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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210154	1/2	Homo sapiens	human invasive proliferative extravillous cytotrophoblast (HIPEC)	DG (d)(U)C	Bergamelli M	2022	100%
Study summary Full title Human Cytomegalovirus Modifies Placental Small Extracellular Vesicle Composition to Enhance Infection of Fetal Neural Cells In Vitro. All authors Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE Journal Viruses Abstract Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide) EV-METRIC 100% (99th 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 Other/ 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: calnexin/ TOM20 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human invasive proliferative extravillous cytotrophoblast (HIPEC) EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,00E+08 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1,7 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 30 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected EV-associated proteins CD63 Not detected contaminants calnexin/ TOM20 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes Particle yield as number of particles per million cells: 1,00E+06 Particle analysis: flow cytometry Flow cytometer type Mascquant VYB Myltenyi Hardware adjustment Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm Calibration bead size 0.16/ 0.2/ 0.25/ 0.5 EV concentration Yes Particle yield as number of particles per million cells: 1,00E+06 EM EM-type Immuno-EM/ Transmission-EM EM protein CD9/ CD81/ CD63 Image type Close-up, Wide-field Report size (nm) 120

	EV210154	2/2	Homo sapiens	human invasive proliferative extravillous cytotrophoblast (HIPEC)	DG (d)(U)C	Bergamelli M	2022	100%
Study summary Full title Human Cytomegalovirus Modifies Placental Small Extracellular Vesicle Composition to Enhance Infection of Fetal Neural Cells In Vitro. All authors Bergamelli M, Martin H, Aubert Y, Mansuy JM, Marcellin M, Burlet-Schiltz O, Hurbain I, Raposo G, Izopet J, Fournier T, Benchoua A, Bénard M, Groussolles M, Cartron G, Tanguy Le Gac Y, Moinard N, D'Angelo G, Malnou CE Journal Viruses Abstract Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pre (show more...)Although placental small extracellular vesicles (sEVs) are extensively studied in the context of pregnancy, little is known about their role during viral congenital infection, especially at the beginning of pregnancy. In this study, we examined the consequences of human cytomegalovirus (hCMV) infection on sEVs production, composition, and function using an immortalized human cytotrophoblast cell line derived from first trimester placenta. By combining complementary approaches of biochemistry, electron microscopy, and quantitative proteomic analysis, we showed that hCMV infection increases the yield of sEVs produced by cytotrophoblasts and modifies their protein content towards a potential proviral phenotype. We further demonstrate that sEVs secreted by hCMV-infected cytotrophoblasts potentiate infection in naive recipient cells of fetal origin, including human neural stem cells. Importantly, these functional consequences are also observed with sEVs prepared from an ex vivo model of infected histocultures from early placenta. Based on these findings, we propose that placental sEVs could be important actors favoring viral dissemination to the fetal brain during hCMV congenital infection. (hide) EV-METRIC 100% (99th 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 infected by Human Cytomegalovirus / clinical strain VHL/E Focus vesicles Other/ 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 Density gradient (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Alix/ CD63/ CD9/ CD81 non-EV: calnexin/ TOM20 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells human invasive proliferative extravillous cytotrophoblast (HIPEC) EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell count 1,00E+08 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Rotor type SW 41 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1,7 Fraction processing Centrifugation Pelleting: volume per fraction 30 Pelleting: duration (min) 60 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 30 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix/ CD81 Not detected contaminants calnexin/ TOM20 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield as number of particles per million cells: 1,00E+06 Particle analysis: flow cytometry Flow cytometer type Macsquant VYB Myltenyi Hardware adjustment Mascquant VYB Myltenyi set up fluorescent beads SSC Megamix, with calibration on gates of 160nm 200nm 250nm and 500nm Calibration bead size 0.16/ 0.2/ 0.25/ 0.5 Report type Mean Reported size (nm) 160 EV concentration Yes Particle yield as number of particles per million cells: 1,00E+06 EM EM-type Immuno-EM/ Transmission-EM EM protein CD9/ CD81/ CD63 Image type Close-up, Wide-field Report size (nm) 110

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EV-TRACK ID	EV210154
species	Homo sapiens
sample type	Cell culture
cell type	human invasive proliferative extravillous cytotrophoblast (HIPEC)
condition	Control condition	infected by Human Cytomegalovirus / clinical strain VHL/E
separation protocol	Density gradient/ dUC	Density gradient/ dUC
Exp. nr.	1	2
EV-METRIC %	100	100