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Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS- CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention., Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen in the worldwide pandemic of coronavirus disease 2019 (COVID-19), is readily transmitted via respiratory droplets during close contact. The [...]

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

StatementThe International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

The article focuses on the International Society of Extracellular Vesicles (ISEV) and its use of social media to enhance communication within the scientific community. It highlights the growing influence of social media platforms and their potential for scientists to share research, connect with peers, and engage in discussions. The article specifically examines ISEV's presence on various platforms, including Facebook, Twitter/X, LinkedIn, and YouTube, and emphasizes the organization's global outreach. It concludes by emphasizing the importance of social media in connecting diverse individuals and facilitating communication in the scientific field. Additionally, the text mentions ISEV's efforts to expand its presence in different regions, particularly in Asia, Africa, and Latin America, to ensure comprehensive geographical representation. The organization plans to utilize social media platforms like Twitter, and potentially Instagram and TikTok, to disseminate scientific advancements and engage with a wider audience. It also encourages young researchers to participate and seeks input from its members to guide its communication strategies. [Extracted from the article]

Background: Extracellular vesicles (EVs) and non-coding RNAs (ncRNAs) are emerging contributors to Alzheimer's disease (AD) pathophysiology. Differential abundance of ncRNAs carried by EVs may provide valuable insights into underlying disease mechanisms. Brain tissue-derived EVs (bdEVs) are particularly relevant, as they may offer valuable insights about the tissue of origin. However, there is limited research on diverse ncRNA species in bdEVs in AD. Objective: This study explored whether the non-coding RNA composition of EVs isolated from post-mortem brain tissue is related to AD pathogenesis. Methods: bdEVs from age-matched late-stage AD patients (n = 23) and controls (n = 10) that had been separated and characterized in our previous study were used for RNA extraction, small RNA sequencing, and qPCR verification. Results: Significant differences of non-coding RNAs between AD and controls were found, especially for miRNAs and tRNAs. AD pathology-related miRNA and tRNA differences of bdEVs partially matched expression differences in source brain tissues. AD pathology had a more prominent association than biological sex with bdEV miRNA and tRNA components in late-stage AD brains. Conclusions: Our study provides further evidence that EV non-coding RNAs from human brain tissue, including but not limited to miRNAs, may be altered and contribute to AD pathogenesis. [ABSTRACT FROM AUTHOR]

Background Brain tissue-derived extracellular vesicles (bdEVs) act locally in the central nervous system (CNS) and may indicate molecular mechanisms in human immunodeficiency virus (HIV) CNS pathology. Using brain homogenate (BH) and bdEVs from a simian immunodeficiency virus (SIV) model of HIV disease, we identified RNA networks in SIV infection and neuroinflammation. Methods Postmortem occipital cortex samples were obtained from uninfected controls and SIV-infected subjects (acute and chronic phases with or without CNS pathology [SIV encephalitis]). bdEVs were separated and characterized per international consensus guidelines. RNAs from bdEVs and BH were sequenced and quantitative polymerase chain reaction (qPCR)-amplified to detect levels of small RNAs (sRNAs, including microRNAs [miRNAs]) and longer RNAs including messenger RNAs (mRNAs) and circular RNAs (circRNAs). Results Dysregulated RNAs in BH and bdEVs were identified in acute and chronic infection with pathology groups, including mRNAs, miRNAs, and circRNAs. Most dysregulated mRNAs in bdEVs reflected dysregulation in source BH. These mRNAs are disproportionately involved in inflammation and immune responses. Based on target prediction, several circRNAs that were differentially abundant in source tissue might be responsible for specific differences in sRNA levels in bdEVs during SIV infection. Conclusions RNA profiling of bdEVs and source tissues reveals potential regulatory networks in SIV infection and SIV-related CNS pathology. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell‐derived EVs into pig‐tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV‐mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV‐based therapies in preclinical studies. [ABSTRACT FROM AUTHOR]

The article "MISEV2023: An updated guide to EV research and applications" is a comprehensive guide to the study of extracellular vesicles (EVs). It covers various aspects of EV research, including terminology, collection and processing, characterization, and studying EVs in vivo. The guide was developed through a survey and input from the International Society for Extracellular Vesicles (ISEV) board, with contributions from 74 scientists and 1051 co-authors from 53 countries. While not a comprehensive review, the guide includes over 500 citations suggested by experts and the community. The authors acknowledge the limitations of the guide and invite critique and collaboration to improve future versions. [Extracted from the article]

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture‐derived EVs are administered intravenously to Macaca nemestrina (pig‐tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 h post‐intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig‐tailed macaques. Using this ex vivo system, we found that labelled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV‐blood cell interactions. [ABSTRACT FROM AUTHOR]