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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200179	3/4	Homo sapiens	Primary human trophoblast	(d)(U)C Filtration DG UF	Ouyang, Yingshi	2016	88%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 88% (98th 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 (Differential) (ultra)centrifugation Filtration Density gradient Ultrafiltration Protein markers EV: TSG101/ CD63/ Syntenin-1 non-EV: Argonaute2 Proteomics yes EV density (g/ml) 1.08-1.10 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary human trophoblast EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight, 100000g or commercial Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 6 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1.5 Orientation Bottom-up Rotor type Not specified Speed (g) 100000 Duration (min) Overnight Fraction volume (mL) . Fraction processing Ultrafiltration Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 kda Membrane type PES Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ Syntenin-1/ TSG101 Not detected contaminants Argonaute2 Proteomics database Yes: Data and Specimen Hub Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mode Reported size (nm) 80-90nm EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV200179	4/4	Homo sapiens	Blood plasma	(d)(U)C Filtration DG UF PEG precipitaton Gelatin-sepharose chromatograhy	Ouyang, Yingshi	2016	75%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 75% (96th 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 Blood plasma Sample origin Healthy pregnant 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 (Differential) (ultra)centrifugation Filtration Density gradient Ultrafiltration PEG precipitaton Gelatin-sepharose chromatograhy Protein markers EV: CD63 non-EV: Fibronectin Proteomics yes EV density (g/ml) 1.08-1.10 Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 6 Highest density fraction 30 Total gradient volume, incl. sample (mL) 14 Sample volume (mL) 0.5 Orientation Top-down Rotor type Not specified Speed (g) 10000 Duration (min) Overnight Fraction volume (mL) . Fraction processing Ultrafiltration Pelleting: volume per fraction . Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 kda Membrane type PES Other Name other separation method PEG precipitaton Other Name other separation method Gelatin-sepharose chromatograhy Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63 Not detected contaminants Fibronectin Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mode Reported size (nm) 80-90nm EV concentration Yes

	EV200179	1/4	Homo sapiens	Primary human trophoblast	(d)(U)C	Ouyang, Yingshi	2016	15%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 15% (52nd 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 apoptotic body 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 Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary human trophoblast EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight, 100000g or commercial Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type Not specified Pelleting: speed (g) 2500 Characterization: Protein analysis Protein Concentration Method BCA Proteomics database Yes: Data and Specimen Hub Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up

	EV200179	2/4	Homo sapiens	Primary human trophoblast	(d)(U)C	Ouyang, Yingshi	2016	15%
Study summary Full title Isolation of human trophoblastic extracellular vesicles and characterization of their cargo and antiviral activity All authors Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky Journal Placenta Abstract Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide) EV-METRIC 15% (52nd 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 (shedding) microvesicle 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 Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary human trophoblast EV-harvesting Medium EV-depleted medium Preparation of EDS Overnight, 100000g or commercial Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 12000 Wash: volume per pellet (ml) 2 Wash: time (min) 30 Wash: Rotor Type Not specified Wash: speed (g) 12000 Characterization: Protein analysis Protein Concentration Method BCA Proteomics database Yes: Data and Specimen Hub Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mode Reported size (nm) 200nm EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV200179
species	Homo sapiens
sample type	Cell culture	Blood plasma	Cell culture	Cell culture
cell type	Primary human trophoblast	NA	Primary human trophoblast	Primary human trophoblast
medium	EV-depleted medium	NA	EV-depleted medium	EV-depleted medium
condition	Control condition	Healthy pregnant	Control condition	Control condition
separation protocol	dUC Filtration Density gradient Ultrafiltration	dUC Filtration Density gradient Ultrafiltration PEG precipitaton Gelatin-sepharose chromatograhy	dUC	dUC
vesicle related term	exosome	exosome	apoptotic body	(shedding) microvesicle
Exp. nr.	3	4	1	2
EV-METRIC %	88	75	15	15