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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210197	1/4	Homo sapiens	Platelets	(d)(U)C	Puhka, Maija	2017	56%
Study summary Full title Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes All authors Maija Puhka, Maarit Takatalo, Maria-Elisa Nordberg, Sami Valkonen, Jatin Nandania, Maria Aatonen, Marjo Yliperttula, Saara Laitinen, Vidya Velagapudi, Tuomas Mirtti, Olli Kallioniemi, Antti Rannikko, Pia R-M Siljander, Taija Maria Af Hällström Journal Theranostics Abstract Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the se (show more...)Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 1010 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials. (hide) EV-METRIC 56% (90th 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 extracellular vesicle 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 Protein markers EV: TSG101/ CD63/ CD9 non-EV: TOMM20 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Platelets EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 75 Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 110000 Wash: volume per pellet (ml) 16 Wash: time (min) 75 Wash: Rotor Type Not specified Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants TOMM20 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 1-1000

	EV210197	3/4	Homo sapiens	Urine	(d)(U)C Filtration	Puhka, Maija	2017	56%
Study summary Full title Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes All authors Maija Puhka, Maarit Takatalo, Maria-Elisa Nordberg, Sami Valkonen, Jatin Nandania, Maria Aatonen, Marjo Yliperttula, Saara Laitinen, Vidya Velagapudi, Tuomas Mirtti, Olli Kallioniemi, Antti Rannikko, Pia R-M Siljander, Taija Maria Af Hällström Journal Theranostics Abstract Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the se (show more...)Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 1010 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials. (hide) EV-METRIC 56% (89th 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 Urine Sample origin Control condition Focus vesicles extracellular vesicle 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 Filtration Protein markers EV: TSG101/ CD59/ CD63/ CD9 non-EV: Calnexin/ TOMM20/ GM130 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD59/ TSG101 Not detected contaminants Calnexin/ TOMM20/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 1-500 EV concentration Yes EM EM-type Immuno-EM/ Transmission-EM EM protein Other;CD63;CD59 Image type Close-up, Wide-field Report size (nm) 1-1000

	EV200153	1/6	Homo sapiens	HTR-8/SVneo	DG (d)(U)C Filtration	Grace Truong	2017	56%
Study summary Full title Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy All authors Grace Truong, Dominic Guanzon, Vyjayanthi Kinhal, Omar Elfeky, Andrew Lai, Sherri Longo, Zarin Nuzhat, Carlos Palma, Katherin Scholz-Romero, Ramkumar Menon, Ben W Mol, Gregory E Rice, Carlos Salomon Journal PLoS One Abstract Our understanding of how cells communicate has undergone a paradigm shift since the recent recogniti (show more...)Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications. (hide) EV-METRIC 56% (90th 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 8% oxygen 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: TSG101/ CD63 non-EV: None Proteomics no EV density (g/ml) 1.13-1.19 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HTR-8/SVneo EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Cell count 6E8 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: time(min) 120 Pelleting: rotor type Surespin 630/36 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 14.5mL Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) Not specified Fraction processing Centrifugation Pelleting: volume per fraction Not spec Pelleting: duration (min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins TSG101 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 108+/-15 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) Around 100nm

	EV170012	1/3	Homo sapiens	adipose tissue mesenchymal stromal cells	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide) EV-METRIC 56% (90th 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 extracellular vesicle 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 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose tissue mesenchymal stromal cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

	EV170012	2/3	Homo sapiens	dermal fibroblasts	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide) EV-METRIC 56% (90th 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 extracellular vesicle 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 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells dermal fibroblasts 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

	EV170012	3/3	Homo sapiens	bone marrow-derived mesenchymal stromal cells	(d)(U)C	Gualerzi, Alice	2017	56%
Study summary Full title Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells All authors Alice Gualerzi, Stefania Niada, Chiara Giannasi, Silvia Picciolini, Carlo Morasso, Renzo Vanna, Valeria Rossella, Massimo Masserini, Marzia Bedoni, Fabio Ciceri, Maria Ester Bernardo, Anna Teresa Brini & Furio Gramatica Journal Scientific Reports Abstract Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeut (show more...)Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves. (hide) EV-METRIC 56% (90th 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 extracellular vesicle 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 158.5 (pelleting) / 158.5 (washing) Protein markers EV: Flotillin-1/ CD63/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stromal cells 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 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 55.2 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 158.5 Wash: time (min) 70 Wash: Rotor Type Type 55.2 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 158.5 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 46.5 ± 15.8 Other particle analysis name(1) Raman spectroscopy Extra information Primary antibodies: - Purified Mouse Anti-Flotillin-1; Clone 18/Flotillin-1; BD Transduction Laboratories™, San Jose, CA, USA - Rabbit anti-CD63; System Biosciences, Palo Alto, CA, USA - Rabbit anti-CD9; System Biosciences, Palo Alto, CA, USA - Rabbit anti-calnexin; clone C5C9, Cell Signaling Technology, Danvers, MA, USA

