Journal of Extracellular Vesicles

Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is central for mediating the EVs role in intercellular communication processes, labelling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of the EVs target cells and tissues. However, the accuracy and specificity of commonly utilized labelling dyes has not been sufficiently analyzed. By combining recent advancements in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant MSC-EV preparations, we explored the EV labelling efficacy of various fluorescent dyes, specifically of CFDA-SE, Calcein AM, PKH67, BODIPY-TR-Ceramide and a novel lipid dye named Exoria. Our analyses qualified Exoria as the only dye which specifically labels EVs within our MSC-EV preparations. Furthermore, we demonstrate Exoria labelling does not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labelled EVs were differentially taken-up by different immune cell types. Overall, our results qualify Exoria as an appropriate dye for the labelling of EVs derived from our MSC-EV preparations, this study also demonstrates the need for the development of next generation EV characterization tools which are able to localize and confirm specificity of EV labelling.

CD4 T helper cells (TH cells) play a vital role in coordinating and amplifying the immune response to specific pathogens. They constitutively produce different kinds of extracellular vesicles (EVs), which mediate cell-to cell communication and play diverse roles in immune regulation and inflammatory processes. Here we provide a resource documenting the composition of activated TH cell EVs and demonstrating their ability to instigate pro-inflammatory response in antigen-presenting cells (APCs). EVs were characterized by lipidomics, proteomics, and NanoFCM. The activated TH cells derived EVs (act-EVs) were found to be enriched in TH cell-specific proteins, transmembrane and cytosolic EV marker proteins, and HLA proteins relative to resting CD4 T cells EVs (rest-EVs). The pro-inflammatory effect of act-EVs vs rest-EVs on donor matched APCs were characterized by chemokine and cytokine profiling and flow cytometry analysis. There was no discernible contrast in endotoxin levels between act-EVs and rest-EVs. Functional distinctions were seen to arise from variations in the content and composition of these EVs. Moreover, we validated our findings with an in-vivo investigation in mice, demonstrating the recruitment of monocytes, dendritic cells (DCs), neutrophils, and NK cells in the spleen, accompanied by the release of pro-inflammatory cytokines in the serum after administering act-EVs. In summary, this study sheds light on the role of TH cell released EVs in modulating the immune response during pro-inflammatory responses and this resource provides a foundation for development of novel therapeutics on EV based scaffolds.

Extracellular vesicles (EVs) are key mediators of intercellular communication, yet their molecular profiles across tissues and species remain poorly characterized, particularly due to currently available methods requiring a large amount of biological material (tissue or biofluids). Here, we established a workflow allowing the deep phenotyping of EV cargos starting from single samples of human and mouse origin. We took advantage of standardised EV isolation procedures and multi-omic techniques for the isolation and analysis of EVs from brain and plasma of human and mouse, integrating flow cytometric profiling, proteomics, miRNA sequencing, and fatty acid profiling. Here we report specific brain-derived EVs proteome, enriched in neuronal and glial proteins, polyunsaturated fatty acids profiles, and distinct miRNAs. At the periphery, we also report plasma-derived EVs signatures reflecting immune, metabolic, and systemic transport functions. Despite these expected material-specific differences, EVs from the same source displayed greater similarity across species than EVs from different material, supporting the translational relevance of mouse models. Importantly, using state-of-the-art miRNA profiling approach, we identified novel EV-specific miRNAs in human and mouse brain EVs, potentially allowing the exploration of new roles in neuronal signalling. Overall, we report here a method enabling deep multi-omic characterization from minimal starting material, offering a practical approach for studies with limited biological samples. These findings also demonstrate that the origin strongly shapes EV composition, highlighting conserved and species-specific molecular features, and provide a scalable framework for multi-omic investigations of EV biology. Summary StatementWe present a standardised workflow allowing multi-omic profiling of brain and plasma-derived EVs from minimal human and mouse material. Our findings reveal both tissue-specific and species specific EV molecular signatures.

