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Extracellular vesicles from II trimester human amniotic fluid as paracrine conveyors counteracting oxidative stress

Authors :
Giorgia Senesi
Laura Guerricchio
Maddalena Ghelardoni
Nadia Bertola
Stefano Rebellato
Nicole Grinovero
Martina Bartolucci
Ambra Costa
Andrea Raimondi
Cristina Grange
Sara Bolis
Valentina Massa
Dario Paladini
Domenico Coviello
Assunta Pandolfi
Benedetta Bussolati
Andrea Petretto
Grazia Fazio
Silvia Ravera
Lucio Barile
Carolina Balbi
Sveva Bollini
Source :
Redox Biology, Vol 75, Iss , Pp 103241- (2024)
Publication Year :
2024
Publisher :
Elsevier, 2024.

Abstract

Background: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress. Methods: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue. Results: Our protocol resulted in a yield of 6.31 ± 0.98 × 109 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H2O2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis. Conclusions: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury.

Details

Language :
English
ISSN :
22132317
Volume :
75
Issue :
103241-
Database :
Directory of Open Access Journals
Journal :
Redox Biology
Publication Type :
Academic Journal
Accession number :
edsdoj.76044c78d75425cbafcc4dbd4d895d6
Document Type :
article
Full Text :
https://doi.org/10.1016/j.redox.2024.103241