	EV180047	1/1	Homo sapiens	mesenchymal stem cells	(d)(U)C Filtration	O'Brien KP	2017	55%
Study summary Full title Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379. All authors O'Brien KP, Khan S, Gilligan KE, Zafar H, Lalor P, Glynn C, O'Flatharta C, Ingoldsby H, Dockery P, De Bhulbh A, Schweber JR, St John K, Leahy M, Murphy JM, Gallagher WM, O'Brien T, Kerin MJ, Dwyer RM Journal Oncogene Abstract Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting pote (show more...)Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer. (hide) EV-METRIC 55% (88th 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 miR-379 expressing Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 98.44 (pelleting) Protein markers EV: CD63 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type S50-A Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 98.44 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170060	1/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other Sample origin Control condition Focus vesicles extracellular vesicle 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 208.3 (pelleting) / 208.3 (washing) Protein markers EV: CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 1.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	2/4	Bos taurus	Serum	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (91st 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 Serum Sample origin Control condition Focus vesicles extracellular vesicle 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 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 2.50E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	3/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other Sample origin Control condition Focus vesicles extracellular vesicle 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 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 3.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170060	4/4	Bos taurus	Other	(d)(U)C	Kornilov R	2017	55%
Study summary Full title Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. All authors Kornilov R, Puhka M, Mannerström B, Hiidenmaa H, Peltoniemi H, Siljander P, Seppänen-Kaijansinkko R, Kaur S Journal J Extracell Vesicles Abstract Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture expe (show more...)Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories. (hide) EV-METRIC 55% (50th 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 Other Sample origin Control condition Focus vesicles extracellular vesicle 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 208.3 (pelleting) / 208.3 (washing) Protein markers EV: HSP70/ CD63/ CD71 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Other Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 121896 Pelleting: adjusted k-factor 208.3 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 121896 Wash: adjusted k-factor 208.3 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, CD71 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield 1.00E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field Extra information Aim of the study was to compare different EV depletion protocols for fetal bovine serum (FBS).

	EV170045	1/1	Homo sapiens	Primary glioblastoma cells	(d)(U)C Filtration	Treps L	2017	55%
Study summary Full title Glioblastoma stem-like cells secrete the pro-angiogenic VEGF-A factor in extracellular vesicles. All authors Treps L, Perret R, Edmond S, Ricard D, Gavard J Journal J Extracell Vesicles Abstract Glioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour ce (show more...)Glioblastoma multiforme (GBM) are mortifying brain tumours that contain a subpopulation of tumour cells with stem-like properties, termed glioblastoma stem-like cells (GSCs). GSCs largely contribute to tumour initiation, propagation and resistance to current anti-cancer therapies. GSCs are situated in perivascular niches, closely associated with brain microvascular endothelial cells, thereby involved in bidirectional molecular and cellular interactions. Moreover, extracellular vesicles are suspected to carry essential information that can adapt the microenvironment to the tumour's needs, including tumour-induced angiogenesis. In GBM, extracellular vesicles produced by differentiated tumour cells and GSCs were demonstrated to disseminate locally and at distance. Here, we report that the pro-angiogenic pro-permeability factor VEGF-A is carried in extracellular vesicles secreted from ex vivo cultured patient-derived GSCs. Of note, extracellular vesicle-derived VEGF-A contributes to the in vitro elevation of permeability and angiogenic potential in human brain endothelial cells. Indeed, VEGF-A silencing in GSCs compromised in vitro extracellular vesicle-mediated increase in permeability and angiogenesis. From a clinical standpoint, extracellular vesicles isolated from circulating blood of GBM patients present higher levels of VEGF-A, as compared to healthy donors. Overall, our results suggest that extracellular vesicle-harboured VEGF-A targets brain endothelial cells and might impact their ability to form new vessels. Thus, tumour-released EV cargo might emerge as an instrumental part of the tumour-induced angiogenesis and vascular permeability modus operandi in GBM. (hide) EV-METRIC 55% (88th 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 Brain cancer Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 138.6 (pelleting) / 138.6 (washing) Protein markers EV: CD63/ TIMP2/ TIMP1/ VEGF-A/ ANXA5/ MMP1 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Primary glioblastoma cells EV-harvesting Medium Serum free medium Cell viability (%) NA 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: time(min) 240 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 138.6 Wash: time (min) 120 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 138.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA ELISA Antibody details provided? No Lysis buffer provided? Yes Flow cytometry Type of Flow cytometry MACSQuant Analyzer Antibody details provided? No Other 1 Protein array Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 109 EV concentration Yes EM EM-type Transmission-EM/ Immune-EM EM protein CD63 Image type Close-up, Wide-field Extra information Wash volume not in original publication.

