Rpmi b27 without insulin

hiPSCs from the DF19–9–11T line were differentiated to cardiac fibroblasts (hiPSC-CFs) using the GiFGF protocol as previously described.^{27 (link)} hiPSCs were dissociated with 1 mL/well Versene solution (Gibco) at 37 °C for 5 min, seeded on Matrigel (GFR, BD Biosciences) and coated 6-well plates at the density of 1.9 × 10⁶ cells/well in mTeSR1 medium (STEMCELL Technologies) supplemented with 10 μM ROCK inhibitor (Y-27632) (Tocris). Cells were subsequently cultured for 5 days in mTeSR1 medium with daily medium changes, and differentiation was started when the cells reached 100% confluency (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). After Day 1, the medium was changed to 2.5 mL RPMI+B27 without insulin (Gibco) and cells were cultured in this medium for 24 h (Day 2). After Day 2 but within 24 h (before Day 3), the medium was changed to 2.5 mL of the CFBM medium^{27 (link)} supplemented with 75 ng/mL bFGF (WiCell Research Institute). For every other day until Day 20, cells were fed with CFBM+75 ng/mL bFGF. The purity of the differentiated hiPSC-CFs were assessed by flow cytometry 20 days after differentiation. The hiPSC-CFs were passaged, expanded, and cryopreserved as previously described.^{27 (link)}