301 Modeling and Rescue of PLN-R14del Cardiomyopathy Phenotype in Human iPSC-Derived Cardiac Spheroids 12 hours. Differentiated hiPSC-CMs were maintained with RPMI + 2% B27 medium. All subsequent experiments were conducted by using hiPSC-CMs differentiated for 20 to 30 days. Differentiation of hiPSC to CFs. Cardiac fibroblast (CF) differentiation was carried out following a small molecule Wnt-activation protocol as previously described.22 Briefly, hiPSCs were first treated with CHIR99021 (12 μM; Tocris) in RPMI+ 2% B27 without insulin (Life Technologies) for 24 hours, and then in RPMI+ 2% B27 without insulin for another 24 hours. Media was replaced every other day with CFBM medium consisting of DMEM, high glucose (Gibco) supplemented with 500 μg/mL HAS (Sciencell), 0.6 μM Linoleic Acid (SigmaAldrich), 0.6 μg/mL Lecithin(Sigma-Aldrich), 50 μg/mL Ascorbic Acid (Sigma-Aldrich), 7.5 mM GlutaMAX (Thermo Scientific), 1.0 μg/mL Hydrocortisone Hemisuccinate (Millipore Sigma), 5 μg/mL recombinant human Insulin (Sigma-Aldrich) and the fresh addition of 75 ng/ml bFGF (Sigma-Aldrich). All subsequent experiments were conducted by using hiPSC-CFs that were differentiated for at least 20 days and passaged 3-6 times. Cardiac spheroid generation. Human cardiac spheroid (hCS) generation was achieved following a protocol that was previously described.23 Briefly, hiPSC-CMs were digested by TrypLE Select Enzyme (10X, Thermo Fisher Scientific) for 15 min at 37°C. Next, 10.000 hiPSCCM were collected and added to a 96- or 384-well ultra-low attachment multiple-well plate (Corning) in RPMI-B27 supplemented with 10% knockout serum replacement (KOSR) and 10 μM Y-27632 (Tocris). The hCSs culture plates were kept on a rotation plate, 70 rpm at 37°C, and 5% CO2. Medium change was performed after 24 hours with a basal medium without KOSR and Y-27632. After 21 days, the hCSs were used for downstream analysis. Spheroid maturation. Spheroid maturation was achieved by a previously described maturation medium used for hiPSC-CMs.24 The maturation medium consists of DMEM without glucose (Thermo Fisher Scientific, 11966025) supplemented with 3mM glucose (Sigma Aldrich, G7021), 10mM L-lactate (Sigma Aldrich, 71718), 5μg/ml Vitamin B12 (Sigma Aldrich, V6629), 0.82μM Biotin (Sigma Aldrich, B4639), 5mM Creatine monohydrate (Sigma Aldrich, C3630), 2mM Taurine (Sigma Aldrich, T0625), 2mM L-carnitine (Sigma Aldrich, C0283), 0.5mM Ascorbic acid (Sigma Aldrich, A8960), 1x NEAA (Thermo Fisher Scientific, 11140), 0.5% (w/v) Albumax (Thermo Fisher Scientific, 11020021), 1x B27 and 1% KOSR (Thermo Fisher Scientific, 10828028). After 48 hours, the medium was replaced by the maturation medium for 1, 2, 3, or 6 weeks, which metabolically matured hCSs. Parse single-cell RNA sequencing. The samples for single-cell RNA sequencing were prepared by dissociating day 75 cardiac spheroids with TrypLE Select Enzyme for 45 minutes
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