	EV170018	2/12	Homo sapiens	osteoclasts	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 3, grown on hydroxyapatite Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 120 EV concentration Yes Particle yield 1.20E+08 particles/ml start sample

	EV170018	4/12	Homo sapiens	osteoclasts	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 4, grown on hydroxyapatite Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 135 EV concentration Yes Particle yield 7.90E+08 particles/ml start sample

	EV170018	6/12	Homo sapiens	primary circulating monocytes	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 3, LPS-activated Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary circulating monocytes EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 133 EV concentration Yes Particle yield 3.60E+08 particles/ml start sample

	EV170018	9/12	Homo sapiens	osteoclasts	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 3, grown on polystyrene Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 134 EV concentration Yes Particle yield 7.40E+08 particles/ml start sample

	EV170018	10/12	Homo sapiens	osteoclasts	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 4, grown on polystyrene Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD63/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells osteoclasts EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 131 EV concentration Yes Particle yield 5.40E+08 particles/ml start sample

	EV170018	11/12	Homo sapiens	primary circulating monocytes	(d)(U)C Filtration	Gebraad A	2017	55%
Study summary Full title Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells. All authors Gebraad A, Kornilov R, Kaur S, Miettinen S, Haimi S, Peltoniemi H, Mannerström B, Seppänen-Kaijansinkko R Journal FEBS J Abstract Intercellular communication is essential in bone remodelling to ensure that new bone is formed with (show more...)Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. (hide) EV-METRIC 55% (88th 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 Donor 4, LPS-activated Focus vesicles extracellular vesicle 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 Filtration Adj. k-factor 211.6 (pelleting) / 211.6 (washing) Protein markers EV: TSG101/ HSP70/ CD90 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary circulating monocytes EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 211.6 Wash: time (min) 120 Wash: Rotor Type SW 28 Wash: speed (g) 120000 Wash: adjusted k-factor 211.6 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins HSP70, TSG101, CD90 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 147 EV concentration Yes Particle yield 3.20E+08 particles/ml start sample

	EV170016	2/2	Mus musculus	adipose tissue-derived macrophages	(d)(U)C Filtration	Ying, Wei	2017	55%
Study summary Full title Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity. All authors Ying W, Riopel M, Bandyopadhyay G, Dong Y, Birmingham A, Seo JB, Ofrecio JM, Wollam J, Hernandez-Carretero A, Fu W, Li P, Olefsky JM Journal Cell Abstract MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we (show more...)MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis. (hide) EV-METRIC 55% (88th 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 Filtration Adj. k-factor 256 (pelleting) / 256 (washing) Protein markers EV: TSG101/ HSP70/ Syntenin1/ CD63/ CD9 non-EV: Grp94 Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells adipose tissue-derived macrophages EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240-360 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 256.0 Wash: time (min) 20 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 256.0 Filtration steps 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes 1.13-1.15 g/ml Used subtypes Yes Characterization: Protein analysis Protein Concentration Method DC protein assay Protein Yield (µg) 9-May Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, HSP70, TSG101, Syntenin1 Not detected contaminants Grp94 Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Extra information Full UC protocol not in original article

	EV200114	1/4	Homo sapiens	Trophoblasts	DG (d)(U)C Filtration	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (87th 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Trophoblasts EV-harvesting Medium Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes

	EV200114	3/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (83rd 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 Overweight (BMI=25-29.9kg/m2) 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 4.20e+7

	EV200114	4/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	50%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 50% (83rd 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 Obese (BMI= >30kg/m2) 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 6.30e+7

	EV170062	1/1	Homo sapiens	Blood plasma	(d)(U)C ExoQuick IAF	Mustapic M	2017	50%
Study summary Full title Plasma Extracellular Vesicles Enriched for Neuronal Origin: A Potential Window into Brain Pathologic Processes. All authors Mustapic M, Eitan E, Werner JK, Berkowitz ST, Lazaropoulos MP, Tran J, Goetzl EJ, Kapogiannis D Journal J Cell Sci Abstract Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin (show more...)Our team has been a pioneer in harvesting extracellular vesicles (EVs) enriched for neuronal origin from peripheral blood and using them as a biomarker discovery platform for neurological disorders. This methodology has demonstrated excellent diagnostic and predictive performance for Alzheimer's and other neurodegenerative diseases in multiple studies, providing a strong proof of concept for this approach. Here, we describe our methodology in detail and offer further evidence that isolated EVs are enriched for neuronal origin. In addition, we present evidence that EVs enriched for neuronal origin represent a more sensitive and accurate base for biomarkers than plasma, serum, or non-enriched total plasma EVs. Finally, we proceed to investigate the protein content of EVs enriched for neuronal origin and compare it with other relevant enriched and non-enriched populations of plasma EVs. Neuronal-origin enriched plasma EVs contain higher levels of signaling molecules of great interest for cellular metabolism, survival, and repair, which may be useful as biomarkers and to follow response to therapeutic interventions in a mechanism-specific manner. (hide) EV-METRIC 50% (83rd 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 Control condition Focus vesicles extracellular vesicle 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 ExoQuick IAF Protein markers EV: TSG101/ MAP2/ gamma-enolase/ L1CAMandCD81/ L1CAM/ pTau23/ CD9/ tubulin-betaIII non-EV: None Proteomics no Show all info Study aim Biomarker, Technical analysis comparing/optimizing EV-related methods 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 Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) L1CAM Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, L1CAM, MAP2, gamma-enolase, pTau23, tubulin-betaIII ELISA Antibody details provided? No Lysis buffer provided? Yes Other 1 Proteome Profiler Human Phospho-Mitogen-activated Protein Kinase (MAPK) Antibody Array (Catalog # ARY002B), Human Kidney Biomarker Antibody Array (Catalog # ARY019), Human Phospho-Kinase Antibody Arra Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Nanoparticle tracking analysis Extra particle analysis NTA Report type Size range/distribution EV concentration Yes EM EM-type Immune-EM EM protein L1CAM and CD81 Image type Close-up, Wide-field

