Edta dissociation reagent

NSC-34 (86 (link)) cells were cultured in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) (Invitrogen), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Bovogen). In order to passage and plate NSC-34 cells, they were washed once with prewarmed DMEM-F12 and treated with 0.25% trypsin and 0.02% EDTA dissociation reagent (Invitrogen) to lift off the adherent cells. The cells were pelleted via centrifugation (300g for 5 min) and resuspended in prewarmed culture media. Following washing, plates were seeded at a confluency of 40% and cultured at 37 °C in a humidified incubator with 5% atmospheric CO₂ for 24 h prior to transfection (∼70–80% confluent). Cells were transfected with plasmid DNA (0.5 μg per well of a 24-well plate, 2.5 μg per well of a 6-well plate) 24 h postplating using TransIT-X2 reagent (Mirus Bioscience) according to the manufacturer's instructions.

The human induced pluripotent stem cell line (P356) was generated by reprogramming of healthy human peripheral blood mononuclear cells using Sendai virus-mediated transduction of Sox2, Oct3/4, KLF4, and c-myc (Carcamo-Orive et al., 2017 (link)). This line was used for subcutaneous implantation studies. The human embryonic stem cell line (H9, WiCell Research Institute, Madison, WI) was used for epicardial transplantation studies. Both lines were passaged every 4 days using ethylenediaminetetraacetic acid (0.1 mM, EDTA) dissociation reagent (Invitrogen). The PSC cells were expanded on tissue culture dishes coated with Geltrex basement membrane matrix extract (1:200 dilution, Sigma) in Essential 8 expansion medium (Invitrogen). All cells were maintained in humidified incubators at 37°C and 5% CO₂.