Fura 2

The increase in intracellular calcium levels after cardiac differentiation of WJMSCs was determined using the fluorescent radiometric Ca²⁺ indicator Fura-2 acetoxymethyl ester (Fura-2, 1 μmol/L; Molecular Probes, Invitrogen, Carlsbad, CA, USA) as reported previously [19 (link)]. After induction of WJMSCs with DC301 + DC302 as described, the cells were washed and Fura-2 (1 μmol/L; Molecular Probes) was added to the cells in plain medium and incubated for 37 °C for 45 min. Fluorescent intracellular Ca²⁺ flux was identified by fluorescence microscopy (450–480 nm) and calorimetrically at 480 nm.

The hiPSC‐CMs were dissociated and seeded in 24mm × 24mm coverslips for calcium imaging. Cells were loaded with 5 μmol/L Fura‐2, AM (Invitrogen) and 0.02% Pluronic F‐127 (Invitrogen) in Tyrodes solution (137 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L glucose, 1.8 mmol/L CaCl2, 1mM NaH2PO4 and 20 mmol/L HEPES at pH 7.4 with NaOH at 25°C) for 10 minutes at 37°C. Following Fura‐2, AM loading, cells were washed three times with Tyrodes solution. Ca2+ transients imaging was collected using the confocal microscope with a 40x lens using Zen software (Carl Zeiss). Cells were perfused with calcium‐free Tyrodes solution containing 0.5 mmol/L EGTA, and then the 5 mmol/L caffeine prepared in the same calcium‐free Tyrodes solution was delivered via perfusion apparatus. The analysis was performed using Zeiss physiology software.

Restriction and modification enzymes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Digitonin, histamine, CPA, DMEM, trypsin, L-glutamine, and penicillin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). BAPTA and Fura-2 were purchased from Invitrogen, Thermo Fisher Scientific (Carlsbad, CA, USA). All other materials were analytical or of the highest available grade.

5‐Methyl‐10,11‐di‐hydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK801) and isolectin‐B4‐FITC were purchased from Tocris (Bristol, United Kingdom). 7‐aminoactinomycin D (7‐AAD), lactate dehydrogenase (LDH), phenylmethanesulfonyl fluoride (PMSF), phosphatase inhibitor, protease inhibitor cocktails 2 and 3, Hoechst 33258, glutamate, β‐nicotinamide adenine dinucleotide (NADH), sodium pyruvate, β‐nicotinamide adenine dinucleotide 2′‐phosphate reduced tetrasodium salt hydrate (NADPH), MgSO₄, and nitro blue tetrazolium were from Sigma Aldrich (Saint‐Quentin Fallavier, France). The ratiometric intracellular calcium probe Fura‐2, pluronic^® F‐127 and Cell Tracker green were from Invitrogen (Cergy Pontoise, France). The Apo‐ONE homogeneous caspase‐3/7 kit was provided by Charbonnières‐les‐Bains, France. Isoflurane was from CSF (Cournon, France). Characteristics of the antibodies used in the present study for immunohistofluorescence and Western blot experiments are summarized in Table S1.

Chelated Ca²⁺/EGTA solutions containing 50 mM HEPES, pH 7.4, 100 mM KCl, and varying concentrations of Na₂EGTA and CaCl₂ (0 to 10 mM) were made by predicting [Ca²⁺]_free with MaxChelator (http://maxchelator.stanford.edu/CaEGTA-TS.htm). Actual [Ca²⁺]_free was verified in each solution by recording fluorescence excitation spectra of fura-2 (Invitrogen, 0.07 µM) at an emission wavelength of 510 nm on a LS55 fluorescence spectrophotometer (Perkin Elmer). [Ca²⁺]_free was calculated as Kd × [(R − R_min)/(R_max − R)] × (F_max³⁸⁰/F_min³⁸⁰), where F³⁸⁰ is the fluorescence intensity at ʎ_excit. = 380 nm and R is the ratio F³⁴⁰/F³⁸⁰. Kd_EGTA was measured at 34.906 nM.
Liposomes were formed by drying chloroform solutions containing 25% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, Avanti Polar Lipids) and 75% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids)under a nitrogen stream. The phospholipids were resuspended in 50 mM HEPES, 100 mM KCl, pH 7.4 to a final concentration of 1 mg/ml, sonicated 5 times for 10 seconds and centrifuged for 90 min at 21,000 × g to clear the liposomes from large aggregates as described previously^{48 (link)}.