Heterogeneous Extracellular Vesicles Populations Research Articles

Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile.

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma

BackgroundPorcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE).MethodsSeminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling.ResultsEach sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04–98.50%).ConclusionsThis study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

Urinary extracellular vesicles in childhood kidney diseases.

Most biological fluids contain extracellular vesicles (EVs). EVs are surrounded by a lipid bilayer and contain biological macromolecules such as proteins, lipids, RNA, and DNA. They lack a functioning nucleus and are incapable of replicating. The physiological characteristics and molecular composition of EVs in body fluids provide valuable information about the status of originating cells. Consequently, they could be effectively utilized for diagnostic and prognostic applications. Urine contains a heterogeneous population of EVs. To date, these urinary extracellular vesicles (uEVs) have been ignored in the standard urinalysis. In recent years, knowledge has accumulated on how uEVs should be separated and analyzed. It has become clear how uEVs reflect the expression of each molecule in cells in nephron segments and how they are altered in disease states such as glomerular/tubular disorders, rare congenital diseases, acute kidney injury (AKI), and chronic kidney disease (CKD). Significant promise exists for the molecular expression signature of uEVs detected by simple techniques such as enzyme-linked immunosorbent assay (ELISA), making them more applicable in clinical settings. This review presents the current understanding regarding uEVs, emphasizing the potential for non-invasive diagnostics, especially for childhood kidney diseases.

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

A heterogenous population of extracellular vesicles mobilize to the alveoli postinjury.

Acute lung injury and subsequent resolution following severe injury are coordinated by a complex lung microenvironment that includes extracellular vesicles (EVs). We hypothesized that there is a heterogenous population of EVs recruited to the alveoli postinjury and that we could identify specific immune-relevant mediators expressed on bronchoalveolar lavage (BAL) EVs as candidate biomarkers of injury and injury resolution. Mice underwent 30% TBSA burn injury and BAL fluid was collected 4 hours postinjury and compared with sham. Extracellular vesicles were purified and single vesicle flow cytometry (vFC) was performed using fluorescent antibodies to quantify the expression of specific cell surface markers on individual EVs. Next, we evaluated human BAL specimens from injured patients to establish translational relevance of the mouse vFC analysis. Human BAL was collected from intubated patients following trauma or burn injury, EVs were purified, then subjected to vFC analysis. A diverse population of EVs were mobilized to the alveoli after burn injury in mice. Quantitative BAL vFC identified significant increases in macrophage-derived CD44+ EVs (preinjury, 10.8% vs. postinjury, 13%; p < 0.05) and decreases in IL-6 receptor alpha (CD126) EVs (preinjury, 19.3% vs. postinjury, 9.3%, p < 0.05). Bronchoalveolar lavage from injured patients also contained a heterogeneous population of EVs derived from myeloid cells, endothelium, and epithelium sources, with CD44+ EVs being highly detected. Injury causes mobilization of a heterogeneous population of EVs to the alveoli in both animal models and injured patients. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit postinjury acute lung injury.

Cryo-electron microscopy of adipose tissue extracellular vesicles in obesity and type 2 diabetes mellitus.

Extracellular vesicles (EVs) are cell-derived membrane vesicles which play an important role in cell-to-cell communication and physiology. EVs deliver biological information from producing to recipient cells by transport of different cargo such as proteins, mRNAs, microRNAs, non-coding RNAs and lipids. Adipose tissue EVs could regulate metabolic and inflammatory interactions inside adipose tissue depots as well as distal tissues. Thus, adipose tissue EVs are assumed to be implicated in obesity-associated pathologies, notably in insulin resistance and type 2 diabetes mellitus (T2DM). In this study we for the first time characterize EVs secreted by visceral (VAT) and subcutaneous adipose tissue (SAT) of patients with obesity and T2DM with standard methods as well as analyze their morphology with cryo-electron microscopy. Cryo-electron microscopy allowed us to visualize heterogeneous population of EVs of various size and morphology including single EVs and EVs with internal membrane structures in samples from obese patients as well from the control group. Single vesicles prevailed (up to 85% for SAT, up to 75% for VAT) and higher proportion of EVs with internal membrane structures compared to SAT was typical for VAT. Decreased size of single and double SAT EVs compared to VAT EVs, large proportion of multilayered EVs and all EVs with internal membrane structures secreted by VAT distinguished obese patients with/without T2DM from the control group. These findings could support the idea of modified biogenesis of EVs during obesity and T2DM.

