Glutamaxtmsupplement hepes

hiPSCs were cultured until 80–90% confluency and washed with dPBS (Gibco, 14190094). The cells were then provided with RMPI++ (bare RPMI-1640-Medium-GlutaMAX^TMSupplement-HEPES (Gibco, 72400-021) supplemented with 0.5 mg/mL human recombinant albumin (Sigma, A9731), 0.2 mg/mL L-ascorbic acid 2-phosphate (Sigma, A8960)), and 4 µM CHIR99021 (Sigma, 361559). After 48 h, the medium was replaced with RMPI++ and 5 µM IWP2 (Sigma, 681671) after a single rinse with bare RMPI-1640. Then, the cells were refreshed every other day with RMPI++, for 4 days. Thereafter, the cells were cultured every 3–4 days with bare RMPI, supplemented with B-27^TM Supplement (Gibco, 17504001). Prior to the optical experiments, the hiPSC-CMs were dissociated with TrypLE Select Enzyme without phenol red (Gibco, A1217703) and seeded on Geltrex^TM coated coverslips.

MLR potency assay was performed according to Nicotra et al. [16 (link)]. MLR assay was performed on UC-MSCs both in a resting state (NT) and after priming for 48 h with IFNγ, TNFα and IFNγ + TNFα. Briefly, peripheral blood mononuclear cells (PBMCs) pooled from 10 healthy donors and labeled with the CellTrace^TM Violet (CTV) Cell Proliferation Kit (Invitrogen, Ref C34557) were co-cultured with UC-MSCs at 0:1 (control), 1:10, 1:30, 1:100, 1:300 and 1:1000 UC-MSCs:PBMC ratio with a constant amount of PBMC (3 × 10⁵) and a decreasing amount of UC-MSCs from 3 × 10⁴ down to 3 × 10².
Cells were co-cultured in a culture medium composed of Roswell Park Memorial Institute (RPMI) Medium 1640, GlutaMAX^TM Supplement, HEPES (Gibco, Ref 72400-013), 10% human A/B serum (Eurobio, Ref CAEHUM010U), 1% Amphotericin B/Penicillin/Streptomycin (Gibco, Ref 15240062), and 10 UI/mL Heparin (PanPharma, Ref 5520508) at 37 °C, 5% CO₂ for 7 days. At day 4 ± 1, 50 µL of culture medium was added. At day 7, cells were labeled with 5 µL of human antibodies anti-CD3 PE (BD Biosciences, Ref 345765), anti-CD45 FITC (BD Biosciences, Ref 345808) and 7-AAD viability dye (Beckman Coulter, Ref A07704) for 15 min at 4 °C, protected from light. Cells were washed in PBS 1X before acquisition on the Attune NxT™ Thermofisher® Flow Cytometer and analyzed using Attune NxT™ software.