Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Exploration of the Noncoding Genome for Human-Specific Therapeutic Targets-Recent Insights at Molecular and Cellular Level.

While it is well known that 98-99% of the human genome does not encode proteins, but are nevertheless transcriptionally active and give rise to a broad spectrum of noncoding RNAs [ncRNAs] with complex regulatory and structural functions, specific functions have so far been assigned to only a tiny fraction of all known transcripts. On the other hand, the striking observation of an overwhelmingly growing fraction of ncRNAs, in contrast to an only modest increase in the number of protein-coding genes, during evolution from simple organisms to humans, strongly suggests critical but so far essentially unexplored roles of the noncoding genome for human health and disease pathogenesis. Research into the vast realm of the noncoding genome during the past decades thus lead to a profoundly enhanced appreciation of the multi-level complexity of the human genome. Here, we address a few of the many huge remaining knowledge gaps and consider some newly emerging questions and concepts of research. We attempt to provide an up-to-date assessment of recent insights obtained by molecular and cell biological methods, and by the application of systems biology approaches. Specifically, we discuss current data regarding two topics of high current interest: (1) By which mechanisms could evolutionary recent ncRNAs with critical regulatory functions in a broad spectrum of cell types (neural, immune, cardiovascular) constitute novel therapeutic targets in human diseases? (2) Since noncoding genome evolution is causally linked to brain evolution, and given the profound interactions between brain and immune system, could human-specific brain-expressed ncRNAs play a direct or indirect (immune-mediated) role in human diseases? Synergistic with remarkable recent progress regarding delivery, efficacy, and safety of nucleic acid-based therapies, the ongoing large-scale exploration of the noncoding genome for human-specific therapeutic targets is encouraging to proceed with the development and clinical evaluation of novel therapeutic pathways suggested by these research fields.

Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart.

BACKGROUND: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. METHODS: We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. RESULTS: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell-derived cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. CONCLUSIONS: Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.

Hydrogel-Embedded Poly(Lactic-co-Glycolic Acid) Microspheres for the Delivery of hMSC-Derived Exosomes to Promote Bioactive Annulus Fibrosus Repair.

OBJECTIVE: Intervertebral disk degeneration is a prevalent postoperative complication after discectomy, underscoring the need to develop preventative and bioactive treatment strategies that decelerate degeneration and seal annulus fibrosus (AF) defects. Human mesenchymal stem cell-derived exosomes (MSC-Exos) hold promise for cell-free bioactive repair; however, their ability to promote AF repair is poorly understood. The objective of this study was to evaluate the ability of MSC-Exos to promote endogenous AF repair processes and integrate MSC-Exos within a biomaterial delivery system. DESIGN: We characterize biophysical and biochemical properties of normoxic (Nx) and hypoxic (Hx) preconditioned MSC-Exos from young, healthy donors and examine their effects on AF cell proliferation, migration, and gene expression. We then integrate a poly(lactic-co-glycolic acid) microsphere (PLGA µSphere) delivery platform within an interpenetrating network hydrogel to facilitate sustained MSC-Exo delivery. RESULTS: Hx MSC-Exos led to a more robust response in AF cell proliferation and migration than Nx MSC-Exos and was selected for a downstream protection experiment. Hx MSC-Exos maintained a healthy AF cell phenotype under a TNFα challenge in vitro and attenuated catabolic responses. In all functional assays, AF cell responses were more sensitive to Hx MSC-Exos than Nx MSC-Exos. PLGA µSpheres released MSC-Exos over a clinically relevant timescale without affecting hydrogel modulus or pH upon initial embedment and µSphere degradation. CONCLUSIONS: This MSC-Exo treatment strategy may offer benefits of stem cell therapy without the need for exogenous stem cell transplantation by stimulating cell proliferation, promoting cell migration, and protecting cells from the degenerative proinflammatory microenvironment.

Extracellular vesicles carry distinct proteo-transcriptomic signatures that are different from their cancer cell of origin.

Circulating extracellular vesicles (EVs) contain molecular footprints-lipids, proteins, RNA, and DNA-from their cell of origin. Consequently, EV-associated RNA and proteins have gained widespread interest as liquid-biopsy biomarkers. Yet, an integrative proteo-transcriptomic landscape of EVs and comparison with their cell of origin remains obscure. Here, we report that EVs enrich distinct proteo-transcriptome that does not linearly correlate with their cell of origin. We show that EVs enrich endosomal and extracellular proteins, small RNA (∼13-200 nucleotides) associated with cell differentiation, development, and Wnt signaling. EVs cargo specific RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, and SRSF1/2). To ensure an unbiased and independent analyses, we studied 12 cancer cell lines, matching EVs (inhouse and exRNA database), and serum EVs of patients with prostate cancer. Together, we show that EV-RNA-protein complexes may constitute a functional interaction network to protect and regulate molecular access until a function is achieved.