Exosome in Crosstalk between Inflammation and Angiogenesis: A Potential Therapeutic Strategy for Stroke.

The endothelial dysfunction, associated with inflammation and vascular permeability, remains the key event in the pathogenesis of cerebral ischemic stroke. Angiogenesis is essential for neuroprotection and neural repair following stroke. The neuroinflammatory reaction plays a vital role in stroke, and inhibition of inflammation contributes to establishing an appropriate external environment for angiogenesis. Exosomes are the heterogeneous population of extracellular vesicles which play critical roles in intercellular communication through transmitting various proteins and nucleic acids to nearby and distant recipient cells by body fluids and circulation. Recent reports have shown that exosomal therapy is a valuable and potential treatment strategy for stroke. In this review, we discussed the exosomes in complex interaction mechanisms of angiogenesis and inflammation following stroke as well as the challenges of exosomal studies such as secretion, uptake, modification, and application.

Male-female communication enhances release of extracellular vesicles leading to high fertility in Drosophila

The female reproductive tract (female-RT) must decipher the repertoire of molecular cues received from the male during copulation in order to activate and coordinate tract functionality necessary for high fertility. In Drosophila, this modulation is partially driven by spermathecal secretory cells (SSC). The SSC are a layer of cuboidal secretory glandular cells surrounding the spermatheca capsule where sperm is stored. It is unclear, however, how the SSC regulate the system’s activity. Here we show that mating activates the secretory machinery of the SSC. The SSC release a heterogeneous population of extracellular vesicles (EVs) which is involved in initiating and managing the increase in egg-laying, and possibly sperm storage. Moreover, sperm and male accessory gland proteins are essential for such mating-mediated SSC activity. Thus, mating regulates secretory/endocytic pathways required for trafficking of vesicles to SSC-female-RT target sites, which modulate and coordinate reproductive tract activity to achieve high fertility.

Acute Myeloid Leukemia Cells Functionally Compromise Hematopoietic Stem/Progenitor Cells Inhibiting Normal Hematopoiesis Through the Release of Extracellular Vesicles.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). It is not well known how leukemia cells alter hematopoiesis promoting tumor growth and leukemic niche formation. In this study, we investigated how AML deregulates the hematopoietic process of HSPCs through the release of extracellular vesicles (EVs). First, we found that AML cells released a heterogeneous population of EVs containing microRNAs involved in AML pathogenesis. Notably, AML-EVs were able to influence the fate of HSPCs modifying their transcriptome. In fact, gene expression profile of AML-EV-treated HSPCs identified 923 down- and 630 up-regulated genes involved in hematopoiesis/differentiation, inflammatory cytokine production and cell movement. Indeed, most of the down-regulated genes are targeted by AML-EV-derived miRNAs. Furthermore, we demonstrated that AML-EVs were able to affect HSPC phenotype, modifying several biological functions, such as inhibiting cell differentiation and clonogenicity, activating inflammatory cytokine production and compromising cell movement. Indeed, a redistribution of HSPC populations was observed in AML-EV treated cells with a significant increase in the frequency of common myeloid progenitors and a reduction in granulocyte-macrophage progenitors and megakaryocyte-erythroid progenitors. This effect was accompanied by a reduction in HSPC colony formation. AML-EV treatment of HSPCs increased the levels of CCL3, IL-1B and CSF2 cytokines, involved in the inflammatory process and in cell movement, and decreased CXCR4 expression associated with a reduction of SDF-1 mediated-migration. In conclusion, this study demonstrates the existence of a powerful communication between AML cells and HSPCs, mediated by EVs, which suppresses normal hematopoiesis and potentially contributes to create a leukemic niche favorable to neoplastic development.

Extracellular Vesicles From LPS-Treated Macrophages Aggravate Smooth Muscle Cell Calcification by Propagating Inflammation and Oxidative Stress

Background: Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. Methods: The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi—7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. Results: LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Conclusion: Our results show that EVs derived from LPS-EK–treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.

