Dissecting human amniotic fluid discrete extracellular vesicle populations with cardioprotective effects against premature cardiac senescence

II-trimester human amniotic fluid (hAF) is an appealing source of stromal progenitor cells (hAFSCs), releasing extracellular vesicles (EVs) with cardioprotective properties. EVs directly isolated from hAF (hAF-EVs) also exhibit antioxidant and antifibrotic effects.

We compared the paracrine potential of hAFSC-derived EVs (hAFSC-EVs) versus hAF-EVs in a model of doxorubicin (Dox)-induced premature cardiac senescence, mimicking late-onset cardiotoxicity.

hAF samples were obtained from leftover samples of routine amniocentesis procedure with previous informed consent. hAF-EVs were isolated by size-exclusion chromatography (SEC) and ultracentrifugation (UC), while hAFSC-EVs were collected from conditioned medium using UC alone (hAFSC-EVs_UC) or UC followed by SEC (hAFSC-EVs_UC+SEC). Human iPSC-derived cardiomyocytes (iCMs) were exposed to Dox with or without EV treatment. Mitochondrial function and oxidative stress were assessed using Seahorse Mito Stress Test, CellROX, and TMRM probes; intracellular ATP was measured by chemiluminescent method. The presence of mitochondrial complexes was analyzed by Western blot; oxygen consumption and ATP synthesis were assessed in all hAF(SC)-EV types.

Dox treatment reduced basal and maximal respiration by 0.54- and 0.47-fold, respectively, relative to untreated control (p<0.0001), increased oxidative stress (1.37-fold, p<0.0001), depolarised mitochondria (0.86-fold, p<0.05), and decreased intracellular ATP (0.53-fold, p<0.001). Treatment with hAFSC-EVs_UC partially reversed these effects, restoring basal (to 0.77-fold, p<0.05) and maximal respiration (to 0.67-fold, p<0.05), reducing Dox-induced oxidative stress to 1.10-fold (p<0.01), preserving mitochondrial potential to 1.05-fold (p<0.01), and increasing ATP content up to 0.78-fold (p<0.01). hAFSC-EVs_UC+SEC improved maximal respiration (p<0.01), mitochondrial potential (p<0.001), and ATP levels (p<0.05), without significantly reducing ROS levels. hAF-EVs lowered ROS levels (p<0.05) but did not considerably restore mitochondrial or energetic parameters. Mitochondrial complexes were detected exclusively in hAFSC-EVs (UC and UC+SEC). Both hAFSC- and hAF-EVs types consumed oxygen and synthesised ATP, which decreased upon rotenone and antimycin A treatment (p<0.0001).

hAFSC-EVs (especially UC) offer superior protection against Dox-induced cardiomyocyte senescence, likely through direct metabolic support involving mitochondrial complexes transfer. These findings underscore how EV origin and purification strategy critically determine therapeutic efficacy, supporting the translational potential of hAFSC-EVs in preventing chemotherapy-related cardiac ageing.