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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180048 1/1 Mus musculus Cell culture supernatant dUC Luther KM 2017 11%

Study summary

Full title
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% (28th 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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
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
Sample Condition
Control condition
EV-producing cells
mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
1.5h at 100,000g
Cell viability
90
Isolation Method
Differential ultracentrifugation
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: 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
Detected EV-associated proteins
TSG101, CD9
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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