In Vitro and In Vivo Analysis of Extracellular Vesicle‐Mediated Metastasis Using a Bright, Red‐Shifted Bioluminescent Reporter Protein (Advanced Genetics 1/03)

Extracellular Vesicle Imaging Cancer cell-derived extracellular vesicles (EVs) promote tumor growth and spread. Studying the distribution of EVs in the body is key to understanding cancer progression and developing therapies. In article number 2100055, Masamitsu Kanada and co-workers develop a highly sensitive EV reporter for tracking heterogeneous EV populations released from cancer cells in live mice.

Role of extracellular vesicles in the propagation of inflammation and oxidative stress during vascular calcification

Background and Aims: During vascular calcification (VC), a cardiovascular complication of atherosclerosis, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs) involved in inflammation and oxidative stress. The objective of our study is to determine the role and the mechanism of action of EVs in the propagation of inflammation and oxidative stress during VC.

Isolation and characterization of extracellular vesicles in saliva of children with asthma.

Aim:To confirm the presence of extracellular vesicles (EVs) in cell-free saliva (CFS) of children with asthma and describe the isolated EV population.Methods:A pooled sample of CFS EVs isolated from 180 participants using ExoQuick-TC was examined in downstream analyses. Transmission electron microscopy (TEM) was used to confirm the presence of EVs. Nanoparticle tracking analysis (NTA) and single particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence were used for sizing, counting, and phenotyping of EVs. Capillary immunoassays were used for protein quantitation.Results:TEM confirmed the presence of EVs of diverse sizes, indicating the prep contained a heterogeneous population of EVs. Capillary immunoassays confirmed the presence of EV-associated proteins (CD9, CD63, CD81, ICAM-1, and ANXA5) and indicated limited cellular contamination. As others have also reported, there were discrepancies in the EV sizing and enumeration across platforms. Fluorescent NTA detected particles with a mode diameter of ~90 nm, whereas SP-IRIS reported sizes of ~55–60 nm that more closely approximated the TEM results. Consistent with protein immunoassay results, SP-IRIS with fluorescence showed that the majority of these EVs were CD9- and CD63-positive, with little expression of CD81.Conclusion:EVs from CFS can be isolated using a high-throughput method that can be scaled to large epidemiological studies. To our knowledge, we are the first to characterize CFS EVs from patients with asthma. The use of CFS EVs as potential novel biomarkers in asthma warrants further investigation and opens a new avenue of research for future studies.

Isolation of cancer-derived extracellular vesicle subpopulations by a size-selective microfluidic platform.

Extracellular vesicles (EVs) play an important role in intercellular communication. Recently, there has been increasing interest in EVs as potential diagnostic biomarkers and therapeutic vehicles. However, the molecular properties and cargo information of EV subpopulations have not yet been fully investigated due to lack of reliable and reproducible EV separation technology. Current approaches have faced difficulties with efficiently isolating EVs from biofluids, especially subpopulations of small EVs. Here, we report an EV isolation method based on a size-selective microfluidic platform (ExoSMP) via nanomembrane filtration and electrophoretic force. This unique platform offers an enhanced approach to sorting a heterogeneous population of EVs based on size, with the additional advantages of being label-free and low-cost, and featuring a short processing time (<1 h), and convenient integration with downstream analysis. In this research, we used ExoSMP to demonstrate the isolation of cancer-derived small EVs (30–120 nm) with high recovery (94.2%) and reproducibility at an optimum sample flow rate. Furthermore, we investigated isolation of EV subpopulations by altering nanomembrane combinations with different pore size combinations (50 and 100 nm, 30 and 100 nm, 30 and 200 nm, and 30 and 50 nm). This ExoSMP technique can serve as a standardized EV isolation/separation tool, facilitating the clinical prospects of EVs and opening up a new avenue for future point-of-care applications in liquid biopsies.

Mesenchymal Stem Cell Derived Extracellular Vesicles for Tissue Engineering and Regenerative Medicine Applications.

