Df19 9 11t h

CPCs were obtained from human right atria appendage myocardial tissue, isolated, and characterized as described elsewhere [14 (link)]. Cells were cultured at 37 °C in humidified incubators (5% CO₂, 3% O₂) in expansion medium (ExpM) composed by DMEM:F12: Neurobasal medium (1:1), supplemented with 1% penicillin streptomycin, 10% fetal bovine serum embryonic stem cell-qualified, N2 supplement (1×), B27 supplement (1× ), 0.9 mM l-glutamine, 50 μM β-mercaptoethanol (Sigma), insulin transferrin selenium (0.5× ), 10 ng/mL bFGF, 20 ng/mL EGF-I, and 30 ng/mL IGF-II (Prepotech) (all percentages in v/v). Medium was replaced by 50% every 3 days. Cells were subcultured when about 80% confluent using Tryple™ Select Enzyme for 5 min at 37 °C. All cell culture reagents were purchased from Gibco, Life Technologies unless otherwise stated.
Human iPSCs (DF19-9-11 T.H, WiCell) were cultured and differentiated to CMs (hiPSC-CMs) as previously described [25 (link), 26 (link)]. Using this protocol, monolayer cultures composed of > 90% of hiPSC-CMs were obtained after 15 days. To further improve the maturation state of this cell population, hiPSC-CMs were cultured for additional 10 days in Pluricyte Medium (NCardia), as described elsewhere [27 ]. Cells were maintained at 37 °C in humidified incubators (5% CO₂, 95% air).

In this study, hiPSC line DF19-9-11T.H from WiCell was used. hiPSCs were cultured on Matrigel^® (Corning)–coated plates in mTESR1 medium (STEMCELL Technologies, hereafter designated as expansion medium) at 37°C, in a humidified atmosphere of 5% CO₂ (vol/vol) in air. Cells were routinely subcultured when reaching 80% confluence using Versene (Thermo Fisher Scientific) as described by our group (Correia et al., 2016 (link)).