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

ABSTRACT: Background. We previously demonstrated that the human amniotic fluid from the II trimester of gestation is a feasible source of stromal progenitors (namely human amniotic-fluid derived stem cells, hAFSC), endowed with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from the hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic, and cardioprotective effects in different rodent preclinical models of cardiac and skeletal muscle injury. Yet, hAFSC-EV isolation may be significantly influenced by in vitro cell culture; thus, growing interest has recently been addressed in profiling of EVs directly concentrated from human amniotic fluid samples (hAF) as an alternative option. Here we provide a detailed characterization of II trimester hAF-EVs and we investigate their paracrine potential against oxidative stress as a hallmark of cardiac and skeletal muscle dysfunction. Methods. II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were then assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot and flow cytometry. hAF-EV 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 two different preclinical models of oxidative stress and dysfunction in vitro on murine C2C12 cells and on 3D human cardiac micro tissue. Results. Our protocol resulted in a yield of 6.31±0.98x109 EV particles per hAF millilitre showing round cup-shaped morphology and 209.63±6.10 nm average size, with a relevant expression of CD81, CD63, and CD9 tetraspannin markers. hAF-EVs were found 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-EVs priming resulted in almost complete rescue in 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 TGFbeta stimulation, showed considerable improvement in viability with remarkable decrease of fibrosis onset. 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Amniotic Fluid

SUBMITTER: Martina Bartolucci

LAB HEAD: Andrea Petretto

PROVIDER: PXD050813 | Pride | 2026-07-16

REPOSITORIES: Pride

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			Action	DRS
	20240320_143606_Bollini_hAF_EVs_gen_2024.sne	Other
	Bollini_hAF_EV_SB184_04.raw	Raw
	Bollini_hAF_EV_SB187_07.raw	Raw
	Bollini_hAF_EV_SB188_05.raw	Raw
	Bollini_hAF_EV_SB189_06.raw	Raw

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Project description:We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, pro-angiogenic, anti-fibrotic and proliferative effects on target cells. hAFS can be also isolated from III trimester clinical waste samples during C-section scheduled procedures (perinatal hAFS), thus offering an easily accessible alternative source. Nonetheless, little is known about their paracrine profile. Here we provide a detailed characterization of the hAFS total secretome (i.e. whole of the soluble paracrine factors released by the cells in their conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it as from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. Their secretome formulations were analyzed in terms of protein and chemokine/cytokine content, with the EV cargo further investigated by RNA sequencing. While the appearance of fetal and perinatal hAFS, along with their corresponding secretome formulations was overlapping, the profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. While both cell sources provided paracrine formulations enriched with neurotrophic, immunomodulatory and endothelial stimulating factors, the more immature fetal hAFS secretome was defined by a more pronounced pro-angiogenic, pro-resolving and anti-aging profile over the perinatal one, while the corresponfing EV…. Moreover, small RNA profiling showed a relevant enrichment of microRNA in both the fetal and perinatal hAFS-EV cargo, with a steadily expressed pro-resolving miRNA core as reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

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

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