Mesenchymal stem cells (MSCs) are being extensively investigated for their potential in tissue engineering and regenerative medicine. However, recent evidence suggests that the beneficial effects of MSCs may be manifest by their released extracellular vesicles (EVs); typically not requiring the administration of MSCs. This evidence, predominantly from pre-clinical in vitro and in vivo studies, suggests that MSC-EVs may exhibit substantial therapeutic properties in many pathophysiological conditions, potentially restoring an extensive range of damaged or diseased tissues and organs. These benefits of MSC EVs are apparently found, regardless of the anatomical or body fluid origin of the MSCs (and include e.g., bone marrow, adipose tissue, umbilical cord, urine, etc). Furthermore, early indications suggest that the favourable effects of MSC-EVs could be further enhanced by modifying the way in which the donor MSCs are cultured (for example, in hypoxic compared to normoxic conditions, in 3D compared to 2D culture formats) and/or if the EVs are subsequently bio-engineered (for example, loaded with specific cargo). So far, few human clinical trials of MSC-EVs have been conducted and questions remain unanswered on whether the heterogeneous population of EVs is beneficial or some specific sub-populations, how best we can culture and scale-up MSC-EV production and isolation for clinical utility, and in what format they should be administered. However, as reviewed here, there is now substantial evidence supporting the use of MSC-EVs in tissue engineering and regenerative medicine and further research to establish how best to exploit this approach for societal and economic benefit is warranted.

Single Extracellular Vesicle Protein Analysis Using Immuno-Droplet Digital Polymerase Chain Reaction Amplification.

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno-droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody-DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read-out by droplet imaging. In these proof-of-principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

Mesenchymal stem cells from different sources and their derived exosomes: A pre-clinical perspective.

Since the introduction of cell therapy as a strategy for the treatment of many diseases, mesenchymal stem cells have emerged as ideal candidates, yet the underlying mechanisms of their beneficial effects are only partially understood. At the start of the 21st century, a paracrine effect was proposed as a mechanism of tissue repair by these cells. In addition, a role was suggested for a heterogeneous population of extracellular vesicles in cell-to-cell communication. Some of these vesicles including exosomes have been isolated from most fluids and cells, as well as from supernatants of in vitro cell cultures. Recent research in the field of regenerative medicine suggests that exosomes derived from mesenchymal stem cells could be a powerful new therapeutic tool. This review examines the therapeutic potential of these exosomes obtained from the sources most used in cell therapy: bone marrow, adipose tissue, and umbilical cord.

Single molecule characterization of individual extracellular vesicles from pancreatic cancer

ABSTRACTBiofluid-accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking analysis, proteomics, transcriptomics, transmission electron microscopy, and quantitative single molecule localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer-enriched EV population. Our goal is to advance single molecule characterization of EVs for early disease detection.Abbreviations: EV: Extracellular Vesicle; qSMLM: quantitative Single Molecule Localization Microscopy; PDAC: Pancreatic Ductal Adenocarcinoma; EGFR: epidermal growth factor receptor 1; CA19-9: carbohydrate antigen 19-9; SEC: size exclusion chromatography; WGA: wheat germ agglutinin; AF647: Alexa Fluor 647; Ab: antibody; HPDEC: Healthy Pancreatic Ductal Epithelial Cell; TEM: Transmission Electron Microscopy.

Picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential.

Several naked virus species, including members of the Picornaviridae family, have recently been described to escape their host cells and spread infection via enclosure in extracellular vesicles (EV). EV are 50–300 nm sized lipid membrane-enclosed particles produced by all cells that are broadly recognized for playing regulatory roles in numerous (patho)physiological processes, including viral infection. Both pro- and antiviral functions have been ascribed to EV released by virus-infected cells. It is currently not known whether this reported functional diversity is a result of the release of multiple virus-containing and non-virus containing EV subpopulations that differ in composition and function. Using encephalomyocarditis virus infection (EMCV, Picornaviridae family), we here provide evidence that EV populations released by infected cells are highly heterogeneous. Virus was contained in two distinct EV populations that differed in physical characteristics, such as sedimentation properties, and in enrichment for proteins indicative of different EV biogenesis pathways, such as the plasma membrane resident proteins Flotillin-1 and CD9, and the autophagy regulatory protein LC3. Additional levels of EV heterogeneity were identified using high-resolution flow cytometric analysis of single EV. Importantly, we demonstrate that EV subsets released during EMCV infection varied largely in potency of transferring virus infection and in their kinetics of release from infected cells. These data support the notion that heterogeneous EV populations released by virus-infected cells can exert diverse functions at distinct time points during infection. Unraveling the compositional, temporal and functional heterogeneity of these EV populations using single EV analysis technologies, as employed in this study, is vital to understanding the role of EV in virus dissemination and antiviral host responses.