H1 hesc line

hiPSC lines 201B7 (female) and 253G1 (female) were obtained from RIKEN BRC (Tsukuba, Japan). hESC line H1 (male) was obtained from Wicell Research Institute (Madison, WI, USA). hiPSCs and hESCs were cultured in mTeSR1 WO 2ME/MV medium (Stemcell Technologies, Vancouver, BC, Canada).
iCell cardiomyocytes (hiPSC-derived cardiomyocytes; female) were obtained from FUJIFILM Cellular Dynamics (Madison, WI, USA) and cultured in accordance with the manufacturer’s instructions.

H1 hESC line (WiCell Research Institute, Madison, WI), H9 hESC line (WiCell Research Institute, Madison, WI), BC1 hiPSCs (from Dr. Linzhao Cheng) [28 (link)], and Z-15 hiPSCs (from Dr. Zhijian Xiao) [29 (link)–31 ] were used in this study. BC1 cells were derived from BM CD34⁺ cells reprogrammed by OCT4, SOX2, KLF4, c-MYC, and LIN28 and characterized with pluripotency markers, karyotyping, in vitro pluripotency assay of embyroid body formation, and in vivo pluripotency assay of teratoma formation [28 (link)]. Z-15 cells were derived from peripheral blood mononuclear cells reprogrammed by OCT4, SOX2, KLF4, c-MYC, and BCL-XL and characterized with pluripotency markers, karyotyping, and in vivo pluripotency assay of teratoma formation [30 (link), 31 ]. hPSCs were cultured on Matrigel-coated plates (Corning) in mTeSR1 (Stem cell Technology) to maintain a pluripotent state. Medium was changed daily, and colonies were passaged every 4 days with 2 U/mL Dispase (Sigma) according to the manufacturer’s instructions.

hiPSCs were generated from human fibroblasts as previously described (Kyrkou et al., 2016 (link)), and the H1 hESC line was purchased from Wicell Research Institute (Madison, WI, United States). hPSCs were cultured on six-well tissue culture plates coated with hESC-qualified Matrigel (Corning, 354277) in mTeSR1 medium (StemCell Technologies, 05850) at 37°C and 5% CO₂. Every 4–6 days, cells were passaged enzymatically using 1 mg/ml dispase (Invitrogen, 17105-041) for 2 min at 37°C. hPSC colonies were then harvested, dissociated into small clumps and replated onto Matrigel-coated 6-well plates (ratio 1:6).

A step-by-step protocol describing the differentiation protocol can be found at Protocol Exchange³⁰ . hESCs are maintained as feeder-free in mTeSR™1 (STEMCELL Technologies, Inc.) according to the manufacturer’s instructions. Before differentiation, adherent hESCs were rinsed with phosphate-buffered saline (PBS) and then incubated with Accutase (Millipore) for 6–8 min at 37 °C. Dissociated single cells were rinsed twice with Dulbecco’s modified Eagle’s medium (DMEM)/F12 and spun at 300 × g for 3 min. The resulting cells were re-suspended in mTeSR™1, which was supplied with 10 μM Y27632 (Sigma-Aldrich), and seeded on 1:30 diluted Matrigel (BD Biosciences)-coated dishes at a density of 55,000 cells/cm². The next day, the medium was exchanged for mTeSR™1 and maintained for one more day prior to differentiation initiation. The H1 hESC line was obtained from WiCell Research Institute, Inc. (Madison, WI) and iPS cell lines were obtained from the Stem Cell Core of the Salk Institute.

hESC line (HUES8) was obtained from Harvard Stem Cell Institute (Cambridge, Madison, USA) and H1-hESC line was obtained from WiCell Research Institute (Maddison, USA). Both cell lines were cultured with mTesR1 medium (Stem Cell Technologies, Canada) and maintained in 5% CO2 at 37 °C on Matrigel-coated (Corning, USA) cell culture plates. Differentiation was started after 48–72 hr. after passaging with a confluency of ~70%. Cells were dissociated with the use of ReLeSR (Stem Cell Technologies, Canada) for 2–4 min or until detachment of the colony borders, and then the cells were collected and resuspended in mTesR1 medium supplemented with 10 μM Y-27632 (Rock inhibitor) (Stemgent, USA) for first 24 h of passaging.