Extracellular vesicles (EVs) are important mediators in intercellular communication. However, understanding the biological origin and functional effects of EVs subtypes has been challenging due to the moderate differences in their physical properties and absence of reliable markers. Here, we characterize the proteomes of ectosomes and exosomes using an improved differential ultracentrifugation protocol and quantitative proteomics. Cytoskeleton and glycolytic proteins are distinctively present in ectosomes, while endosomal sorting complexes proteins and tetraspanins are enriched in exosomes. Furthermore, annexin-A2 was identified as a specific marker for ectosomes derived from cell media and human cerebrospinal fluid. Expression of EGFP as a cytosolic reporter leads to its incorporation in EVs and enables their imaging with higher resolution. Importantly, ectosomes and exosomes internalization in neuronal cells results in the modulation of neuronal spontaneous activity. Our findings suggest that EVs cargoes reflect core intracellular processes, and their functional properties might regulate basic biological and pathological processes.

Extracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The study of EV secretion and EV characterization remains challenging due to the small size of these particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV separation techniques. We developed a method for in-cell EV labeling with fluorescent lipids (DiI), followed by DiI-labelled EV characterization in the conditioned medium by imaging flow cytometry (IFC). Direct IFC analysis of EVs in the conditioned medium, after removal of apoptotic bodies and cellular debris, significantly reduces sample processing and loss compared to established methods for EV separation, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by ultracentrifugation. In conclusion, our optimized protocol for EV labeling and analysis reduces EV sample processing and loss, and is well suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin.

Despite immense interest in biomarker applications of extracellular vesicles (EVs) from blood, our understanding of their physiological population in healthy humans remains limited. Using imaging and multiplex bead-based flow cytometry, we comprehensively quantified circulating EVs with respect to their cellular origin in a large cohort of healthy blood donors. We assessed coefficients of variations to characterise their biological variability and explored demographic, clinical, and lifestyle factors contributing to this variability. Cell-specific circulating EV subsets show a wide range of concentrations, which do not directly reflect concentrations of blood cells, indicating diverse patterns of EV subset release and/or uptake, even for EVs originating from the same cell type. Interestingly, tetraspanin+ circulating EVs largely originate from platelets and to a lesser extent from lymphocytes. PCA and association analyses demonstrate high biological inter-individual variability in circulating EVs across healthy humans, which can be only partly explained by the influence of sex, menopausal status, age and smoking on specific circulating EV and/or tetraspanin+ circulating EV subsets. No global influence of the explored subjects factors on circulating EVs was detected. Our findings provide the first comprehensive, quantitative data towards the cell-origin atlas of blood EVs, with important implications in the clinical use of EVs as biomarkers of disease.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by most cell types, which can transfer signals and cargoes between cells. EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous, in terms of size, intracellular origin, and cargo composition. Analyzing large numbers of EVs at the single vesicle level is therefore the only way to truly decipher their heterogeneity. Here, we developed a reliable pipeline of single EV analysis using a nanoparticle-dedicated flow cytometer, which detects particles and measures their size down to 55 nm in diameter, without the need for vesicle pre-immobilization or fluorescent label. We show that titrating each antibody, eliminating unbound antibodies and using EVs devoid of the analyzed markers as negative controls are required to reliably quantify the proportion of EVs bearing none or any combination of two markers, as well as to measure their sizes. We thus observed, depending on the cell source (human cell lines MDA-MB-231, HeLa, A549), variable proportions of EVs bearing none of the CD9, CD81 and CD63 tetraspanins often used to define EVs, and of single- and double-positive EVs for each of these markers. We also observed CD29 (ITGB1) as a protein detected as frequently on EVs as CD9, while other transmembrane proteins (CD44, SSEA-4, CD98), were detected in a small proportion of EVs, and mostly of relatively large size. Finally, we used this pipeline to uncover differential effects of small molecule drugs on subtypes of EVs, and showed that Homosalate increased the proportion of CD9+/CD63+ EVs while two other drugs, Dipivefrin hydrochloride and Metaraminol bitartrate, instead increased the proportion of CD9-/CD63+ EVs. Overall, nano-flow cytometry allows to reliably quantify proportions of EV subpopulations suggested by bulk analyzes of EV markers, at single EV resolution.

Extracellular vesicles (EVs) form through regulated biogenesis processes involving sphingomyelinases (SMases), enzymes that metabolize sphingomyelin to produce ceramide--a lipid influencing membrane rigidity and essential for EV generation. This study explores the impact on EV protein and RNA cargoes resulting from inhibiting neutral SMase (NSM) and acid SMase (ASM) in human MCF7 cells. Our results revealed that NSM inhibition reduces EV nanoparticles and diminishes RNA and protein cargoes, including endosomal, spliceosomal, and translation-related proteins. Conversely, ASM inhibition increased RNA-binding proteins within and enhanced the expression of ribonucleoprotein complex-associated RNA in released EVs, including several snRNAs and 7SL RNA. Intriguingly, ASM-inhibited EVs enhanced the migration and translational activity of recipient MCF10A cells. These findings suggest an important role for SMase-dependent vesiculation in governing RNA and protein trafficking to the extracellular space, unveiling potential implications for cellular communication and function.

