Gibco™ MEM α, GlutaMAX™ Supplement, no nucleosides

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Cardiac Differentiation: Cardiac cells were produced using recently developed protocols where cardiomyocytes and stromal cells are produced in the same differentiation culture (Hudson et al., 2012, Mills et al., 2017a, Mills et al., 2017b, Voges et al., 2017); multi-cellular cultures are critical for function (Hudson et al., 2011, Tiburcy et al., 2017). hESCs were seeded at 2 × 104 cells/cm2 in Matrigel-coated flasks and cultured for 4 days using mTeSR-1. They were then differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement without insulin, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (all ThermoFisher Scientific unless otherwise indicated)) containing 5 ng/mL BMP-4 (RnD Systems), 9 ng/mL Activin A (RnD Systems), 5 ng/mL FGF-2 (RnD Systems) and 1 μM CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days. Subsequently, they were specified into a hPSC-CM/stromal cell mixture using RPMI B27- containing 5 μM IWP-4 (Stem Cell Technologies) followed by another 7 days of RPMI B27+ (RPMI1640 GlutaMAX + 2% B27 supplement with insulin, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin) with medium exchange every 2-3 days. The differentiated cells were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca2+ and Mg2+) for 60 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min. The cells were filtered using a 100 μm mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in CTRL medium: α-MEM GlutaMAX, 10% FBS, 200 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin. Based on flow cytometry the cells generated and used for tissue engineering were ∼70% α-actinin+/CTNT+ hPSC-CMs with the rest being predominantly CD90+ stromal cells (Voges et al., 2017), which are critical for function (Hudson et al., 2011, Tiburcy et al., 2017).

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