Cardiac Fibroblasts Differentiation from iPSCs

Human cardiac fibroblasts were differentiated from a human‐induced pluripotent stem cell (iPSC) line iPS‐DF19‐9‐11T (male) (WiCell) as described previously (Zhang et al., 2019 (link)). Briefly, iPSCs were dissociated with 1 ml/well Versene solution (Invitrogen) at 37℃ for 5 min, and seeded on Matrigel (GFR, BD Biosciences) ‐coated six‐well plates at the density of 2 × 10⁶ cells/well in mTeSR1 medium supplemented with 10 μM ROCK inhibitor (Y‐27632) (Tocris). Cells were cultured for 5 days in mTeSR1 medium with medium change daily until 100% confluence was reached and differentiation was started (day 0). At Day 0, the medium was changed to 2.5 ml RPMI+B27 without insulin and supplemented with 12 µM CHIR99021 (Tocris) and cells were treated in this medium for 24 h (day 1). At Day 1 medium was changed to 2.5 ml RPMI+B27 without insulin (Invitrogen) and cells were cultured in this medium for another day (day 2). At Day 2, the medium was changed to 2.5 ml of the defined fibroblast culture medium (CFBM) (Zhang et al., 2019 (link)) supplemented with 75 ng/ml bFGF (WiCell). Cells were fed every other day with CFBM supplemented with 75 ng/ml bFGF and cultured until day 20 for flow cytometry analysis and subculture of cardiac fibroblasts.