	EV170061	2/3	Homo sapiens	BEAS2B	(d)(U)C	Benedikter BJ	2017	50%
Study summary Full title Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. All authors Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM Journal J Cell Sci Abstract Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide) EV-METRIC 50% (87th 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 extracellular vesicle 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 133.2 (pelleting) / 48.17 (washing) Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Wash: time (min) 150 Wash: Rotor Type NVT 90 Wash: speed (g) 110656 Wash: adjusted k-factor 48.17 Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS EV concentration Yes Particle yield 2.00E+00

	EV170061	3/3	Homo sapiens	BEAS2B	(d)(U)C	Benedikter BJ	2017	50%
Study summary Full title Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. All authors Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM Journal J Cell Sci Abstract Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide) EV-METRIC 50% (87th 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 extracellular vesicle 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 133.2 (pelleting) Protein markers EV: CD81/ CD63 non-EV: None Proteomics yes Show all info Study aim Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Characterization: Protein analysis Protein Concentration Method Bradford Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD63 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Median Reported size (nm) 74.6 EV concentration Yes Particle yield 1.50E+01 EM EM-type Cryo-EM Image type Close-up Report size (nm) 25-200

	EV170002	1/1	Homo sapiens	Umbilical cord mesenchymal stem cells	(d)(U)C SEC UF	Monguió-Tortajada M	2017	50%
Study summary Full title Nanosized UCMSC-derived extracellular vesicles but not conditioned medium exclusively inhibit the inflammatory response of stimulated T cells: implications for nanomedicine. All authors Monguió-Tortajada M, Roura S, Gálvez-Montón C, Pujal JM, Aran G, Sanjurjo L, Franquesa M, Sarrias MR, Bayes-Genis A, Borràs FE Journal Theranostics Abstract Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation (show more...)Undesired immune responses have drastically hampered outcomes after allogeneic organ transplantation and cell therapy, and also lead to inflammatory diseases and autoimmunity. Umbilical cord mesenchymal stem cells (UCMSCs) have powerful regenerative and immunomodulatory potential, and their secreted extracellular vesicles (EVs) are envisaged as a promising natural source of nanoparticles to increase outcomes in organ transplantation and control inflammatory diseases. However, poor EV preparations containing highly-abundant soluble proteins may mask genuine vesicular-associated functions and provide misleading data. Here, we used Size-Exclusion Chromatography (SEC) to successfully isolate EVs from UCMSCs-conditioned medium. These vesicles were defined as positive for CD9, CD63, CD73 and CD90, and their size and morphology characterized by NTA and cryo-EM. Their immunomodulatory potential was determined in polyclonal T cell proliferation assays, analysis of cytokine profiles and in the skewing of monocyte polarization. In sharp contrast to the non-EV containing fractions, to the complete conditioned medium and to ultracentrifuged pellet, SEC-purified EVs from UCMSCs inhibited T cell proliferation, resembling the effect of parental UCMSCs. Moreover, while SEC-EVs did not induce cytokine response, the non-EV fractions, conditioned medium and ultracentrifuged pellet promoted the secretion of pro-inflammatory cytokines by polyclonally stimulated T cells and supported Th17 polarization. In contrast, EVs did not induce monocyte polarization, but the non-EV fraction induced CD163 and CD206 expression and TNF-α production in monocytes. These findings increase the growing evidence confirming that EVs are an active component of MSC's paracrine immunosuppressive function and affirm their potential for therapeutics in nanomedicine. In addition, our results highlight the importance of well-purified and defined preparations of MSC-derived EVs to achieve the immunosuppressive effect. (hide) EV-METRIC 50% (87th 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 extracellular vesicle 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 SEC UF Adj. k-factor 138.6 (pelleting) Protein markers EV: CD63/ CD73/ CD90/ MHC2/ CD9/ MHC1 non-EV: None Proteomics no Show all info Study aim Function, Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Umbilical cord mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 138.6 Ultra filtration Cut-off size (kDa) 100 Membrane type NS Size-exclusion chromatography Total column volume (mL) 1 Sample volume/column (mL) 0.1 Resin type Sepharose CL-2B Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean;Median and size distribution Reported size (nm) 160-230 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV210220	1/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 DG (d)(U)C Commercial method Protein markers EV: CD63/ PLAP/ sENG/ sFLT-1 non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Not detected EV-associated proteins sENG/ sFLT-1 Characterization: Lipid analysis Lipi Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-400 EM EM-type Transmission-EM Image type Close-up Report size (nm) 100nm