Extracellular Vesicles and Their Emerging Roles as Cellular Messengers in Endocrinology: An Endocrine Society Scientific Statement.

During the last decade, there has been great interest in elucidating the biological role of extracellular vesicles (EVs), particularly, their hormone-like role in cell-to-cell communication. The field of endocrinology is uniquely placed to provide insight into the functions of EVs, which are secreted from all cells into biological fluids and carry endocrine signals to engage in paracellular and distal interactions. EVs are a heterogeneous population of membrane-bound vesicles of varying size, content, and bioactivity. EVs are specifically packaged with signaling molecules, including lipids, proteins, and nucleic acids, and are released via exocytosis into biofluid compartments. EVs regulate the activity of both proximal and distal target cells, including translational activity, metabolism, growth, and development. As such, EVs signaling represents an integral pathway mediating intercellular communication. Moreover, as the content of EVs is cell-type specific, it is a "fingerprint" of the releasing cell and its metabolic status. Recently, changes in the profile of EV and bioactivity have been described in several endocrine-related conditions including diabetes, obesity, cardiovascular diseases, and cancer. The goal of this statement is to highlight relevant aspects of EV research and their potential role in the field of endocrinology.

Updating MISEV: Evolving the minimal requirements for studies of extracellular vesicles.

The minimal information for studies of extracellular vesicles (EVs, MISEV) is a field-consensus rigour initiative of the International Society for Extracellular Vesicles (ISEV). The last update to MISEV, MISEV2018, was informed by input from more than 400 scientists and made recommendations in the six broad topics of EV nomenclature, sample collection and pre-processing, EV separation and concentration, characterization, functional studies, and reporting requirements/exceptions. To gather opinions on MISEV and ideas for new updates, the ISEV Board of Directors canvassed previous MISEV authors and society members. Here, we share conclusions that are relevant to the ongoing evolution of the MISEV initiative and other ISEV rigour and standardization efforts.

EV Cargo Sorting in Therapeutic Development for Cardiovascular Disease.

Cardiovascular disease remains the leading cause of morbidity and mortality in the world. Thus, therapeutic interventions to circumvent this growing burden are of utmost importance. Extracellular vesicles (EVs) actively secreted by most living cells, play a key role in paracrine and endocrine intercellular communication via exchange of biological molecules. As the content of secreted EVs reflect the physiology and pathology of the cell of their origin, EVs play a significant role in cellular homeostasis, disease pathogenesis and diagnostics. Moreover, EVs are gaining popularity in clinics as therapeutic and drug delivery vehicles, transferring bioactive molecules such as proteins, genes, miRNAs and other therapeutic agents to target cells to treat diseases and deter disease progression. Despite our limited but growing knowledge of EV biology, it is imperative to understand the complex mechanisms of EV cargo sorting in pursuit of designing next generation EV-based therapeutic delivery systems. In this review, we highlight the mechanisms of EV cargo sorting and methods of EV bioengineering and discuss engineered EVs as a potential therapeutic delivery system to treat cardiovascular disease.

Regulation of the Methylation and Expression Levels of the BMPR2 Gene by SIN3a as a Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension.

BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.

Therapeutic Delivery of Pip4k2c-Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart.

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF-ß, respectively. Type-2-phosphatidylinositol-5-phosphate-4-kinase-gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non-injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c-/- mice has higher rates of CH, CF, and sudden death than wild-type mice. In a gain-of-function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGFß1 via its N-terminal motif, Pip5k1α, phospho-AKT 1/2/3, and phospho-Smad3. In sum, loss-and-gain-of-function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.

Therapeutic and Diagnostic Translation of Extracellular Vesicles in Cardiovascular Diseases: Roadmap to the Clinic.

Exosomes are small membrane-bound vesicles of endocytic origin that are actively secreted. The potential of exosomes as effective communicators of biological signaling in myocardial function has previously been investigated, and a recent explosion in exosome research not only underscores their significance in cardiac physiology and pathology, but also draws attention to methodological limitations of studying these extracellular vesicles. In this review, we discuss recent advances and challenges in exosome research with an emphasis on scientific innovations in isolation, identification, and characterization methodologies, and we provide a comprehensive summary of web-based resources available in the field. Importantly, we focus on the biology and function of exosomes, highlighting their fundamental role in cardiovascular pathophysiology to further support potential applications of exosomes as biomarkers and therapeutics for cardiovascular diseases.

Bioinspired artificial exosomes based on lipid nanoparticles carrying let-7b-5p promote angiogenesis in vitro and in vivo.

MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34+ cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.

Targeted delivery of therapeutic agents to the heart.

