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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV231018	1/2	Homo sapiens	Wharton's jelly MSC	(d)(U)C qEVsingle Gen2	Tscherrig V	2023	78%
Study summary Full title MicroRNA Cargo in Wharton's Jelly MSC Small Extracellular Vesicles: Key Functionality to In Vitro Prevention and Treatment of Premature White Matter Injury. All authors Tscherrig V, Cottagnoud S, Haesler V, Renz P, Surbek D, Schoeberlein A, Joerger-Messerli MS Journal Stem Cell Rev Rep Abstract Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matt (show more...)Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matter injury (WMI), leading to long-term neurological disabilities with global health burden. Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEV) are a promising therapeutic agent for treating perinatal neurological injury. They carry microRNAs (miRNAs) predicted to be involved in the onset of premature WMI. We hypothesize that miRNAs have a key function in the beneficial effects of MSC-sEV. We isolated MSC from umbilical cord tissue, the Wharton's jelly (WJ), and purified small extracellular vesicles (sEV) from WJ-MSC culture supernatant by ultracentrifugation and size exclusion chromatography. The miRNA content was quantified by real-time polymerase chain reaction. A luciferase gene assay validated silencing of TP53 and TAOK1, which we previously identified as predicted target genes of MSC-sEV miRNAs by Next Generation Sequencing and pathway enrichment analysis. The impact of sEV miRNAs on oligodendroglial maturation and neuronal apoptosis was evaluated using an in vitro oxygen-glucose deprivation model (OGD/R) by knocking-down DROSHA in WJ-MSC, which initiates miRNA processing. WJ-MSC-sEV contained miRNAs involved in WMI, namely hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-27b-3p, and the hsa-let-7 family. The luciferase assay strongly indicated an inhibitory effect of sEV miRNAs on the gene expression of TP53 and TAOK1. Small EV initiated oligodendrocyte maturation and reduced OGD/R-mediated neuronal apoptosis. Knocking-down DROSHA in WJ-MSC reduced the expression of sEV miRNAs and led to the loss of their beneficial effects. Our in vitro study strongly indicates the key function of miRNAs in the therapeutic potential of WJ-MSC-sEV in premature WMI. (hide) EV-METRIC 78% (97th 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 small extracellular vesicles 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ CD81/ Syntenin/ CD9 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Wharton's jelly MSC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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: rotor type TLA-100.3 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 110000 Commercial kit qEVsingle Gen2 Characterization: Protein analysis Protein Concentration Method NanoView Spectrometry Protein Yield (µg) per microliter of recovered sample Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin Not detected contaminants GM130/ Calnexin Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV231018	2/2	Homo sapiens	Wharton's jelly MSC	(d)(U)C qEVsingle Gen2	Tscherrig V	2023	78%
Study summary Full title MicroRNA Cargo in Wharton's Jelly MSC Small Extracellular Vesicles: Key Functionality to In Vitro Prevention and Treatment of Premature White Matter Injury. All authors Tscherrig V, Cottagnoud S, Haesler V, Renz P, Surbek D, Schoeberlein A, Joerger-Messerli MS Journal Stem Cell Rev Rep Abstract Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matt (show more...)Preterm birth is the leading cause of childhood morbidity and mortality and can result in white matter injury (WMI), leading to long-term neurological disabilities with global health burden. Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEV) are a promising therapeutic agent for treating perinatal neurological injury. They carry microRNAs (miRNAs) predicted to be involved in the onset of premature WMI. We hypothesize that miRNAs have a key function in the beneficial effects of MSC-sEV. We isolated MSC from umbilical cord tissue, the Wharton's jelly (WJ), and purified small extracellular vesicles (sEV) from WJ-MSC culture supernatant by ultracentrifugation and size exclusion chromatography. The miRNA content was quantified by real-time polymerase chain reaction. A luciferase gene assay validated silencing of TP53 and TAOK1, which we previously identified as predicted target genes of MSC-sEV miRNAs by Next Generation Sequencing and pathway enrichment analysis. The impact of sEV miRNAs on oligodendroglial maturation and neuronal apoptosis was evaluated using an in vitro oxygen-glucose deprivation model (OGD/R) by knocking-down DROSHA in WJ-MSC, which initiates miRNA processing. WJ-MSC-sEV contained miRNAs involved in WMI, namely hsa-miR-22-3p, hsa-miR-21-5p, hsa-miR-27b-3p, and the hsa-let-7 family. The luciferase assay strongly indicated an inhibitory effect of sEV miRNAs on the gene expression of TP53 and TAOK1. Small EV initiated oligodendrocyte maturation and reduced OGD/R-mediated neuronal apoptosis. Knocking-down DROSHA in WJ-MSC reduced the expression of sEV miRNAs and led to the loss of their beneficial effects. Our in vitro study strongly indicates the key function of miRNAs in the therapeutic potential of WJ-MSC-sEV in premature WMI. (hide) EV-METRIC 78% (97th 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 DROSHA siRNA modified Focus vesicles small extracellular vesicles 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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD63/ CD81/ Syntenin non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Wharton's jelly MSC EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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: rotor type TLA-100.3 Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-100.3 Wash: speed (g) 110000 Commercial kit qEVsingle Gen2 Characterization: Protein analysis Protein Concentration Method NanoView Spectrometry Protein Yield (µg) per microliter of recovered sample Western Blot Detected EV-associated proteins CD63/ CD81/ Syntenin Not detected contaminants GM130/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 2000 RNAse treatment Yes RNAse type RNase A RNAse concentration 0.02 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 148 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.00E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV231018
species	Homo sapiens
sample type	Cell culture
cell type	Wharton's jelly MSC
condition	Control condition	DROSHA siRNA modified
separation protocol	dUC/ qEVsingle Gen2	dUC/ qEVsingle Gen2
Exp. nr.	1	2
EV-METRIC %	78	78