Optimized Cardiac Differentiation of Human iPSCs

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.