	EV210220	2/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick Immunoaffinity capture	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 DG (d)(U)C Commercial method Immunoaffinity capture Protein markers EV: CD63/ TGFBR1/ TGFBR2/ sENG non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) PLAP Other Name other separation method Immunoaffinity capture Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TGFBR1/ TGFBR2 Not detected EV-associated proteins sENG Characterization: Lipid analysis Lipi Characterization: Particle analysis None

	EV210220	3/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 Preeclampsia 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 DG (d)(U)C Commercial method Protein markers EV: CD63/ PLAP/ sENG/ sFLT-1 non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins sENG/ CD63/ PLAP Not detected EV-associated proteins sFLT-1 Flow cytometry Type of Flow cytometry Beckman Coulter Gallios 10/3 Antibody details provided? No Detected EV-associated proteins PLAP Characterization: Lipid analysis Lipi Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-400

	EV210220	4/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick Immunoaffinity capture	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 Preeclampsia 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 DG (d)(U)C Commercial method Immunoaffinity capture Protein markers EV: CD63/ TGFBR1/ TGFBR2/ sENG non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) PLAP Other Name other separation method Immunoaffinity capture Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TGFBR1/ TGFBR2/ sENG Characterization: Lipid analysis Lipi Characterization: Particle analysis None

	EV210220	5/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 pre-term birth 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 DG (d)(U)C Commercial method Protein markers EV: CD63/ PLAP/ sENG/ sFLT-1 non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ PLAP Not detected EV-associated proteins sENG/ sFLT-1 Flow cytometry Type of Flow cytometry Beckman Coulter Gallios 10/3 Antibody details provided? No Detected EV-associated proteins PLAP Characterization: Lipid analysis Lipi Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-400

	EV210220	6/6	Homo sapiens	Blood plasma	DG (d)(U)C ExoQuick Immunoaffinity capture	Ermini, Leonardo	2017	45%
Study summary Full title A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia All authors Leonardo Ermini, Jonathan Ausman, Megan Melland-Smith, Behzad Yeganeh, Alessandro Rolfo, Michael L Litvack, Tullia Todros, Michelle Letarte, Martin Post 10 11 , Isabella Caniggia 12 Journal Sci Rep Abstract Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a (show more...)Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women. (hide) EV-METRIC 45% (79th 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 pre-term birth 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 DG (d)(U)C Commercial method Immunoaffinity capture Protein markers EV: CD63/ TGFBR1/ TGFBR2/ sENG non-EV: None Proteomics no EV density (g/ml) * Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 1200 Pelleting: rotor type TLA-55 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers Not specified Lowest density fraction 0.25M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) Not specified Sample volume (mL) Not spec Orientation Not specified Rotor type TLA-100 Speed (g) 110000 Duration (min) 1200 Fraction volume (mL) 10 fractions, 0.3mL per fraction Fraction processing None Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) PLAP Other Name other separation method Immunoaffinity capture Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TGFBR1/ TGFBR2 Not detected EV-associated proteins sENG Characterization: Lipid analysis Lipi Characterization: Particle analysis None

	EV210202	6/7	Mus musculus	Primary adipocytes	(d)(U)C	Durcin, Maëva	2017	45%
Study summary Full title Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles All authors Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide) EV-METRIC 45% (86th 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 Large 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary adipocytes EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type MLA-50 Pelleting: speed (g) 13000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type MLA-50 Wash: speed (g) 13000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Detected EV-associated proteins Mfge8/ Flotillin2/ caveolin-1 Not detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Reported size (nm) 110-150 EV concentration Yes Particle yield EV sercreted per adipocyte;Yes, other: 100 EM EM-type Transmission-EM Image type Close-up

	EV210202	7/7	Mus musculus	Primary adipocytes	(d)(U)C	Durcin, Maëva	2017	45%
Study summary Full title Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles All authors Maëva Durcin, Audrey Fleury, Emiliane Taillebois, Grégory Hilairet, Zuzana Krupova, Céline Henry, Sandrine Truchet, Martin Trötzmüller, Harald Köfeler, Guillaume Mabilleau, Olivier Hue, Ramaroson Andriantsitohaina, Patrice Martin, Soazig Le Lay Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells (show more...)Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues. (hide) EV-METRIC 45% (86th 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 (d)(U)C Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary adipocytes EV-harvesting Medium Serum free medium 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) 60 Pelleting: rotor type MLA-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 60 Wash: Rotor Type MLA-50 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ Mfge8/ Flotillin2/ caveolin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Reported size (nm) 80-100 EV concentration Yes Particle yield EV sercreted per adipocyte;Yes, other: 200 EM EM-type Transmission-EM Image type Close-up

