Differentiation and Maintenance of hiPSC-Cardiac Fibroblasts

Mouse embryonic fibroblast cell line (NIH3T3), purchased from the Bioresource Collection and Research Center, Taiwan, was cultured in Dulbecco's Modified Eagle Medium, high glucose (Gibco) supplemented with 10% bovine serum (Gibco) and 1x penicillin/streptomycin (Corning). Differentiation and maintenance of human iPSC-Cardiac fibroblasts (hiPSC-CFs) were carried out as described in Zhang et al 32 (link). In brief, human iPSCs were dissociated and seeded on Matrigel (GFR, BD Biosciences) coated 6-well plates in mTeSR1 medium supplemented ROCK inhibitor (Y-27632) (Tocris). Cells were cultured for 5-6 days in mTeSR1 medium with medium changes daily until they reached 100% confluence when differentiation started. The medium was then changed to RPMI+B27 (Gibco) without insulin and supplemented with CHIR99021 (Tocris) for 24 h, followed with CFBM medium supplemented with bFGF until day 20 when they were used for flow cytometry analysis and passaged. The hiPSC-CFs were fed every other day with the FibroGRO+2% FBS medium and passaged every 4-6 days using 0.05% Trypsin-EDTA. For the hypoxia experiments, cells were incubated in a hypoxia chamber supplemented with 1% O₂ and 5% CO₂ for 48 h.