prepared from said culture medium by adjusting the level of one or more differentiation factors in said culture medium at amounts determined to be appropriate for cardiac differentiation of the selected cell clone and culture media batch employed.
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 1 , wherein from about 10 6 to about 10 10 of the pluripotent stem cells are first incubated in the suspension culture in step a).
6 . The method of claim 1 , wherein the conditions to promote aggregate formation comprises externally added ROCK inhibitor, myosin II inhibitor, fibroblast growth factor (FGF) or hepatic growth factor (HGF).
7 . The method of either of claim 4 or 6 , wherein the myosin II inhibitor is blebbistatin.
8 . The method of claim 1 , wherein the aggregates formed from the pluripotent stem cells prior to cardiac differentiation are about 10 nm to 400 μm in diameter.
9 . The method of claim 1 , wherein the suspension culture for differentiating the stem cells has a volume of from 5 milliliters to 25 liters.
10 . The method of claim 1 , wherein the pluripotent stem cells and/or cardiomyocytes differentiated therefrom contain one or more transgenes.
11 . The method of claim 10 , wherein the one or more transgenes encode a selectable and/or screenable marker under the control of a cardiomyocyte-specific promoter.
12 . The method of claim 1 , further comprising enriching or purifying the differentiated cardiomyocytes.
13 . The method of claim 1 , wherein the cardiac differentiation medium comprises externally added fibroblast growth factor (FGF) in an amount of from 5 to 200 ng/ml.
14 . The method of claim 1 , wherein the suspension culture is rotated or shaken at a speed of about 15 rpm to 100 rpm.
15 . The method of claim 1 , wherein the culture medium is TeSR, mTeSR, RPMI medium, supplemented DMEM-F12 or dilutions thereof.
16 . The method of claim 1 , wherein the differentiation factors whose level are adjusted in the culture medium comprise one or more of modulators of signaling pathways of bone morphogenetic protein, ActivinA/Nodal, vascular endothelial growth factor (VEGF), dickkopf homolog 1 (DKK1), basic fibroblast growth factor (bFGF), insulin growth factor (IGF), and/or epidermal growth factor (EGF).
17 . The method of claim 16 , wherein the differentiation factors comprises BMP2, BMP4, BMP10, Activin A, bFGF, IGF, EGF, BMP signaling inhibitor, Activin signaling inhibitor, or a combination thereof.
18 . The method of claim 17 , wherein the BMP signaling inhibitor comprises dorsomorphin.
19 . The method of claim 17 , wherein the Activin A signaling inhibitor comprises SB431542.
20 . An isolated cell population of about 5×10 8 to 10 10 cells comprising at least 99% cardiomyocytes.