Dataset Information

Characterizations of molecularly distinct subpopulations of conditioned medium derived extracellular vesicles prior to be used as medicinal product

ABSTRACT: Mesenchymal stromal cells (MSCs) derived EVs are greatly studied in disease models but lack of manifested characterization rather than generic EVs characterization in different EV subpopulations, greatly limits our ability to bring the most reliable EV types into our studies. We provide a comparative evaluation between three most well-reputed EVs subpopulations isolated by ultracentrifuge (EV-110K), sucrose gradient (EV-S110K), and 20,000g centrifugation only (EV-20K). We delved into purity, membrane perfectness, genuine content and apoptotic markers as prior factors need to be checked before functional application. Based on our observations, EV-SUC confirmed higher membrane integrity compare to EV-20K (P<0.01) and EV-110K (P<0.05) and carried the parental content like as acetylcholinesterase (AChE), however it requires to be adjusted in yield of purity. Based on these findings, EV-S110K as selected EV, also showed promising functional results on healthy and diabetic islet derived cell. Proteome profiling also confirmed distinct protein content of these three sub-populations. Our results attempt to stand up for the necessity of determining the most practical EV subpopulations prior to check its functional tests in in vitro models or even in disease models.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yunqi Wu

LAB HEAD: Ghasem Hosseini Salekdeh

PROVIDER: PXD022174 | Pride | 2026-03-23

REPOSITORIES: Pride

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			Action	DRS
	200708_P90068_Exosome_QE1_HpH_1.raw	Raw
	200708_P90068_Exosome_QE1_HpH_10.raw	Raw
	200708_P90068_Exosome_QE1_HpH_11.raw	Raw
	200708_P90068_Exosome_QE1_HpH_12.raw	Raw
	200708_P90068_Exosome_QE1_HpH_13.raw	Raw

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Publications

Proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Impact of isolation methods on protein cargo.

Abyadeh Morteza M Mirshahvaladi Shahab S Kashani Sara Assar SA Paulo Joao A JA Amirkhani Ardeshir A Mehryab Fatemeh F Seydi Homeyra H Moradpour Niloufar N Jodeiryjabarzade Sheyda S Mirzaei Mehdi M Gupta Vivek V Shekari Faezeh F Salekdeh Ghasem Hosseini GH

Journal of extracellular biology 20240606 6

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we ...[more]

PMID: 38947171

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.