	EV200153	2/6	Homo sapiens	HTR-8/SVneo	DG (d)(U)C Filtration	Grace Truong	2017	45%
Study summary Full title Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells - Liquid biopsies for monitoring complications of pregnancy All authors Grace Truong, Dominic Guanzon, Vyjayanthi Kinhal, Omar Elfeky, Andrew Lai, Sherri Longo, Zarin Nuzhat, Carlos Palma, Katherin Scholz-Romero, Ramkumar Menon, Ben W Mol, Gregory E Rice, Carlos Salomon Journal PLoS One Abstract Our understanding of how cells communicate has undergone a paradigm shift since the recent recogniti (show more...)Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications. (hide) EV-METRIC 45% (86th 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 1% oxygen 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63 non-EV: None Proteomics no EV density (g/ml) 1.13-1.19 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HTR-8/SVneo EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Cell count 6E8 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: time(min) 120 Pelleting: rotor type Surespin 630/36 Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 14.5mL Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) Not specified Fraction processing Centrifugation Pelleting: volume per fraction Not spec Pelleting: duration (min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? No Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 108+/-15 EV concentration Yes

	EV210480	1/4	Homo sapiens	MDA-MB-231	(d)(U)C DG	Li XJ	2017	44%
Study summary Full title Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer. All authors Li XJ, Ren ZJ, Tang JH, Yu Q Journal Cell Physiol Biochem Abstract Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microR (show more...)Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. (hide) EV-METRIC 44% (84th 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 extracellular vesicle 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 Density gradient Protein markers EV: TSG101/ Alix/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW40 Ti Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: Rotor Type SW40 Ti Wash: speed (g) 110000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Orientation Top-down Rotor type SW 40 Ti Speed (g) 110000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: duration (min) 70 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins CD9/ CD63/ TSG101/ Alix Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution

	EV210457	1/2	Bos taurus	Primary oviduct epithelial cells	(d)(U)C	Almiñana C	2017	44%
Study summary Full title Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. All authors Almiñana C, Corbin E, Tsikis G, Alcântara-Neto AS, Labas V, Reynaud K, Galio L, Uzbekov R, Garanina AS, Druart X, Mermillod P Journal Reproduction Abstract Successful pregnancy requires an appropriate communication between the mother and the embryo. Recent (show more...)Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by and oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between and oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when and EVs were compared ( < 0.01). Interestingly, 97 were exclusively expressed in EVs, 47 were present only in and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in EVs. Moreover, we showed that -produced embryos were able to internalize EVs during culture with a functional effect in the embryo development. EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under conditions. (hide) EV-METRIC 44% (84th 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 extracellular vesicle 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: HSPA8/ HSP70/ MYH9/ OVGP non-EV: Grp78 Proteomics yes Show all info Study aim Function Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells Primary oviduct epithelial cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins MYH9/ HSPA8/ HSP70 Not detected EV-associated proteins OVGP Not detected contaminants Grp78 Proteomics database Yes: Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-250

	EV210457	2/2	Bos taurus	Oviduct flushing	(d)(U)C	Almiñana C	2017	44%
Study summary Full title Oviduct extracellular vesicles protein content and their role during oviduct-embryo cross-talk. All authors Almiñana C, Corbin E, Tsikis G, Alcântara-Neto AS, Labas V, Reynaud K, Galio L, Uzbekov R, Garanina AS, Druart X, Mermillod P Journal Reproduction Abstract Successful pregnancy requires an appropriate communication between the mother and the embryo. Recent (show more...)Successful pregnancy requires an appropriate communication between the mother and the embryo. Recently, exosomes and microvesicles, both membrane-bound extracellular vesicles (EVs) present in the oviduct fluid have been proposed as key modulators of this unique cross-talk. However, little is known about their content and their role during oviduct-embryo dialog. Given the known differences in secretions by and oviduct epithelial cells (OEC), we aimed at deciphering the oviduct EVs protein content from both sources. Moreover, we analyzed their functional effect on embryo development. Our study demonstrated for the first time the substantial differences between and oviduct EVs secretion/content. Mass spectrometry analysis identified 319 proteins in EVs, from which 186 were differentially expressed when and EVs were compared ( < 0.01). Interestingly, 97 were exclusively expressed in EVs, 47 were present only in and 175 were common. Functional analysis revealed key proteins involved in sperm-oocyte binding, fertilization and embryo development, some of them lacking in EVs. Moreover, we showed that -produced embryos were able to internalize EVs during culture with a functional effect in the embryo development. EVs increased blastocyst rate, extended embryo survival over time and improved embryo quality. Our study provides the first characterization of oviduct EVs, increasing our understanding of the role of oviduct EVs as modulators of gamete/embryo-oviduct interactions. Moreover, our results point them as promising tools to improve embryo development and survival under conditions. (hide) EV-METRIC 44% (50th 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 Oviduct flushing Sample origin Control condition Focus vesicles extracellular vesicle 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: HSPA8/ HSP70/ MYH9/ OVGP non-EV: Grp78 Proteomics yes Show all info Study aim Function Sample Species Bos taurus Sample Type Oviduct flushing Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins OVGP/ MYH9/ HSPA8/ HSP70 Not detected contaminants Grp78 Proteomics database Yes: Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-250

