Fig. 2.

EV-derived-miR-106a–363 cluster improves iCM viability, decreases oxidative stress, and preserves contractile rate after ischemic injury. a The number of EVs secreted from normoxic and hypoxic conditions was measured. EVs were analyzed by Nanosight (vesicles/mL). Statistical significance was determined by unpaired two-tailed t test and mean data ± SD are shown (n = 4). b Venn Diagram of microarray profiling of EV derived-miRNAs shows that 53 miRNAs were down-regulated and 136 miRNAs were up-regulated in the hEVs vs. nEVs from a total of 6631 homo sapience probe set (> twofold change). The red dots indicate 6 miRNAs of miR-106a–363 cluster, which are consistently, and significantly increased in hEV. c Transfection of the contractile iCMs by FAM-labeled miR-mimic was confirmed (immunostained with cTnT antibody). Green fluorescence from FAM in the cytoplasm was captured 48 h post-transfection (White arrows). Scale bars: 50 μm. d Survival rate of hypoxia-injured iCMs was measured by MTT assay after 18 h of hypoxia. iCMs were transfected with 50 nM of the following: (1) negative control miR-mimic (Ctrl); (2) individual miR-92a, miR-20b, and miR-363 mimics; and (3) combined miR-92a, −20b, and −363 mimics. Statistical significance was determined by one-way ANOVA with multiple comparisons and mean data ± SD (n = 5). e The number of viable and dead cells in the Core-vs. Ctrl-miR transfected group was measured by ApoLive-Glo™ Multiplex Assay after exposure to 18 h of hypoxic condition. Statistical significance was determined by unpaired two-tailed t test and mean ± SD (n = 4). f Oxidative stress was evaluated by lactate dehydrogenase (LDH) release assay after 18 h of hypoxia. iCMs were transfected with 50 nM of the following: (1) negative control miR-mimic (Ctrl); (2) individual miR-92a, miR-20b, and miR-363 mimics; and (3) combined miR-92a, −20b, and −363 mimics. Statistical significance was determined by one-way ANOVA with multiple comparisons and mean data ± SD (n = 4). g Survival rate of hypoxia-injured iCMs was measured by MTT assay after 18 h of hypoxia. iCMs were either treated with hEVs or transfected with the following: (1) negative control miR inhibitors (Ctrl); (2) Core-miR inhibitors. Statistical significance was determined by one-way ANOVA with multiple comparisons and mean data ± SD (n = 4). h Measurements of the peak divergence (magnitude of contractile movement and rate) of the control (normoxia) iCMs, hypoxia-injured iCM transfected with Ctrl-miR, and Core-miRs (n = 6) were performed. Statistical significance was determined by one-way ANOVA with multiple comparisons. i Contractile rate of the iCMs in the following treatment conditions was measured: (1) Normoxic, (2) Hypoxic, (3) Ctrl-miR transfection, and (4) Core-miR transfection. Data were normalized with the normoxic control iCMs. Statistical significance was determined by one-way ANOVA with multiple comparisons and mean ± SD (n = 6). Statistically non-significant comparisons are not shown. * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001