128 Chapter 6 have shown that three weeks of culture in a medium with physiological levels of glucose and Ca2+, supplemented with a fine-tuned composition of additives, induced metabolic, structural, electrophysiological and mechanical iPSC-CM maturation characterized by lower resting membrane potential, rapid depolarization, increased sarcoplasmic reticulum calcium cycling, increased contractile force and enhanced fatty acid oxidation, in line with cardiomyocyte maturation during cardiac development.18 In this study, we sought to investigate the utilization of metabolically matured iPSC-CMs to model cardiac ischemic damage and exemplarily evaluate the cardioprotective effect of the RIP1 kinase inhibitor necrostatin-1. METHODS Cell culture. The female iPSC line SCVI-273 (Sendai virus reprogrammed) was kindly provided by Joseph Wu, Stanford University.26 The female iPSC line NP0141-31B was generated in the Šarić group from peripheral blood mononuclear cells, also using the Sendai virus reprogramming method.27,28 This line is deposited as cell line UKKi032-C at the European Bank for induced Pluripotent Stem Cells (EBiSC, www.ebisc.org) and is listed at the international online registry hPSCreg (www.hpscreg.eu). iPSCs were cultured in Essential 8 medium (Gibco, A1517001) in 0,1 mg/mL matrigel (Corning, CLS356252) coated-6 well plates. When reaching 80-90% confluency, directed differentiation to CMs was initiated by changing medium to RPMI 1640 (ThermoFisher Scientific, 11875085) supplemented with 2% B27 minus insulin (ThermoFisher Scientific, A1895601) (B27- medium) and 7 μM CHIR99021 (Selleck Chemicals, S2924). After 3 days, B27-medium was changed and supplemented with 2 μM Wnt-C59 (R&D systems, 5148) to inhibit canonical Wnt signaling. On day 7, the medium was changed to RPMI 1640 and 2% B27 plus insulin supplement (ThermoFisher Scientific, 17504001) (B27+ medium) and on day 9 to RPMI 1640 without glucose (ThermoFisher Scientific, 118979020) and 2% B27 plus insulin for purification (depletion of non-CM cells). On day 11 cells were re-plated in RPMI/B27 plus insulin supplemented with 10% KnockOut serum replacement (KOSR, ThermoFisher Scientific, 108280028) and 10 µM selective Rock-1 inhibitor Y-27632 (Selleck Chemicals, S1049). After the second purification on day 14 for two days in RPMI 1640 without glucose, the medium was changed to RPMI/B27 plus insulin. On day 20, the purification medium was changed to maturation medium18 or cells were kept in RPMI/B27 plus insulin. Cells were matured in maturation medium for three weeks before re-plating with medium changes every four days. Damage induction. 4 days before applying hypoxic conditions at day 20, the medium was changed to medium containing variable nutrient compositions (Supplementary table 1). Ischemia was induced by incubation at 5% O2 in a hypoxia incubator (In-VitroCell NU-5800, NuAire) providing continuous oxygen monitoring or ~1% O2 using the GasPak EZ Pouch system (BD, 260683) with indicator strips to confirm O2 concentrations below 1%.
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