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.

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02049-1) contains supplementary material, which is available to authorized users.

The pandemic of COVID-19 is continuously spreading, becoming a worldwide emergency. Early and fast identification of subjects with a current or past infection must be achieved to slow down the epidemiological widening. Here we report a Raman-based approach for the analysis of saliva, able to significantly discriminate the signal of patients with a current infection by COVID-19 from healthy subjects and/or subjects with a past infection. Our results demonstrated the differences in saliva biochemical composition of the three experimental groups, with modifications grouped in specific attributable spectral regions. The Raman-based classification model was able to discriminate the signal collected from COVID-19 patients with accuracy, precision, sensitivity and specificity of more than 95%. In order to translate this discrimination from the signal-level to the patient-level, we developed a Deep Learning model obtaining accuracy in the range 89–92%. These findings have implications for the creation of a potential Raman-based diagnostic tool, using saliva as minimal invasive and highly informative biofluid, demonstrating the efficacy of the classification model.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (“liquid biopsies”). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non‐EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single‐centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence‐based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves.

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases.

Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application.