O sativa derived recombinant human albumin

Purification of SANLCs referenced a metabolic-selection method of cardiomyocytes as previous described [2 (link), 9 (link)]. Briefly, SANLCs induced by three small molecule chemicals were performed in RPMI 1640 medium without d-glucose (11,879,020, Life Technologies) supplemented with 213 µg/ml l-ascorbic acid 2-phosphate (66,170–10-3, Sigma-Aldrich), 500 µg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich) for 2 days and then in RPMI 1640 medium without d-glucose supplemented with 213 µg/ml l-ascorbic acid 2-phosphate, 500 µg/ml O. sativa-derived recombinant human albumin, and 5 mM sodium dl-lactate (L4263, Sigma-Aldrich) for 3–4 days. Medium was changed to CDM3 medium for maintenance of SANLCs for 2 days. The markers of pacemaker cells were evaluated by analysis of RT-PCR and flow cytometry at day 32. Electrophysiological characteristics were analyzed using action potential (AP) recording at day 32.

Cardiomyocyte differentiation was performed in CardioEasy medium (Cellapy China) by temporal modulation of the canonical WNT signaling pathway with GSK3 inhibitor and WNT inhibitor, as described in previous protocol [2 (link)]. CardioEasy medium, consisting of RPMI 1640 medium, 500 µg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich), and 213 µg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich), is abbreviated as CDM3 medium. Briefly, at day 0, 80–90% confluent hiPSCs were cultured in CDM3 medium with 6 µM CHIR99021 (SML1046, Sigma, USA) for 48 h. At day 2, medium was changed to CDM3 medium supplemented with 2 µM WNT-C59 (S7037, Selleck Chemicals) and continued to incubate for 48 h. Medium was refreshed on day 4 and every other day for CDM3 medium. The contracting cardiomyocytes can be noted from day 7.

Healthy human-induced pluripotent stem cell line (named—HS1M) was generated and maintained as previously described (27 (link)). Cardiomyocytes were differentiated using a protocol based upon previous work published by Burridge et al. (20 (link)). In brief, hiPSCs were seeded at 150 000 cells/cm² in a 24-well plate and allowed to reach ~ 80% confluence over 48 h. Media was changed to ‘CDM3’ (RPMI1640 (Thermofisher) supplemented with 500 μg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich), 213 μg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich)) with 6 μM CHIR99021 (Sigma-Aldrich). After 48 h, media was changed to CDM3 supplemented with 2 μM C59 for a further 2 days before regular CDM3 media changes every 2–3 days. For patch-clamp analysis, at 15 days post-differentiation, cells were treated with TrypLE Express (Thermofisher) for 10 min at 37°C and seeded onto glass coverslips at a density of 10 000 cells/cm².
For TRPM7 siRNA knockdown experiments, 48 h before patch-clamp analysis, adhered cells were transfected with either SMARTpool: ON-TARGETplus TRPM7 targeting siRNA or a scrambled control (L-005393-00-0005 or L-006233-00-0005, respectively, Dharmacon). DharmaFECT transfection reagent 1, 2.5 μl/well (T-2001-02, Dharmacon) was used to transfect 2.5 μl/well of 2 μM siRNA.