Fig. 3: Placental EVs promote fetal heart development and cardiomyocyte growth.

a Schematic showing procedure used to isolate EVs from unconditioned media (CTRL) and media conditioned by E15.5 placental tissue (Placental CM). b Western blot for EV markers TSG101 and HSP70 in media supernatants and EVs isolated from CTRL and Placental CM. Ponceau S was used to verify total protein. c Quantification of fetal heart rate and epicardial cell outgrowth from embryonic hearts exposed to supernatant from either unconditioned media (M-C) or Placental CM (M-P); or varying concentrations of CTRL (from FBS) or Placental EVs (1, 5, or 10 µg/mL) for 48 h. d Expression of six genes (Tead1, Myh7, Myl7, Acta2, Nppa, and Nppb) associated with cardiac development in cardiomyocytes treated with EVs (10 µg/mL) isolated from CTRL or Placental CM. e Western blot showing levels of Troponin T (TnT) and NKX2-5 in cardiomyocytes treated with CTRL or Placental EVs (10 µg/mL). ACTB was used as a loading control. Quantification of relative band intensity is found to the right of the representative blots. f Immunofluorescence for TnT (green) in cardiomyocytes treated with CTRL or Placental EVs (10 µg/mL). DAPI was used to demarcate nuclei. Values significantly different from CTRL media supernatants (N = 5 independent experiments, P < 0.05), or from CTRL EVs (N = 3 independent experiments, P < 0.05) are denoted with an asterisk (*), using one-way ANOVA followed by Tukey’s post-hoc test (c) or two-tailed Student’s t-test (d and e). Scale bar = 100 µm. Graphs represent means ± SEM. Uncropped images of western blots are provided in Supplementary Fig. 8.