10xtryple select

We utilized primary CEnCs isolated from human donor corneas (CorneaGen, Seattle Eye Bank, WA, USA). The hCEnCs were cultured following established protocols with slight modifications [32 (link), 41 (link)]. Briefly, the Descemet’s membranes with the CEnCs were stripped from donor corneas and digested at 37°C using 1 mg/mL collagenase A (Roche Applied Science, Penzberg, Germany) for 12 h. Subsequently, the hCEnCs obtained from a single donor cornea were collected following two washes with OptiMEM-I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then seeded in one well of a 6-well plate that had been pre-coated with laminin E8 fragments (iMatrix-511; Nippi, Incorporated, Tokyo, Japan) [42 (link)].
hCEnCs were cultured at 37°C with 95% humidity and 5% CO2 in complete hCEnC medium, which consisted of OptiMEM-I (Thermo Fisher Scientific), 8% fetal bovine serum (FBS), 20 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 200 mg/L calcium chloride, 0.08% chondroitin sulfate (Sigma-Aldrich), Rho-kinase inhibitor Y-27632 (10 μM) (Selleck Chemicals, Houston, TX), and penicillin-streptomycin (50 IU/ml) (Nacalai Tesque, Kyoto, Japan). Cultured hCEnCs were passaged after harvest with 10xTrypLE Select (Thermo Fisher Scientific, Inc.) treatment at 37°C for 12 min when they reached confluence and used for subsequent experiments. hCEnCs at passage 2 were used for all experiments.

HiPSCs (LUMC0020iCTRL-06, female) and hESC double Reporter mRubyII-ACTN2 and GFP-NKX2.5 DRRAGN, female) were maintained in Essential 8 medium on vitronectin (5 μg/mL) (Thermo Fisher)¹⁴ (link) maintained in a humidified incubator at 37°C and 5% CO₂. Cardiomyocytes were differentiated as described previously. At day 13 of differentiation, hiPSC-cardiomyocytes were purified in glucose-free-lactate-containing medium for 4 days. The fraction of Troponin-T positive cardiomyocytes was assessed by flow cytometry after staining with Cardiac Troponin T Antibody-VioBlue (Miltenyi Biotech) on a MACSQuant VYB flow cytometer (Miltenyi Biotech) at ex405nm, em450/50nm. Plots were analyzed with FlowLogic software. DRRAGN-cardiomyocytes were dissociated around day 14 using 10x TrypLe Select (Thermo Fisher) and were FACS sorted for double expression of actinin^mRybyII/w-NKX2.5^eGFP/w on a Sony SH800S Cell Sorter (Sony Biotechnology) (mRubyII at ex561nm, em617/30nm. After purification, all cardiomyocytes were cultured in cardiomyocyte-TDI medium.¹ (link) Stem cells and cardiomyocytes were maintained in a humidified incubator at 37°C and 5% CO₂ and were refreshed daily with E8 medium, or with cardiomyocyte-TDI medium twice a week, respectively. Approximately 10 days after seeding, cardiomyocytes were treated with dimethylsulfoxide (DMSO 4.23 mM) as control, DOXO or its analogues.