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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220127	1/2	Homo sapiens	lung tissue	(d)(U)C DG Filtration	Liu, Bowen/ Jin, Yuan	2022	100%
Study summary Full title Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation All authors Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide) EV-METRIC 100% (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 lung tissue 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 Filtration Adj. k-factor 20553 (pelleting) / 17842 (washing) Protein markers EV: Alix/ CD9/ CD81 non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.1 Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type lung tissue 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) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 20553 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 110,000 Wash: adjusted k-factor 17842 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 0.25 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.45 Orientation Bottom-up Rotor type MLS-50 Speed (g) 180,000 Duration (min) 780 Fraction volume (mL) 0.45 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 17842 Pelleting-wash: volume per pellet (mL) 1.5 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) TLA-55 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.8 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 131.7 Particle analysis: flow cytometry Flow cytometer type BD LSRFortessa Hardware adjustment use calibration beads Calibration bead size 0.05/ 0.1/ 0.2/ 0.3/ 0.5 Report type Size range/distribution Reported size (nm) 100 - 200 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV220127	2/2	Mus musculus	lung tissue	(d)(U)C DG Filtration	Liu, Bowen/ Jin, Yuan	2022	100%
Study summary Full title Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation All authors Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide) EV-METRIC 100% (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 lung tissue 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 Filtration Adj. k-factor 20553 (pelleting) / 17842 (washing) Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101 non-EV: GM130/ Calnexin/ Albumin/ Argonaute-2/ Calreticulin/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.1 Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type lung tissue 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) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 20553 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 110,000 Wash: adjusted k-factor 17842 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 0.25 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.45 Orientation Bottom-up Rotor type MLS-50 Speed (g) 180,000 Duration (min) 780 Fraction volume (mL) 0.45 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 17842 Pelleting-wash: volume per pellet (mL) 1.5 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) TLA-55 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.7 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101 Not detected contaminants GM130/ Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124.9 Particle analysis: flow cytometry Flow cytometer type BD LSRFortessa Hardware adjustment use calibration beads Calibration bead size 0.05/ 0.1/ 0.2/ 0.3/ 0.5 Report type Size range/distribution Reported size (nm) 100 - 200 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV210151	1/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 95 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2 Not detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 2.23E7 +- 1.35E7 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	2/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CPC (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2/ TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 8.27E6 +- 3.53E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	3/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMi (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2/ TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 2.95E7 +- 1.19E7 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	4/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	100%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin2 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.08 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMm (IMR90)-4 EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin2 Not detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 nm EV concentration Yes Particle yield number of particles per million cell per 24h;Yes, other: 4.10E7 +- 9.75E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	5/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hiPSC (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 90 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 1.99E7 +- 1.99E6 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	6/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CPC (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 6.43E06 +- 5.40E05 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	7/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMi (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 3.49E07 +- 2.85E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210151	8/8	Homo sapiens	Cell culture supernatant	DG (d)(U)C Filtration	Louro, Ana Filipa	2022	89%
Study summary Full title Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation All authors Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra Journal Advanced Science Abstract Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide) EV-METRIC 89% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles 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 DG (d)(U)C Filtration Protein markers EV: TSG101 non-EV: Argonaute2 Proteomics no EV density (g/ml) 1.083 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CMm (DF19-9-11T.H) EV-harvesting Medium Serum free medium Cell viability (%) 85 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) 180 Pelleting: rotor type SW 28 Pelleting: speed (g) 110000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 0.8 Orientation Bottom-up Rotor type SW 28.1 Speed (g) 110000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants Argonaute2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes Particle yield number of particles per million cells per 24h;Yes, other: 2.13E07 +- 2.25E06 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190029	1/8	Homo sapiens	Cell culture supernatant	(d)(U)C	Bhome R	2022	78%
Study summary Full title Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles. All authors Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE Journal J Extracell Vesicles Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium EV-depleted medium 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TFT 50.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type TFT 50.38 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Alix/ CD81 Not detected contaminants cytochrome c/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 110 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190029	2/8	Homo sapiens	Cell culture supernatant	(d)(U)C	Bhome R	2022	78%
Study summary Full title Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles. All authors Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE Journal J Extracell Vesicles Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT-116 EV-harvesting Medium EV-depleted medium 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TFT 50.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type TFT 50.38 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101/ CD81 Not detected contaminants cytochrome c/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 98 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190029	3/8	Homo sapiens	Cell culture supernatant	(d)(U)C	Bhome R	2022	78%
