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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180048	1/1	Mus musculus	mesenchymal stem cells	(d)(U)C	Luther KM	2017	11%
Study summary Full title Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells. All authors Luther KM, Haar L, McGuinness M, Wang Y, Lynch Iv TL, Phan A, Song Y, Shen Z, Gardner G, Kuffel G, Ren X, Zilliox MJ, Jones WK Journal J Mol Cell Cardiol Abstract Though experimental, stem cell transplantation has the potential to improve the condition of the hea (show more...)Though experimental, stem cell transplantation has the potential to improve the condition of the heart after myocardial infarction. It does so by reducing infarct size and inducing repair of heart muscle and its blood supply. Mesenchymal stem cells (MSC) have been found to be effective in pre-clinical animal models and clinical trials, but the mechanisms by which they induce cardioprotection and repair are still not fully understood. Small extracellular vesicles known as exosomes are now recognized to be key mediators of beneficial MSC paracrine effects, and the concept that they transfer miRNA to change gene expression in recipient cells is of current therapeutic interest. We present complete deep miRNA sequencing of MSC exosome cargo, and found that of several cardioprotective miRNAs, miR-21a-5p was the most abundant. Because miR-21a-5p is a well-known cardioprotective miRNA, we investigated the hypothesis that MSC exosomes can cardioprotect the heart by increasing the level of miR-21a-5p in recipient cardiac cells, thereby downregulating expression of the pro-apoptotic gene products PDCD4, PTEN, Peli1 and FasL in the myocardium. Using miR-21 mimic transfection and treatment with wild type and miR-21a knockout MSC exosomes, we confirmed that exosomal miR-21a-5p is transferred into myocardium and is a major cardioprotective paracrine factor produced by MSCs acting via synergistic activity on multiple pathways. The data supports that residual cardioprotective effect may be due to other ncRNA or protein cargo. In silico analyses support that MSC exosomes may also contribute to angiogenesis, cell proliferation and other aspects of cardiac repair. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 209.7 (pelleting) / 60.38 (washing) Protein markers EV: TSG101/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS 1.5h at 100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 100000 Wash: adjusted k-factor 60.38 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins TSG101, CD9 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Amnis ImageStream Hardware adjustment Calibration bead size 100-150 EV concentration Yes Extra information Number of particles per microgram protein in final sample was 4.5 X 10E4

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EV-TRACK ID	EV180048
species	Mus musculus
sample type	Cell culture
cell type	mesenchymal stem cells
condition	Control condition
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	11