Extracellular vesicles from II trimester human amniotic fluid as paracrine conveyors counteracting oxidative stress
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
Affiliations
Giorgia Senesi
Cardiovascular Theranostics, Istituto Cardiocentro Ticino and Laboratories for Traslational Research Ente Ospedaliero Cantonale, CH-6500, Bellinzona, Switzerland; Euler Institute, Faculty of Biomedical Sciences, Università della Svizzera Italiana, CH-6900, Lugano, Switzerland
Laura Guerricchio
Department of Experimental Medicine (DIMES), University of Genova, 16132, Genova, Italy
Maddalena Ghelardoni
IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy
Nadia Bertola
IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy
Stefano Rebellato
Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, 20900, Monza, Italy; School of Medicine and Surgery, University of Milano-Bicocca, 20900, Monza, Italy
IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy
Andrea Raimondi
Institute for Research in Biomedicine, Università della Svizzera Italiana, CH-6500, Bellinzona, Switzerland
Cristina Grange
VEXTRA Facility and Department of Medical Sciences, University of Turin, 10126, Turin, Italy
Sara Bolis
Cardiovascular Theranostics, Istituto Cardiocentro Ticino and Laboratories for Traslational Research Ente Ospedaliero Cantonale, CH-6500, Bellinzona, Switzerland
Valentina Massa
Department of Health Sciences, University of Milan, 20146, Milan, Italy
Dario Paladini
Fetal Medicine and Surgery Unit, IRCCS Istituto Giannina Gaslini, 16147, Genova, Italy
Domenico Coviello
Human Genetics Laboratory, IRCCS Istituto Giannina Gaslini, 16147, Genova, Italy
Assunta Pandolfi
Department of Medical, Oral and Biotechnological Sciences, University ''G. d'Annunzio'' Chieti-Pescara and Center for Advanced Studies and Technology – CAST, 66100, Chieti, Italy
Benedetta Bussolati
Department of Molecular Biotechnology and Health Sciences, University of Turin, 10126, Turin, Italy
Tettamanti Center, Fondazione IRCCS San Gerardo dei Tintori, 20900, Monza, Italy; School of Medicine and Surgery, University of Milano-Bicocca, 20900, Monza, Italy
Silvia Ravera
Department of Experimental Medicine (DIMES), University of Genova, 16132, Genova, Italy
Lucio Barile
Cardiovascular Theranostics, Istituto Cardiocentro Ticino and Laboratories for Traslational Research Ente Ospedaliero Cantonale, CH-6500, Bellinzona, Switzerland; Euler Institute, Faculty of Biomedical Sciences, Università della Svizzera Italiana, CH-6900, Lugano, Switzerland; Corresponding author. Istituto Cardiocentro Ticino and Laboratories for Translation Research, EOC, CH-6500, Bellinzona, Switzerland.
Carolina Balbi
Center for Molecular Cardiology, University of Zurich, 8952, Schlieren, Switzerland; Department of Internal Medicine, Cantonal Hospital Baden, Baden, Switzerland; Corresponding author. Center for Molecular Cardiology, University of Zurich, Zurich, Switzerland.
Sveva Bollini
Department of Experimental Medicine (DIMES), University of Genova, 16132, Genova, Italy; IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy; Corresponding author. Dept. Experimental Medicine (DIMES), Largo R. Benzi 10, 16132, Genova, Italy.
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.