Study summary Full title Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles. All authors Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE Journal J Extracell Vesicles Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW620 EV-harvesting Medium EV-depleted medium 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TFT 50.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type TFT 50.38 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101/ CD81 Not detected contaminants cytochrome c/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 115 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190029	4/8	Homo sapiens	Cell culture supernatant	(d)(U)C	Bhome R	2022	78%
Study summary Full title Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles. All authors Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE Journal J Extracell Vesicles Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW480 EV-harvesting Medium EV-depleted medium 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TFT 50.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type TFT 50.38 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101/ CD81 Not detected contaminants cytochrome c/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 124 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV190029	5/8	Homo sapiens	Cell culture supernatant	(d)(U)C	Bhome R	2022	78%
Study summary Full title Epithelial to mesenchymal transition influences fibroblast phenotype in colorectal cancer by altering miR-200 levels in extracellular vesicles. All authors Bhome R, Emaduddin M, James V, House LM, Thirdborough SM, Mellone M, Tulkens J, Primrose JN, Thomas GJ, De Wever O, Mirnezami AH, Sayan AE Journal J Extracell Vesicles Abstract Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for (show more...)Colorectal cancer (CRC) with a mesenchymal gene expression signature has the greatest propensity for distant metastasis and is characterised by the accumulation of cancer-associated fibroblasts in the stroma. We investigated whether the epithelial to mesenchymal transition status of CRC cells influences fibroblast phenotype, with a focus on the transfer of extracellular vesicles (EVs), as a controlled means of cell-cell communication. Epithelial CRC EVs suppressed TGF-β-driven myofibroblast differentiation, whereas mesenchymal CRC EVs did not. This was driven by miR-200 (miR-200a/b/c, -141), which was enriched in epithelial CRC EVs and transferred to recipient fibroblasts. Ectopic miR-200 expression or ZEB1 knockdown, in fibroblasts, similarly suppressed myofibroblast differentiation. Supporting these findings, there was a strong negative correlation between miR-200 and myofibroblastic markers in a cohort of CRC patients in the TCGA dataset. This was replicated in mice, by co-injecting epithelial or mesenchymal CRC cells with fibroblasts and analysing stromal markers of myofibroblastic phenotype. Fibroblasts from epithelial tumours contained more miR-200 and expressed less ACTA2 and FN1 than those from mesenchymal tumours. As such, these data provide a new mechanism for the development of fibroblast heterogeneity in CRC, through EV-mediated transfer of miRNAs, and provide an explanation as to why CRC tumours with greater metastatic potential are CAF rich. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin ZEB-1 knock down 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: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW480 EV-harvesting Medium EV-depleted medium 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type TFT 50.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 20 Wash: time (min) 70 Wash: Rotor Type TFT 50.38 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101/ Alix/ CD81 Not detected contaminants cytochrome c/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 105 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	1/17	Homo sapiens	Cell culture supernatant	(d)(U)C	Osteikoetxea X	2022	78%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield number of particles per million cells: 4.00e+4 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	3/17	Homo sapiens	Cell culture supernatant	(d)(U)C	Osteikoetxea X	2022	78%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin MysPalm-PHYB-PIF6-Cas9 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: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Not detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ syntenin-1/ spCas9 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield number of particles per million cells: 2.00e+4 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	5/17	Homo sapiens	Cell culture supernatant	(d)(U)C	Osteikoetxea X	2022	78%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin MysPalm-CIBN-CRY2-Cas9 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: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ Syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ Syntenin-1/ spCas9 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield number of particles per million cells: 4.00e+4 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210199	1/1	Homo sapiens	Urine	(d)(U)C Filtration	Correll, Vanessa	2022	78%
Study summary Full title Optimization of small extracellular vesicle isolation from expressed prostatic secretions in urine for in-depth proteomic analysis All authors Vanessa L. Correll, Joseph J. Otto, Cristina M. Risi, Brian P. Main, Paul C. Boutros, Thomas Kislinger, Vitold E. Galkin, Julius O. Nyalwidhe, O. John Semmes, Lifang Yang Journal J Extracell Vesicles Abstract The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient (show more...)The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin prostate 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 Protein markers EV: TSG101/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods/Biomarker/New methodological development Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 175000 Wash: volume per pellet (ml) 12.5 Wash: time (min) 130 Wash: Rotor Type SW 40 Ti Wash: speed (g) 175000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per 1E10 particles: 1.16 Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Detected contaminants Tamm-Horsfall protein Not detected contaminants Calnexin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.20E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210187	1/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Swatler, Julian	2022	78%
Study summary Full title 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells All authors Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka Journal Blood advances Abstract Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide) EV-METRIC 78% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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/ Alix/ CD63/ CD81 non-EV: APOA1/ GM130 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells K562 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 3.00E+08 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 Type 45 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 60 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Alix/ CD81 Not detected contaminants APOA1/ GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94 EV concentration Yes Particle yield Yes, as number of particles per million cells 3.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210002	1/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	78%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 78% (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 dissociated tissues 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 No extra separation steps Protein markers EV: Alix/ CD63/ Flotillin1/ CD9/ CD81 non-EV: Calnexin/ PDI Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type dissociated tissues Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type Type 80 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 7 Wash: time (min) 90 Wash: Rotor Type Type 80 Ti Wash: speed (g) 150000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin1/ CD9/ CD63/ Alix/ CD81 Not detected contaminants Calnexin/ PDI Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database Yes Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 0,01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Per dissociated thyroid;Yes, other: 1,50E+09 EM EM-type Transmission-EM/ Scanning-EM Image type Close-up, Wide-field

