B27 plus insulin

Human iPSCs were differentiated into cardiomyocytes using a new optimized protocol derived from previous publications [13 (link),14 (link),15 (link)] (Figure 1A). In short, a confluent monolayer of human iPSCs was differentiated by adding RPMI media (Thermofisher Scientific) supplemented with B27 minus insulin (Thermofisher Scientific), 50 μg/mL of ascorbic acid (Thermofisher Scientific), 20 ng/mL of BMP4 (R&D Systems, Minneapolis, MN, USA), 20 ng/mL of activinA (StemCell Technologies), and 1.5 μM of CHIR99021 (TOCRIS, Bristol, UK) for 3 days. Next, the medium was changed to an RPMI medium supplemented with B27 minus insulin, 50 μg/mL of ascorbic acid, and 5 μM of XAV939 (TOCRIS) for 3 more days; later, the cells were cultured with an RPMI medium supplemented with B27 minus insulin and 50 μg/mL of ascorbic acid for 2 days. From day 7, the first beating areas appeared and the medium was refreshed with an RPMI medium supplemented with B27 plus insulin (Thermofisher Scientific) and 50 μg/mL of ascorbic acid, with a media change every other day.

Differentiation into CMs was performed using a PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. On differentiation day 12, hiPSC-derived cells were incubated with glucose and glutamine-free DMEM (Thermo Fisher Scientific) supplemented with 4 mM l-lactic acid (Sigma-Aldrich) and 0.1% bovine serum albumin (Thermo Fisher Scientific) for 3 days. On day 15, the medium was changed to RPMI with B27 plus insulin (Thermo Fisher Scientific). After metabolic selection on day 17, purified hiPSC-CMs were used for immunostaining with cardiac troponin T.

Directed differentiation of human iPS cells into iCMs was performed via WNT signaling pathway modulation. Differentiation was initiated at 90% confluence in 12-well Matrigel-coated plates with a differentiation medium composed of RPMI 1640 supplemented with B-27 minus insulin (Thermo Fisher Scientific) with 4 μM CHIR99021 (Merck Millipore) for 48 h and subsequently 5 μM IWP4 (Merck Millipore) for 48 hours 33 (link). The medium was changed to a maintenance medium composed of RPMI 1640 with B-27 plus insulin (Thermo Fisher Scientific) at day 7. Metabolic selection of CMs was performed using a selection medium composed of RPMI 1640 without glucose (Thermo Fisher Scientific), 0.5 mg/ml human recombinant albumin, 0.2 mg/ml L-ascorbic acid 2-phosphate, and 4 mM lactate (Sigma-Aldrich) from days 10 to 17. Afterwards, iCMs were cultured in maintenance medium at least to day 30 for further maturation.