Salivary extracellular vesicles (EVs) represent a powerful, non-invasive source of biomarkers for disease diagnosis and monitoring. Their molecular cargo reflects systemic and local physiological states, offering a window into neurological and inflammatory disorders. However, the diversity of EV isolation protocols and the possibility that each enriches distinct EV subpopulations remains a major barrier to reproducibility and data comparability. We conducted a comprehensive comparison of three EV isolation methods: ultracentrifugation (UC), PEG-based precipitation (Q), and immunoaffinity capture (M) to evaluate their impact on EV yield, purity, and molecular composition. Salivary EVs from healthy volunteers were analysed using proteomic and small-RNA sequencing approaches. Principal component analysis revealed clear isolation method-dependent clustering, where M-derived EVs displayed the most distinct profile. UC and Q produced broader proteomic repertoires with higher total protein content, whereas M-isolated EVs exhibited greater purity and enrichment of trafficking-and lysosome-associated proteins. Over 731 miRNAs selected, 28 were consistently altered across methods and 65 uniquely enriched in M isolates. RT-qPCR confirmed key directional trends. These 93 method-dependent miRNAs have predicted targets associated with synaptic structure and neurodegenerative pathways. These findings show that isolation methodology deeply shapes salivary EV cargo and suggest that immunoaffinity capture can isolate specific EV populations meeting diagnostic requirements.

Release and uptake of extracellular vesicles (EVs) is a highly conserved means of communication used by cells across all kingdoms of life. These nano-sized vesicles transfer messages encoded in proteins and nucleic acids and play a role in numerous (patho)physiological processes. A longstanding question in the field is whether interaction and uptake of EVs is a stochastic process or whether specific EV subpopulations target different types of recipient cells. Here we present EVtrace, a proteomics-based approach that accommodates stable isotopic labeling of amino acids in culture (SILAC) to trace back labeled EV-associated proteins in unlabeled recipient cell types. We describe the optimization of EV labeling conditions and EV-cell cultures, and introduce a proteomics data analysis pipeline to confidently identify sparse internalized EV proteins among unlabeled recipient cell proteins. As a proof of concept, we studied the targeting of prostate cancer (PCa) EVs to bone cells and non-bone cells, considering that bone is a common metastatic site of PCa. Using EVtrace we demonstrate that interaction of PCa EVs with recipient cell types was not stochastic, and that different subpopulations of EVs targeted different cells. Proteins of EV subpopulations that were traced in bone cells, but not in non-bone cells, were strongly enriched for pathways involved in cancer progression and metastasis. With super-resolution microscopy we confirmed that EV proteins traced in different targeted cell types co-occurred on specific EV subpopulations. EVtrace is a valuable new tool for EV research because it supports identification of EV subpopulations that are transferred to recipient cells and discloses candidate proteins potentially involved in EV binding/uptake and functional modification of target cells.

Most approaches to extracellular vesicle (EV) characterization focus on EV size or density. However, such approaches provide few clues regarding EV origin, molecular composition, and function. New methods to characterize the EV surface proteins may aid our understanding of their origin, physiological roles, and biomarker potential. Recently developed immunoassays for intact EVs based on ELISA, NanoView, SIMOA and MesoScale platforms are highly sensitive, but have limited multiplexing capabilities, whereas MACSPlex FACS enables the detection of multiple EV surface proteins, but requires significant quantities of purified EVs, which limits its adoption. Here, we describe a novel Luminex-based immunoassay, which combines multiplexing capabilities with high sensitivity and, importantly, bypasses the enrichment and purification steps that require larger sample volumes. We demonstrate the methods specificity for detecting EV surface proteins using multiple EV depletion techniques, EVs of specific cellular origin isolated from culture media, and by co-localization with established EV surface markers. Using this novel approach, we elucidate differences in the tetraspanin profiles of the EVs carrying erythrocyte and neuron markers. Using size exclusion chromatography, we show that plasma EVs of putative neuronal and tissue macrophage origin are eluted in fractions distinct from those derived from erythrocytes, or from their respective cultured cells. In conclusion, our novel multiplexed assay differentiates between EVs from erythrocytes, macrophages, and neurons, and offers a new means for capture, classification, and profiling of EVs from diverse sources.

