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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220020	1/2	Homo sapiens	Bone marrow derived mesenchymal stromal cells	(d)(U)C	Fischer S	2016	33%
Study summary Full title Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles. All authors Fischer S, Cornils K, Speiseder T, Badbaran A, Reimer R, Indenbirken D, Grundhoff A, Brunswig-Spickenheier B, Alawi M, Lange C Journal PLoS One Abstract The biological relevance of extracellular vesicles (EV) in intercellular communication has been well (show more...)The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development. (hide) EV-METRIC 33% (75th 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: CD81/ HSP70/ CD63/ CD9/ GAPDH non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bone marrow derived mesenchymal stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins CD9/ CD63/ GAPDH/ HSP70/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 146 EV concentration Yes Particle analysis: flow cytometry Flow cytometer type FACSAriaIIIu (Becton Dickinson) Hardware adjustment Calibration bead size 0.2-0.5-0.75-1-2-3 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-150

	EV220020	2/2	Homo sapiens	Bone marrow derived mesenchymal stromal cells	(d)(U)C	Fischer S	2016	0%
Study summary Full title Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles. All authors Fischer S, Cornils K, Speiseder T, Badbaran A, Reimer R, Indenbirken D, Grundhoff A, Brunswig-Spickenheier B, Alawi M, Lange C Journal PLoS One Abstract The biological relevance of extracellular vesicles (EV) in intercellular communication has been well (show more...)The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development. (hide) EV-METRIC 0% (median: 14% 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 Transduction Arabidopsis thaliana-DNA 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 Bone marrow derived mesenchymal stromal cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method Lowry-based assay Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type FACSAriaIIIu (Becton Dickinson) Hardware adjustment Calibration bead size 0.2-0.5-0.75-1-2-3 Report type Not Reported EV concentration Yes

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EV-TRACK ID	EV220020
species	Homo sapiens
sample type	Cell culture
cell type	Bone marrow derived mesenchymal stromal cells
condition	Control condition	Transduction Arabidopsis thaliana-DNA
separation protocol	dUC	dUC
Exp. nr.	1	2
EV-METRIC %	33	0