	EV200146	1/2	Homo sapiens	Blood plasma	"DG (d)(U)C Filtration"	Salomon, Carlos	2017	44%
Study summary Full title Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation All authors Carlos Salomon, Dominic Guanzon, Katherin Scholz-Romero, Sherri Longo, Paula Correa, Sebastian E Illanes, Gregory E Rice Journal J Clin Endocrinol Metab Abstract Context: There is a need to develop strategies for early prediction of patients who will develop pre (show more...)Context: There is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications. Objective: The objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE. Design, setting, patients, and interventions: A retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples. Results: In presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs. Conclusions: Although the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE. (hide) EV-METRIC 44% (77th 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 "Density gradient (Differential) (ultra)centrifugation Filtration" Protein markers EV: "TSG101/ PLAP" non-EV: None Proteomics no EV density (g/ml) 1.12-1.19 Show all info Study aim "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 Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 120 Wash: Rotor Type T-8100 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 14.5mL Sample volume (mL) 0.5mL Orientation Top-down Rotor type T-8100 Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.05 Fraction processing Centrifugation Pelleting: volume per fraction 0.05 Pelleting: duration (min) 120 Pelleting: rotor type T-8100 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Other Name other separation method "Density gradient Other Name other separation method Filtration" Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins PLAP Characterization: RNA analysis RNA analysis Type RNA sequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 40-130nm EM EM-type Transmission-EM Image type Close-up

	EV200114	2/4	Homo sapiens	Blood plasma	DG (d)(U)C	Elfeky O	2017	44%
Study summary Full title Influence of maternal BMI on the exosomal profile during gestation and their role on maternal systemic inflammation. All authors Elfeky O, Longo S, Lai A, Rice GE, Salomon C Journal Placenta Abstract Recent studies report that 35% of women are either overweight or obese at reproductive age. The plac (show more...)Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy. (hide) EV-METRIC 44% (77th 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 Density gradient (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD63/ PLAP/ IgG1 non-EV: None Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Sample volume (mL) 0.5mL Orientation Top-down Rotor type Not specified Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) Not specified Fraction processing Not specified Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Fluorescent NTA Relevant measurements variables specified? NA Antibody details provided? Yes Detected EV-associated proteins PLAP/ IgG1/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Used for determining EV concentration? Yes NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 4.70e+7 EM EM-type Transmission-EM Image type Close-up

	EV170053	1/1	Homo sapiens	HUVEC	(d)(U)C	Pérez-Boza J	2017	44%
Study summary Full title Exploring the RNA landscape of endothelial exosomes. All authors Pérez-Boza J, Lion M, Struman I Journal RNA Abstract Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. (show more...)Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes. (hide) EV-METRIC 44% (84th 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 232.7 (pelleting) / 232.7 (washing) Protein markers EV: CD81/ ANXA2/ CD63/ CD9 non-EV: CytochromeC Proteomics no Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 232.7 Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 232.7 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81, ANXA2 Not detected contaminants CytochromeC Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 110

