Wnt inhibitor iwr1

Feeder-free hiPSCs (iPS-DF19-9-7T and iPS-DF6-9-9T, WiCell) were cultured on hESC-qualified matrigel (Corning) with StemMACS iPSC-Brew XF media (Miltenyi Biotec). Conditions to differentiate hiPSCs into CMs in this work were tested against an established WNT-based cardiogenesis protocol (Lian et al., 2012 (link), 2013 (link)). Briefly, confluent hiPSCs were differentiated in RPMI 1640 media supplemented with B-27 supplement minus insulin (ThermoFisher Scientific) from days 0–7. To promote cardiogenesis, RPMI media was supplemented with 6 μM GSK3 inhibitor CHIR99021 (Tocris), and 5 μM WNT inhibitor IWR1 (Tocris) from differentiation days 0–2 and 3–5, respectively. Small molecule Nodal inhibitor SB431542 (Tocris) was added either at days 0–2, 3–5 or 5–7 as specified in the results section.

HiPSC line, iPS-DF6–9-9T.B (WiCell), was cultured in StemMACS iPS-Brew XF (Miltenyi Biotec) in dishes coated with hESC-Qualified Matrigel (Corning) at 37°C with 5% CO₂ and subsequently differentiated into PCMs as previously described.^{11 (link)} Briefly, confluent hiPSCs were differentiated in RPMI 1640 media supplemented with B-27 supplement minus insulin (ThermoFisher Scientific, 11875119 and A1895601) from days 0–7. To promote cardiogenesis, RPMI media was supplemented with 6 μM GSK3 inhibitor CHIR99021 (Tocris) from differentiation days 0–2 and with 5 μM WNT inhibitor IWR1 (Tocris) from differentiation days 3–5. Small molecule Nodal inhibitor SB431542 (Tocris) was added at days 3–5 to differentiate PCMs (Figure 1A). After D7 post-differentiation, hiPSC-CMs were maintained in RPMI 1640 media supplement with B-27 supplement with insulin (ThermoFisher Scientific, 17504044). Differentiated hiPSC-PCM culture were characterized by flow cytometry for CM yield by cTNT, immunostained for cTNT and pacemaking HCN4 channel, and classified into CM subtypes by optically recorded APs of isolated single cells by FluoVolt. hiPSC-PCMs were used to recellularize the porcine ECM scaffolds to test the role ECM in PCM phenotype and function.