Efficient, aggregation-free extracellular vesicles (EVs) labeling is essential for studying their dynamics in-vitro and in-vivo. However, traditional dyes introduce limitations including aggregation, membrane intercalation, fluorescence transfer and inconsistent performance across EV sources thus distorting quantification, altering surface properties and confounding uptake and biodistribution analyses. Here, we systematically evaluated CytoLight, a luminal dye traditionally used for live-cell imaging, as an alternative for EV quantification, characterization, uptake analysis and in-vivo tracking, benchmarking it against PKH26, CFSE and ExoBrite across multiple platforms. CytoLight generated stable, intravesicular fluorescence without aggregation or membrane alteration, eliminating artifacts characteristic of conventional dyes. Using fluorescence-NTA and single-EV flow cytometry, CytoLight showed more consistent labeling across EV types than CFSE or ExoBrite, while avoiding PKH-related micelle-driven artifacts and exhibited compatibility with CD81 dual-detection. In uptake assays, CytoLight produced EV-specific endocytosis-dependent internalization signals exceeding labeled-BPS/protein controls. In-vivo, CytoLight-labeled EVs enabled fluorescent biodistribution mapping showing conventional EV tropism patterns distinguishable from labeled-PBS controls. These findings establish CytoLight as an effective, aggregation-free EV-labeling strategy. Its stability, specificity, compatibility with single-EV platforms and reliable performance in both cellular uptake and biodistribution studies position CytoLight as a practical, scalable alternative to current dyes, providing a stronger foundation for standardized and reproducible EV research.

Extracellular vesicles (EVs) mediate intercellular transfer of lipids, proteins, and nucleic acids between nearly all cell types. We previously showed that astrocyte-derived EVs modulate neuronal mitochondria in vitro, but whether endogenous astrocytic EVs are trafficked to neuronal mitochondria in vivo remained unknown. To address this, we generated an EV reporter mouse, Aldh1l1-Cre; CD9-tGFPfl/fl, in which astrocyte-secreted EVs are labeled with a CD9-turboGFP fusion protein (CD9-tGFP). Astrocyte-specific expression of CD9-tGFP was verified in brain tissue and isolated EVs, comprising 13.2 {+/-} 1.6% of total brain EVs. In primary glial cultures, CD9-tGFP was restricted to astrocytes, localizing to vesicular compartments and cell protrusions (filopodia and cilia), with 89.3 {+/-} 2.2% of astrocyte-derived EVs carrying the label. These EVs were enriched with the sphingolipid ceramide, consistent with its co-distribution with CD9-tGFP in astrocytic cell protrusions. In the cortex, hippocampus, and cerebellum, CD9-tGFP was predominantly detected in astrocytic processes co-labeled with GLAST1 and GFAP, forming contacts with laminin-positive capillaries and parvalbumin-positive neurons. CD9-tGFP-labeled EVs were detected inside capillaries and neurons, and super-resolution STED microscopy revealed partial overlap with neuronal mitochondria. Live-cell spinning disk confocal imaging and AI-assisted proximity analysis confirmed uptake of CD9-tGFP EVs by neuronal cells and trafficking of their cargo to mitochondria in vitro. Biochemical isolation of synaptic and non-synaptic mitochondria confirmed EV-derived cargo on mitochondria in vivo, with 3-fold higher association of CD9-tGFP with synaptic than non-synaptic mitochondria. Together, these findings validate the Aldh1l1-Cre; CD9-tGFPfl/fl reporter mouse as a powerful tool for tracking astrocyte-derived EVs in vivo and provide direct evidence that their cargo is preferentially trafficked to synaptic mitochondria. Graphical AbstractAstrocyte-derived extracellular vesicles target neuronal mitochondria in vivo O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/718987v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@174d92aorg.highwire.dtl.DTLVardef@5d8248org.highwire.dtl.DTLVardef@114483borg.highwire.dtl.DTLVardef@924d55_HPS_FORMAT_FIGEXP M_FIG C_FIG

Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cells vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting towards EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/003145v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1bf7c79org.highwire.dtl.DTLVardef@85098dorg.highwire.dtl.DTLVardef@362e33org.highwire.dtl.DTLVardef@11349ab_HPS_FORMAT_FIGEXP M_FIG C_FIG Inflammation stimulates release of a heterogeneous population of beta EV with differential expression of immunogenic substances involved in immune cell recruitment and activation. HighlightsO_LIStress engenders an up to four-fold increase in the volume of the vesiculome and enhanced auto-antigen release C_LIO_LICytokines are selectively sorted into EV subspecies C_LIO_LITLR-binding microRNAs are enriched in sEV C_LIO_LIEV from stressed beta cells promote dendritic and macrophage cell activation C_LI

Extracellular vesicles (EVs) are increasingly recognized for their roles in orchestrating embryonic development. Emerging preclinical evidence further suggests that EVs from young organisms possess innate regenerative potential for adult or injured tissues. Here we show that small extracellular vesicles (sEVs) isolated from the mouse embryonic cortex exert neuroprotective effects in vitro and in vivo. Proteomic profiling revealed that embryonic sEVs are enriched with effectors of receptor tyrosine kinase activation, anti-inflammatory responses, and protein synthesis. Notably, we identified BDNF as a surface-bound cargo on embryonic sEVs, displaying superior stability and receptor activation kinetics than its non-vesicular form. Phospho-proteomic analysis further revealed that sEVmediated neuroprotection is driven primarily by the CaMKII signaling axis, which targets downstream effectors of microtubule stability, synaptic plasticity, and membrane-cytoskeleton interactions. Critically, embryonic sEVs, but not those from aged mice, restored microtubule stability and mitochondrial respiration in aged neurons in vitro. Our findings identify embryonic cortical sEVs as significant regulators of neuronal resilience and provide a molecular blueprint for EV-based strategies in neurodegeneration and aging research.

Tuberculous meningitis (TB meningitis), the deadliest form of Mycobacterium tuberculosis infection, leads to mortality and severe neurological disability despite standard therapy. Brain injury and microglial activation are major determinants of outcome, yet the mechanisms linking infection, inflammation and neuronal injury remain poorly understood. Extracellular vesicles (EVs), key mediators of cell-to-cell communication, have been investigated in pulmonary TB but their role in TB meningitis remains unexplored. We used our young rabbit model of TB meningitis to isolate pure, intact EVs from brain tissue (i.e., brain-derived EVs) from infected and uninfected rabbits and used nanoflow cytometry, transmission electron microscopy and protein quantification to characterize the EVs. Comparative proteomic profiling was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by in silico pathway, cell-type and protein-protein interaction analyses using DAVID, Enrichr, and STRING databases. We found that EV isolation from fresh and frozen tissue was equivalent and demonstrated that M. tuberculosis infection activated EV biogenesis. Despite preserved vesicle morphology, EVs from infected brain showed a significant proteomic shift characterized by enrichment of TB host defense, microglial and immune activation, metabolic excitotoxicity, and neuronal injury. These proteome dysregulations suggest that infection reprograms brain EV cargo toward proinflammatory and metabolic stress responses while depleting neuronal and mitochondrial components. Collectively, these data demonstrate that M. tuberculosis infection alters the cargo and abundance of brain-derived EV, highlighting their potential as biomarkers and mediators of host-pathogen interactions in TB meningitis.

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

Extracellular vesicles (EVs) can transport functional RNA between cells and therefore hold great potential for diagnostics and RNA-based therapeutics. Classically, RNA is believed to be encapsulated in the EV lumen. However, it has recently been demonstrated that cells present RNA on their surface. This RNA was found to be glycosylated, and although glycosylated tRNA was also found in EVs, its exact location remained elusive. Here, we demonstrate the presence of RNA on the surface of mesenchymal stem cell (MSC) derived EVs. By combining single-vesicle measurements with direct and selective visualization of RNA, we introduce surface RNA (surfRNA) as a new inherent component of EVs. RNA sequencing supports the surface localization of this RNA and further identifies tRNA fragments as primary constituent of surfRNA. Importantly, surfRNA is co-delivered to target cells together with EVs, suggesting a yet unrecognized uptake route of extracellular RNA. A deeper understanding of the surface-associated RNA may have significant implications for EV biogenesis, targeting, and downstream functional effects. We further envision that these findings are transferable to other nanoparticles and will thereby advance the field of therapeutic RNA delivery.