	EV170047	1/8	Homo sapiens	LNCaP	(d)(U)C	Soekmadji C	2017	44%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 44% (84th 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 Enzalutamide Focus vesicles extracellular vesicle 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 Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: GAPDH Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Not detected contaminants GAPDH Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 120 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170047	3/8	Homo sapiens	LNCaP	(d)(U)C	Soekmadji C	2017	44%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 44% (84th 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 DHT Focus vesicles extracellular vesicle 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 Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: GAPDH Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.017 Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Not detected contaminants GAPDH Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170047	4/8	Homo sapiens	LNCaP	(d)(U)C	Soekmadji C	2017	44%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 44% (84th 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 Charcoal stripped serum Focus vesicles extracellular vesicle 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 Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: GAPDH Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.012 Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Not detected contaminants GAPDH Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 120 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170047	5/8	Homo sapiens	LNCaP	(d)(U)C	Soekmadji C	2017	44%
Study summary Full title Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer. All authors Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C, Hovens CM, Corcoran NM, Hill MM, Nelson CC Journal Oncotarget Abstract Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by (show more...)Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. (hide) EV-METRIC 44% (84th 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 extracellular vesicle 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 Protein markers EV: Alix/ TSG101/ PSA/ CD9 non-EV: GAPDH Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LNCaP EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g 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) 120 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.005 Western Blot Antibody details provided? No Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ TSG101 Not detected EV-associated proteins PSA Not detected contaminants GAPDH Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution,Mode Reported size (nm) 150 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170026	1/1	Mus musculus	bone marrow-derived mesenchymal stem cells	DC (d)(U)C Filtration	Prakash Gangadaran	2017	44%
Study summary Full title Extracellular vesicles from mesenchymal stem cells activates VEGF receptors and accelerates recovery of hindlimb ischemia. All authors Gangadaran P, Rajendran RL, Lee HW, Kalimuthu S, Hong CM, Jeong SY, Lee SW, Lee J, Ahn BC Journal J Control Release Abstract Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for (show more...)Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are potential therapies for various diseases, but their angiogenic mechanisms of therapeutic efficacy remain unclear. Here, we describe how MSC-EVs, activates VEGF receptors and downstream angiogenesis pathways. Mouse MSC-EVs were isolated from cell culture medium and characterized using transmission electron microscopy, nanoparticle analysis, and western blotting. In vitro migration, proliferation, and tube formation assays using endothelial cells were used to assess the angiogenic potential of MSC-EVs, and revealed higher levels of cellular migration, proliferation, and tube formation after treatment. qRT-PCR and western blotting (WB) revealed higher protein and mRNA expression of the angiogenic genes VEGFR1 and VEGFR2 in mouse SVEC-4 endothelial cells after MSC-EVs treatment. Additionally, other vital pro-angiogenic pathways (SRC, AKT, and ERK) were activated by in vitro MSC-EV treatment. WB and qRT-PCR revealed enriched presence of VEGF protein and miR-210-3p in MSC-EV. The hindlimb ischemia mouse model was established and MSC-EVs with or without Matrigel (EV-MSC+Gel) were injected into the ischemic area and blood reperfusion was monitored using molecular imaging techniques. The in vivo administration of MSC-EVs increased both blood reperfusion and the formation of new blood vessels in the ischemic limb, with the addition of matrigel enhancing this effect further by releasing EVs slowly. MSC-EVs enhance angiogenesis in ischemic limbs, most likely via the overexpression of VEGFR1 and VEGFR2 in endothelial cells. These findings reveal a novel mechanism of activating receptors by MSC-EVs influence the angiogenesis. (hide) EV-METRIC 44% (84th 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 extracellular vesicle 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 DC (d)(U)C Filtration Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: Alix/ CD63 non-EV: calnexin/ cytochrome c/ GM130/ cytochromec Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived mesenchymal stem cells EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type SW 28 Wash: speed (g) 100000 Wash: adjusted k-factor 253.9 Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix, CD63 Not detected contaminants GM130, calnexin, cytochrome c Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 135 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV170021	1/2	Homo sapiens	LIM1215	(d)(U)C	Liem, Michael	2017	44%
Study summary Full title Insulin mediated activation of PI3K/Akt signalling pathway modifies the proteomic cargo of extracellular vesicles. All authors Liem M, Ang CS, Mathivanan S Journal Proteomics Abstract Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 44% (84th 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 Insulin induced Focus vesicles extracellular vesicle 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 253.9 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ TSG101,AKT,pAKT,beta-actin,FAT1,p-cadherin/ AKT/ Alix/ FAT1/ pAKT/ beta-actin/ p-cadherin non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LIM1215 EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Densitometry (SYPRO Ruby) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101,AKT,pAKT,beta-actin,FAT1,p-cadherin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes Particle yield 3.08E+07 particles/million cells

	EV170021	2/2	Homo sapiens	LIM1215	(d)(U)C	Liem, Michael	2017	44%
Study summary Full title Insulin mediated activation of PI3K/Akt signalling pathway modifies the proteomic cargo of extracellular vesicles. All authors Liem M, Ang CS, Mathivanan S Journal Proteomics Abstract Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (C (show more...)Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression. This article is protected by copyright. All rights reserved. (hide) EV-METRIC 44% (84th 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 extracellular vesicle 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 253.9 (pelleting) / 89.2 (washing) Protein markers EV: TSG101/ AKT/ Alix/ FAT1/ beta-actin/ p-cadherin/ TSG101,AKT,beta-actin,FAT1,p-cadherin non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LIM1215 EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 60 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 89.20 Characterization: Protein analysis Protein Concentration Method Densitometry (SYPRO Ruby) Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix, TSG101,AKT,beta-actin,FAT1,p-cadherin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-150 EV concentration Yes Particle yield 1.85E+07 particles/million cells

	EV170003	1/1	Mus musculus	mIMCD3	(d)(U)C	Nager AR	2017	44%
Study summary Full title An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling. All authors Nager AR, Goldstein JS, Herranz-Pérez V, Portran D, Ye F, Garcia-Verdugo JM, Nachury MV Journal Cell Abstract Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. H (show more...)Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts. (hide) EV-METRIC 44% (84th 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 GPCR signaling in BBS mutant Focus vesicles Ciliary Ectosome 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 253.9 (pelleting) / 99.86 (washing) Protein markers EV: CD81/ Arl13B/ BiotinylatedGPCRs non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells mIMCD3 EV-harvesting Medium Serum-containing 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: time(min) 90 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Wash: time (min) 90 Wash: Rotor Type TLS-55 Wash: speed (g) 100000 Wash: adjusted k-factor 99.86 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD81, Arl13B Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM/ Immune-EM EM protein Biotinylated GPCRs Image type Close-up, Wide-field Report size (nm) 70-120

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