Rpmi 1640 basal medium

The differentiation of iPSCs into cardiomyocytes was performed according to the method previously described [37 (link)]. Briefly, before differentiation, iPSCs were passaged at a 1:10 ratio, and cardiomyocytes differentiation was initiated when the cells reached about 80% confluence. During the differentiation, the medium was changed every other day. First, iPSCs were treated with CDM3 (a chemical-defined medium with three components) supplemented with CHIR99021 (Selleck, Shanghai, China) for 2 days. The CDM3 included recombinant human albumin (Sciencell Research Laboratories, Carlsbad, CA, USA), L-ascorbic acid 2-phosphate (Wako Chemicals, Osaka, Japan), and Roswell Park Memorial Institute (RPMI) 1640 basal medium (Thermo Fisher). Second, cells were cultured for another 2 days in CDM3 supplemented with WNT-C59 (Selleck). The medium was then replaced with the RPMI 1640 basal medium supplemented with a B27-supplement (Thermo Fisher) for 6 days, and the metabolic purification of the iPSC-CMs was carried out by replacing the medium with a RPMI 1640 medium with no glucose (Thermo Fisher), supplemented with a B27-supplement for 10 days. After being maintained in the RPMI 1640 basal medium supplemented with a B27-supplement for 6 days, the cardiomyocytes were dissociated with the STEMdiff™ Cardiomyocyte Dissociation Kit (STEMCELL Technologies).

As the method previously described [48 (link)], iPSCs were dissociated to aggregates by 0.5 mM EDTA (Thermo Fisher Scientific) and seeded on a Matrigel-coated 6-well plates in mTesR1 containing 10 µM Y27632 (STEMCELL Technologies). The medium was refreshed daily with mTesR1. After 5 days, confluence reached ~90%, the medium was changed to CDM3 medium (including L-ascorbic acid 2-phosphate (Wako Chemicals USA, Richmond, VA, USA) and recombinant human albumin (Sciencell Research Laboratories, Carlsbad, CA, USA) dissolved in the RPMI basal medium (Gibco)) supplemented with 4 μM CHIR99021 (Selleck, Houston, TX, USA) for two days, then the media was switched to CDM3 medium supplemented with 2 μM WNT-C59 (Selleck) for two days. Subsequently, the cells were cultured in the RPMI 1640 basal medium supplemented with B27-supplement (Thermo Fisher Scientific) for 8 days, during which spontaneous contraction can be observed. The medium was changed every other day during the differentiation.

Human iPSC lines (iPS IMR90-1) were obtained from the Wicell Research Institute (https://www.wicell.org/). Differentiation of hiPSCs to cardiomyocytes (hiPSC-CMs) employed a protocol published previously (Dewar et al., 2017 (link)). In brief, hiPSCs were maintained on Corning Matrigel (0.5 mg/six-well plate, dissolved in DMEM/F-12 medium) cultured in mTeSR1 medium (StemCell Technologies, Vancouver, BC). Cells were passaged every 4 days using Versene solution (Thermo Fisher, Waltham, MA) and were then seeded on a six-well Matrigel-coated plate at a density of 100,000 cells cm⁻² in mTeSR1 medium. The medium was changed daily, and after 3–4 days, when the monolayer of cells reached >90% confluency, the mTeSR1 medium was replaced with Roswell Park Memorial Institute (RPMI) 1640 basal medium (Thermo Fisher, Waltham, MA) plus B27 without insulin supplement (Thermo Fisher) containing 12 µM CHIR99021 (R&D Systems) for 24 h, followed by 5 mM IWP4 (Tocris) for 2 days without medium change. The medium was changed to RPMI 1640 plus B27 complete supplement, and then subsequently, the medium was changed every 2–3 days. Robust spontaneous beating of the monolayer was observed by day 12.