	EV220003	1/2	Homo sapiens	Blood plasma	DG SEC (non-commercial)	Karimi, Nasibeh	2022	75%
Study summary Full title Tetraspanins distinguish separate extracellular vesicle subpopulations in human serum and plasma - Contributions of platelet extracellular vesicles in plasma samples All authors Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer Journal J Extracell Vesicles Abstract Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide) EV-METRIC 75% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin non-EV: Calnexin Proteomics no EV density (g/ml) 1.06-1.16 Show all info Study aim Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 12 ml Sample volume (mL) 6 ml Orientation Top-down Rotor type SW 41 Ti Speed (g) 178,000 Duration (min) 180 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) 27.8 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin Not detected contaminants Calnexin Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Detected EV-associated proteins CD9/ CD63/ CD81/ CD41a Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield as total number of particles in pooled SEC fractions from 6 mL of sample: 2.90e+9 Report type Size range/distribution Report size 50-70 EV-concentration No

	EV210383	1/2	Mus musculus	Serum	ExoQuick	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide) EV-METRIC 75% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 ExoQuick Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Not detected contaminants Albumin Proteomics database Yes Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210383	2/2	Mus musculus	Serum	IAF	Mkrtchian, Soiuren	2022	75%
Study summary Full title Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles All authors Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán Journal Front Immunol Abstract Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide) EV-METRIC 75% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Immunoaffinity capture (non-commercial) Protein markers EV: CD63/ CD81/ HSP70 non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Serum Separation Method Immunoaffinity capture Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) 0.75 Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ HSP70 Detected contaminants Albumin Proteomics database Yes Characterization: RNA analysis RNA analysis Type (RT)?(q)PCR/ RNA?sequencing Database NGI Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210251	1/1	Sus scrofa	Follicular fluid	Exo-spin	Gad A	2022	75%
Study summary Full title Small-extracellular vesicles and their microRNA cargo from porcine follicular fluids: the potential association with oocyte quality. All authors Gad A, Murin M, Bartkova A, Kinterova V, Marcollova K, Laurincik J, Prochazka R Journal J Anim Sci Biotechnol Abstract Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable (show more...)Ovarian follicular fluids (FFs) contain several kinds of regulatory factors that maintain a suitable microenvironment for oocyte development. Extracellular vesicles (EVs) are among the factors that play essential roles in regulating follicle and oocyte development through their cargo molecules that include microRNAs (miRNAs). This study aimed to investigate small-EV (s-EV) miRNAs in porcine FFs and their potential association with oocyte quality. (hide) EV-METRIC 75% (95th 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 Follicular fluid Sample origin Control condition Focus vesicles small 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 Commercial method Protein markers EV: Alix/ CD63/ TSG101 non-EV: ATP5A/ CytC Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Follicular fluid Separation Method Commercial kit Exo-spin Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) particles per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ TSG101 Not detected contaminants ATP5A/ CytC Characterization: RNA analysis RNA analysis Type Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 135 EV concentration Yes Particle yield particles per milliliter of starting sample: 9.00e+9 EM EM-type Transmission EM Image type Close-up, Wide-field

	EV210237	1/2	Homo sapiens	Blood plasma	qEV	Gelibter S	2022	75%
Study summary Full title The impact of storage on extracellular vesicles: A systematic study. All authors Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R Journal J Extracell Vesicles Abstract Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (hide) EV-METRIC 75% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 qEV Protein markers EV: Alix/ Flotillin-1/ Lamp1/ ANXA1 non-EV: GM130/ ApoE/ H3 Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) particles per milliliter of starting sample Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ Flotillin-1/ Lamp1/ ANXA1 Not detected contaminants GM130/ ApoE/ H3 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 50-350 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.00e+11 EM EM-type Immuno-EM EM protein CD63 Image type Wide-field

