Extracellular vesicles from second trimester human amniotic fluid as candidate therapeutics against myocardial injury

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Abstract Background We have previously reported that human amniotic fluid (hAF) from II trimester of gestation is an appealing source of stromal progenitor cells (hAFSCs) that release in their secretome extracellular vesicles (EVs) with pro-survival, proliferative, anti-fibrotic effects in preclinical skeletal and cardiac muscle injury models. Purpose Considering hAFSC-EVs yield may be significantly influenced by in vitro culture conditions, we characterized EVs directly separated and concentrated from hAF (hAF-EVs) as alternative approach and explored their paracrine potential in vitro against oxidative stress and oncological treatment-related cardiotoxicity. Method hAF was obtained as leftover material from prenatal screening amniocentesis with informed consent from healthy women (37.5±0.6 years-old) and local ethical committee approval. EVs were separated from hAF (hAF-EVs) by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were characterized by nanoparticle tracking analysis, transmission electron microscopy, super-resolution microscopy, Western Blot, and proteomics. EVs paracrine potential was evaluated in in vitro on a H2O2-triggered oxidative stress model on 3D human cardiac microtissue (hMT) and on a Doxorubicin (Dox)-induced premature ageing model in cardiomyocytes differentiated form human induced pluripotent stem cells (iCMs). Results hAF was enriched with 5.03±0.50*10^9particles/ml, with round cup-shaped morphology and 224.10±4.97 nm average size and relevant expression of CD81, CD63 and CD9 markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 indicating a heterogeneous origin. hMT treated with hAF-EVs and experiencing H2O2 stress and TGFβ stimulation showed improved survival (from 84% to 95%****p<0.0001) with a decreased onset of fibrosis (from 2% to 7%, ****p<0.0001). While Dox induced a 30% increase in p21-positive senescent in iCMs (**p<0.01), hAF-EVs priming counteracted such effect (**p<0.01). EV cargo was found containing proteins related to energetic metabolism pathways (glycolysis, Krebs cycle, oxidative phosphorylation) and antioxidant response (glutathione metabolism, catalase, superoxide dismutase pathway). Conclusions II trimester hAF can be exploited as starting material to isolate hAFSC endowed with broad regenerative potential, but also as straightforward source of EVs showing promising therapeutic cardioprotective capacity to counteract oxidative stress and cardiotoxicity.

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