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.

Prion diseases are infectious neurodegenerative disorders linked to the accumulation in the central nervous system of the abnormally folded prion protein (PrP) scrapie (PrPsc), which is thought to be the infectious agent. Once present, PrPsc catalyzes the conversion of naturally occurring cellular PrP (PrPc) to PrPsc. Prion infection is usually initiated in peripheral organs, but the mechanisms involved in infectious spread to the brain are unclear. We found that both PrPc and PrPsc were actively released into the extracellular environment by PrP-expressing cells before and after infection with sheep prions, respectively. Based on Western blot with specific markers, MS, and morphological analysis, our data revealed that PrPc and PrPsc in the medium are associated with exosomes, membranous vesicles that are secreted upon fusion of multivesicular endosomes with the plasma membrane. Furthermore, we found that exosomes bearing PrPsc are infectious. Our data suggest that exosomes may contribute to intercellular membrane exchange and the spread of prions throughout the organism. Infectious prion diseases include Kuru and variant CreutzfeldtJakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (1, 2). In these diseases, infectious prions enter the host through the gastrointestinal tract and migrate to the spleen, after which they cause pathology in the central nervous system (3). Different cell types, including immune cells, contribute to the replication and transfer of infectious prions from peripheral sites of replication to the brain (4). The mechanisms underlying this intercellular transfer are not elucidated (2), but close cell contact may be involved (5). Nevertheless, cell-free conversion data (6) indicate that additional pathways involving non-cell-associated forms of infectious agent may participate in the propagation of prions. Consistent with this notion, the culture medium of scrapie-infected GT1 cells was infectious (7), suggesting that PrPsc may be released from cells and induce transconformation of PrPc in neighboring cells. Noninfected PrP-expressing cells may also have the ability to release PrPc, given that PrPc has been shown to be transferred between cells (8). Thus, release of PrPc and PrPsc by PrP-expressing cells may provide for a potential cellular mechanism underlying propagation and replication of prions. In this study, we further explored the possibility that PrPsc and PrPc may occur in a non-cell-associated form and analyzed their nature in the culture medium of infected and noninfected cell cultures. Our studies indicate that PrPsc and PrPc are associated with exosomes, secreted intralumenal contents of multivesicular bodies (MVB). These findings open the possibility that exosomes may provide for intercellular carriers of both PrPc and PrPsc. Materials and MethodsCells, Reagents, and Antibodies. Rov cells are derived from the RK13 cell line and express the ovine VRQ allele of PrP in a doxycyclinedependent manner (9). Mov cells are immortalized neuroglial ...