	EV210165	1/2	Homo sapiens	Cell culture supernatant	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (94th 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 Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 100 EV concentration Yes Particle yield as number of particles per million cells: 6.00e+0

	EV210165	2/2	Homo sapiens	Cell culture supernatant	DG DC	Phu TA	2022	75%
Study summary Full title IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity. All authors Phu TA, Ng M, Vu NK, Bouchareychas L, Raffai RL Journal Mol Ther Abstract Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macr (show more...)Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6C monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity. (hide) EV-METRIC 75% (94th 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 IL-4 cytokine treated 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 Density cushion Protein markers EV: CD81/ CD63/ CD9 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell count 1,00E+06 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5 Highest density fraction 40 Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 3 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Density cushion Density medium Iodixanol Sample volume 98 Cushion volume 2 Density of the cushion 60% Centrifugation time 180 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0,4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 90,5 EV concentration Yes Particle yield as number of particles per million cells: 5.00e+0

	EV200029	1/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (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 Bovine embryo culture medium 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 Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV 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 CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94.5±1.7 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV200029	2/2	Bos taurus	Bovine embryo culture medium	qEV	Pavani KC	2022	75%
Study summary Full title Hatching is modulated by microRNA-378a-3p derived from extracellular vesicles secreted by blastocysts. All authors Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K Journal Proc Natl Acad Sci U S A Abstract SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (hide) EV-METRIC 75% (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 Bovine embryo culture medium 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 Commercial method Protein markers EV: TSG101/ CD63/ CD9 non-EV: AGO2/ APOA1 Proteomics no Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Bovine embryo culture medium Separation Method Commercial kit qEV 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 CD9/ CD63/ TSG101 Detected contaminants APOA1 Not detected contaminants AGO2 Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment Before RNAse type RNase A RNAse concentration 10 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 76.5±8.2 E08 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field

	EV210345	7/17	Homo sapiens	Cell culture supernatant	(d)(U)C	Osteikoetxea X	2022	67%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 67% (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 Cell culture supernatant Sample origin MysPalm-pMag-nMag-Cas9 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: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ Syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210345	9/17	Homo sapiens	Cell culture supernatant	(d)(U)C	Osteikoetxea X	2022	67%
Study summary Full title Engineered Cas9 extracellular vesicles as a novel gene editing tool. All authors Osteikoetxea X, Silva A, Lázaro-Ibáñez E, Salmond N, Shatnyeva O, Stein J, Schick J, Wren S, Lindgren J, Firth M, Madsen A, Mayr LM, Overman R, Davies R, Dekker N Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic app (show more...)Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. (hide) EV-METRIC 67% (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 Cell culture supernatant Sample origin MysPalm-FKBP-FRB-Cas9 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: Alix/ CD63/ CD81/ Flotillin1/ TSG101/ B-actin/ Syntenin-1 non-EV: Calnexin Proteomics no Show all info Study aim Function/Drug delivery Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Expi293F EV-harvesting Medium Serum free medium Cell viability (%) 95.4 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: time(min) 70 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 38 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) number of particles per million cells Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix/ CD63/ CD81/ Flotillin1/ TSG101/ syntenin-1/ B-actin Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210187	3/5	Homo sapiens	Cell culture supernatant	(d)(U)C	Swatler, Julian	2022	67%
Study summary Full title 4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells All authors Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka Journal Blood advances Abstract Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide) EV-METRIC 67% (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 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 non-EV: APOA1/ GM130 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 MOLM-14 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 98 Cell count 2.80E+08 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 Type 45 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 60 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Not detected contaminants APOA1/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 95 EV concentration Yes Particle yield Yes, as number of particles per million cells 5.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	1/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th 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 Commercial method Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 102.9 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.17E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	2/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th 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 NAFL 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 Commercial method Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 113.3 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.34E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210168	3/3	Homo sapiens	Blood plasma	qEV IAF	Newman, Lauren	2022	67%
Study summary Full title Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease All authors Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland Journal biomedicines Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide) EV-METRIC 67% (95th 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 NASH 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 Commercial method Immunoaffinity capture (non-commercial) Protein markers EV: None non-EV: albumin/ calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Commercial kit qEV Immunoaffinity capture Selected surface protein(s) ASGR1 Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 110.1 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 2.73E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV210002	2/3	Mus musculus	dissociated tissues	(d)(U)C	Delcorte, Ophélie	2022	67%
Study summary Full title BRAF V600E Induction in Thyrocytes Triggers Important Changes in the miRNAs Content and the Populations of Extracellular Vesicles Released in Thyroid Tumor Microenvironment All authors Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux Journal Biomediscines Abstract Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide) EV-METRIC 67% (33rd 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 quantitat

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