hESC-Derived Cardiomyocyte Culture and Differentiation

Undifferentiated RUES2 hESCs were maintained as previously described5 on Matrigel (Corning Life Sciences) in mouse embryonic fibroblast (MEF)-conditioned media supplemented with 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech). Cardiomyocyte differentiation was performed as described previously using a combination of small molecules and growth factors.5 A high-density cell monolayer was pre-treated with 1 μM CHIR99021 for 6–24 h (Cayman Chemical) and induced with 100 ng/mL activin A (R&D Systems) and 1× Matrigel in RPMI 1640 with B27 supplement minus insulin (Life Technologies). Medium was changed after 18 h to RPMI 1640 with B27 supplement (minus insulin) supplemented with 1 μM CHIR99021 and 5 ng/mL BMP4 (R&D Systems). After 2 days, media were changed and supplemented with XAV939 (Tocris) for an additional 48 h. After day 7 of differentiation, RPMI 1640 with B27 supplement containing insulin (Life Technologies) was used to maintain cells. Beating was typically observed between days 7 and 10, and medium was changed every 2–3 days thereafter. Unless otherwise noted, cardiomyocytes were cryopreserved on day 21–24 of differentiation for long-term storage and thawed 2–3 days prior to implantation to allow for recovery.48 For cryopreservation, cells were heat shocked for 1 h at 42°C the day prior. After a 1-h pretreatment with 10 μM ROCK inhibitor Y-27632 (Tocris), cardiomyocytes were harvested by a brief incubation with EDTA and dispersed into single cells using 0.25% trypsin-EDTA (Life Technologies). Cells were washed and resuspended in CryoStor (Sigma), added to cryovials, and frozen to −80°C in a controlled rate freezer with a decrease in temperature of 1°C/min before transfer to liquid nitrogen for long-term storage. To thaw, cryovials were agitated briefly at 37°C, collected in RPMI 1640 supplemented with 200 U/mL DNase (VWR), and washed with basal medium. Cardiomyocytes were replated onto Matrigel-coated culture dishes in RPMI 1640 with B27 containing insulin and supplemented with 10 μM Y-27632 for the first 24 h. Flow cytometry for cardiac troponin T (1:100, Thermo Scientific) was used to characterize cardiomyocyte population purity after cardiac differentiation and again at the time of harvest for implantation.