Mission mirna mimic

hMSCs were seeded on a culture plate and allowed to attach overnight. On the next day (termed Day 0), synthetic hsa‐miR‐9 (Mission miRNA Mimic; Sigma‐Aldrich) was encapsulated in 306‐O16B‐3 bioreducible lipid and then delivered to the hMSCs. The concentrations of miRNA and lipid were 30 pmol/ml culture medium and 2.25 µg/ml culture medium, respectively. The cells were cultured for 1 week after the first delivery. The culture medium was changed every 2 days. For the multiple dosage experiment, miR‐9 encapsulated in the bioreducible lipid was added to the culture medium on Days 0, 2, and 5 (Figure 5C). As a negative control, ath‐miR‐416 (MISSION miRNA, Negative Control 1; Sigma‐Aldrich), which does not interact with any human genes, was delivered using the bioreducible lipid.

To incorporate miR-4732-3p into EVs, the later were resuspended in an electroporation buffer after the last ultracentrifugation step of isolation and were incubated with an artificial mimic of the chosen miRNA (MISSION miRNA mimic, Merck) at a concentration of 40 nM. The suspension was loaded into a Gene Pulser/MicroPulser Electroporation Cuvette and treated with 3 pulses of 300 V with 5 s between each pulse. Subsequently, the transduced EVs were incubated at 4°C for 30 min. To eliminate the miRNA not loaded in the EVs, the volume was increased with PBS to 25 mL and the solution was ultracentrifuged as described. The final pellet was resuspended in 100 μL of PBS.

To test whether the selected miRNAs showed a cardioprotective effect against doxorubicin, we established a transfection protocol using corresponding synthetic miRNA mimics (MISSION miRNA Mimic, Merck KGaA, Darmstadt, Germany) and doxorubicin treatment. NRCM or cFib, HCF or hiPSC-CMs were seeded and incubated for 2–3 days. The mimics of hsa-miR-4732-3p or cel-miR-243-3p from Caenorhabditis elegans were then transfected individually at a final concentration of 40 nM using Lipofectamine 3000 (ThermoFisher Scientific, Waltham, MA, USA). Equal volumes of mimic diluted in OptiMem (ThermoFisher Scientific) and Lipofectamine 3000 1:25 in OptiMem were mixed and allowed to form Lipofectamine-mimic complexes for 15 min at RT. The Lipofectamine-mimic complexes were then added to the cells to give a final mimic concentration of 40 nM, and cells were incubated for 24 h. To induce cardiac damage, the cells were treated with 1 µM doxorubicin for 48 h to imitate an acute dose of doxorubicin. To induce stable expression of miR-4732-3p and to validate target genes, HEK 293 cells were transduced with LentimiRa-GFP-hsa-miR-4732-3p Vector (Abm, Vancouver, BC, Canada), and cells expressing the vector were selected using puromycin at 1 μg/mL.