For therapeutic materials to be successfully delivered to the heart, several barriers need to be overcome, including the anatomical challenges of access, the mechanical force of the blood flow, the endothelial barrier, the cellular barrier and the immune response. Various vectors and delivery methods have been proposed to improve the cardiac-specific uptake of materials to modify gene expression. Viral and non-viral vectors are widely used to deliver genetic materials, but each has its respective advantages and shortcomings. Adeno-associated viruses have emerged as one of the best tools for heart-targeted gene delivery. In addition, extracellular vesicles, including exosomes, which are secreted by most cell types, have gained popularity for drug delivery to several organs, including the heart. Accumulating evidence suggests that extracellular vesicles can carry and transfer functional proteins and genetic materials into target cells and might be an attractive option for heart-targeted delivery. Extracellular vesicles or artificial carriers of non-viral and viral vectors can be bioengineered with immune-evasive and cardiotropic properties. In this Review, we discuss the latest strategies for targeting and delivering therapeutic materials to the heart and how the knowledge of different vectors and delivery methods could successfully translate cardiac gene therapy into the clinical setting.

Hydroxylation of N-acetylneuraminic Acid Influences the in vivo Tropism of N-linked Sialic Acid-Binding Adeno-Associated Viruses AAV1, AAV5, and AAV6.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy. However, a number of recent preclinical large animal studies failed to translate into the clinic. This illustrates the formidable challenge of choosing the animal models that promise the best chance of a successful translation into the clinic. Several of the most common AAV serotypes use sialic acid (SIA) as their primary receptor. However, in contrast to most mammals, humans lack the enzyme CMAH, which hydroxylates cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) into cytidine monophosphate-N-glycolylneuraminic acid (CMP-Neu5Gc). As a result, human glycans only contain Neu5Ac and not Neu5Gc. Here, we investigate the tropism of AAV1, 5, 6 and 9 in wild-type C57BL/6J (WT) and CMAH knock-out (CMAH-/-) mice. All N-linked SIA-binding serotypes (AAV1, 5 and 6) showed significantly lower transduction of the heart in CMAH-/- when compared to WT mice (5-5.8-fold) and, strikingly, skeletal muscle transduction by AAV5 was almost 30-fold higher in CMAH-/- compared to WT mice. Importantly, the AAV tropism or distribution of expression among different organs was also affected. For AAV1, AAV5 and AAV6, expression in the heart compared to the liver was 4.6-8-fold higher in WT than in CMAH-/- mice, and for AAV5 the expression in the heart compared to the skeletal muscle was 57.3-fold higher in WT than in CMAH-/- mice. These data thus strongly suggest that the relative abundance of Neu5Ac and Neu5Gc plays a role in AAV tropism, and that results obtained in commonly used animal models might not translate into the clinic.

Native and bioengineered extracellular vesicles for cardiovascular therapeutics.

Extracellular vesicles (EVs) are a heterogeneous group of natural particles that are relevant to the treatment of cardiovascular diseases. These endogenous vesicles have certain properties that allow them to survive in the extracellular space, bypass biological barriers and deliver their biologically active molecular cargo to recipient cells. Moreover, EVs can be bioengineered to increase their stability, bioactivity, presentation to acceptor cells and capacity for on-target binding at both cell-type-specific and tissue-specific levels. Bioengineering of EVs involves the modification of the donor cell before EV isolation or direct modification of the EV properties after isolation. The therapeutic potential of native EVs and bioengineered EVs has been only minimally explored in the context of cardiovascular diseases. Efforts to harness the therapeutic potential of EVs will require innovative approaches and a comprehensive integration of knowledge gathered from decades of research into molecular-compound delivery. In this Review, we outline the endogenous properties of EVs that make them natural delivery agents as well as the features that can be improved by bioengineering. We also discuss the therapeutic applications of native and bioengineered EVs to cardiovascular diseases and examine the opportunities and challenges that need to be addressed to advance this research area, with an emphasis on clinical translation.

Analysis of extracellular vesicle miRNA profiles in heart failure.

Extracellular vesicles (EVs) have recently emerged as an important carrier for various genetic materials including microRNAs (miRs). Growing evidences suggested that several miRs transported by EVs were particularly involved in modulating cardiac function. However, it has remained unclear what miRs are enriched in EVs and play an important role in the pathological condition. Therefore, we established the miR expression profiles in EVs from murine normal and failing hearts and consecutively identified substantially altered miRs. In addition, we have performed bioinformatics approach to predict potential cardiac outcomes through the identification of miR targets. Conclusively, we observed approximately 63% of predicted targets were validated with previous reports. Notably, the predicted targets by this approach were often involved in both beneficial and malicious signalling pathways, which may reflect heterogeneous cellular origins of EVs in tissues. Lastly, there has been an active debate on U6 whether it is a proper control. Through further analysis of EV miR profiles, miR-676 was identified as a superior reference control due to its consistent and abundant expressions. In summary, our results contribute to identifying specific EV miRs for the potential therapeutic targets in heart failure and suggest that miR-676 as a new reference control for the EV miR studies.

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop.

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.