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.

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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.

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Limbal epithelial stem cells (LESC) maintenance requires communication between stem cells and neighboring stromal keratocytes. Extracellular vesicles (EVs) are important for intercellular communication in various stem cell niches. We explored the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos), an EV sub-population, in limbal epithelial cells (LEC) in normal and diabetic limbal niche and determined differences in Exo cargos from normal and diabetic LSC. Wound healing and proliferation rates in primary normal LEC were significantly enhanced upon treatment by normal Exos (N-Exos), but not by diabetic Exos (DM-Exos). Western analysis showed increased Akt phosphorylation in wounded LECs and organ-cultured corneas treated with N-Exos, compared to untreated wounded cells and DM-Exos treated fellow corneas, respectively. N-Exos treated organ-cultured corneas showed upregulation of putative LESC markers, keratin 15 (K15) and Frizzled-7, compared to the DM-Exos treated fellow corneas. By next generation sequencing, we identified differentially expressed small RNAs including microRNAs in DM-Exos vs. N-Exos. Overall, N-Exos have greater effect on LEC proliferation and wound healing than DM-Exos, likely by activating Akt signaling. The small RNA differences in Exos from diabetic vs. normal LSC could contribute to the disease state. Our study suggests that exosomes may serve as novel therapeutic tools for diabetic cornea.