Inhibition of HSC70 alleviates hypertrophic cardiomyopathy pathology in human induced pluripotent stem cell‐derived cardiomyocytes with a MYBPC3 mutation

Abstract

Culture and maintenance of iPSCs iPSCs were cultured in feeder-free mTeSR (STEMCELL Technologies) media on matrigel-coated (Corning) plates at 37°C with 5% (vol/vol) CO2. The media were daily changed and cells were passaged every 3-4 days using Accutase (STEMCELL Technologies). Alkaline phosphatase staining Alkaline phosphatase (ALP) staining was performed using the VECTOR Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories) following the manufacturer's instructions. Cardiac differentiation The iPSC-CMs were generated using a 2D monolayer differentiation protocol as previously described 28, 29. Briefly, ~10 5 undifferentiated cells were dissociated and re-plated into matrigel-coated 6-well plates. Cells were cultured and expanded to 85% cell confluence, and then treated for 2 days with 6 μM CHIR99021 (Axon Medchem) in RPMI and B27 supplement minus insulin (RPMI+B27-Insulin) (Gibco) to activate Wnt signaling pathway. On day 2, cells were placed in RPMI+B27-Insulin with CHIR99021 removal. On days 3-4, cells were treated with 5 μM IWR-1 (Sigma-Aldrich) to inhibit Wnt signaling pathway. On days 5-6, cells were removed from IWR-1 treatment and placed in RPMI+B27-Insulin. From day 7 onwards, cells were placed and cultured in RPMI and B27 supplement with insulin (RPMI+B27+Insulin) (Gibco) until beating was observed. Cells were glucose-starved