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 vesicles (EVs), including the nano‐sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS‐EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS‐derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18‐hour centrifugation period eliminates approximately 95% of RNA‐containing FBS EVs, whereas a 1.5‐hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18‐hour ultracentrifugation protocol.

After the initial investigations into applications of mesenchymal stem cells (MSCs) for cell therapy, there was increased interest in their secreted soluble factors. Following studies of MSCs and their secreted factors, extracellular vesicles (EVs) released from MSCs have emerged as a new mode of intercellular crosstalk. MSC-derived EVs have been identified as essential signaling mediators under both physiological and pathological conditions, and they appear to be responsible for many of the therapeutic effects of MSCs. In several in vitro and in vivo models, EVs have been observed to have supportive functions in modulating the immune system, mainly mediated by EV-associated proteins and nucleic acids. Moreover, stimulation of MSCs with biophysical or biochemical cues, including EVs from other cells, has been shown to influence the contents and biological activities of subsequent MSC-derived EVs. This review provides on overview of the contents of MSC-derived EVs in terms of their supportive effects, and it provides different perspectives on the manipulation of MSCs to improve the secretion of EVs and subsequent EV-mediated activities. In this review, we discuss the possibilities for manipulating MSCs for EV-based cell therapy and for using EVs to affect the expression of elements of interest in MSCs. In this way, we provide a clear perspective on the state of the art of EVs in cell therapy focusing on MSCs, and we raise pertinent questions and suggestions for knowledge gaps to be filled.

Extracellular vesicles have the capacity to transfer lipids, proteins, and nucleic acids between cells, thereby influencing the recipient cell's phenotype. While the role of RNAs in EVs has been extensively studied, the function of DNA remains elusive. Here, we distinguished novel heterogeneous subpopulations of small extracellular vesicles (sEVs) based on their DNA content and topology. Low‐ and high‐density sEV subsets from a human mast cell line (HMC‐1) and an erythroleukemic cell line (TF‐1) were separated using high‐resolution iodixanol density gradients to discriminate the nature of the DNA cargo of the sEVs. Paired comparisons of the sEV‐associated DNA and RNA molecules showed that RNA was more abundant than DNA and that most of the DNA was present in the high‐density fractions, demonstrating that sEV subpopulations have different DNA content. DNA was predominately localised on the outside or surface of sEVs, with only a small portion being protected from enzymatic degradation. Whole‐genome sequencing identified DNA fragments spanning all chromosomes and mitochondrial DNA when sEVs were analysed in bulk. Our work contributes to the understanding of how DNA is associated with sEVs and thus provides direction for distinguishing subtypes of EVs based on their DNA cargo and topology.

Extracellular vesicles such as exosomes convey biological messages between cells, either by surface‐to‐surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor β‐1 (TGFβ‐1) on their surfaces. The latent form of TGFβ‐1 is associated with the exosomes via heparinase‐II and pH‐sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes‐associated TGFβ‐1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free‐TGFβ‐1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD‐dependent pathways. Our results show that mast cell‐derived exosomes are decorated with latent TGFβ‐1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.