New Guidelines to Uncover the Physiology of Extracellular Vesicles.

Extracellular vesicles (EVs)—nanosized membrane-enclosed particles containing cellular RNA, lipids, and proteins—are secreted from cells into body fluids such as urine and plasma. The EVs provide exciting opportunities to understand human and animal physiology in that they can be used as liquid biopsies to gain mechanistic insight into complex conditions, such as hypertension.1 Moreover, EVs may serve as vehicles for intercellular communication, potentially adding new layers to the understanding of physiology. However, challenges to purify, characterize, and determine, for example, the intercellular transfer of EVs, have remained a hurdle to realizing the potential of EVs. To address these challenges, the International Society of Extracellular Vesicles (ISEV) has published the "Minimal Information for Studies of Extracellular Vesicles" (MISEV) as a guideline for best practices in EV research. An updated version—MISEV20232—has been published and contains essential information on the advantages and limitations of current methods to study EV biology. This article will highlight new additions to the MISEV2023 critical for determining the physiological role of EVs. The current MISEV2023 guidelines stand on the shoulders of the two previous versions: MISEV2014 and MISEV2018. The MISEV2023 document was updated by expert task forces within the specific sections and through online feedback from over 1000 ISEV members. This community effort has resulted in a rich source of detailed and comprehensive information on EVs and important sample-specific recommendations and caveats for collecting, preprocessing, and characterizing EVs isolated from biofluids and tissue samples. MISEV2023 also contains updated nomenclature and characterization sections to integrate the greater complexity and diversity of EVs and nonvesicular extracellular particles, such as exomeres, uncovered by recent EV research. Importantly, MISEV2023 values transparency highly and should be considered a guide to rigorous and reproducible EV science. A significant challenge for studying EV biology in living organisms is the low EV concentration in body fluids. For example, the EV concentration in human blood—one of the most studied body fluids—is seven orders of magnitude lower than albumin and approximately one-tenth of the fasting insulin level (Figure 1). In contrast to hormones and plasma proteins, often secreted from a small number of cells,3 EVs in body fluids are derived from various cell types, and ~90% of the circulating plasma EVs are derived from blood cells.4 The low abundance challenges not only cell type-specific EV cargo analyses but also the determination of how they are taken up by other cells and their biodistribution, for example, whether plasma EVs are filtered into the urine. The EV biodistribution has been primarily assessed by bolus injection of EVs isolated from a single in vitro cultured cell type.5 Although the bulk plasma EV concentration after injection may be within the normal physiological EV range,5 the cell type-specific EV abundance may greatly exceed the physiological EV-to-cell ratio and affect biodistribution (Figure 1). Moreover, the injected EVs are rapidly cleared (half-life ~7 min) from the circulation by macrophage-dependent mechanisms6; however, it is still unknown whether all endogenously produced EVs have similar short half-life or if some cell types produce more long-lived EVs. To address these unknowns, the MISEV2023 guidelines now encourage using multiple doses and time points to study EV biology in vivo. For example, the acutely increased plasma EV concentration after exercise in humans7 could be extended with various time points to show how well the clearance of injected EVs recapitulates endogenously produced EVs at physiological concentrations. In addition to biodistribution and clearance of EVs, cell type-specific EV secretion rates span several orders of magnitude,4 and in vitro studies of cultured cells have demonstrated that EV secretion rates are not constant. For example, conditions that increase mitochondria-derived reactive oxygen species production, such as hypoxia, are associated with higher EV secretion rates.8 This adds extra challenges to the physiological interpretation of EV data; the RNA and protein levels measured in isolated EVs are the product of two dynamically regulated parameters: the cargo level within each EV and the EV abundance. The dynamic cell type-specific EV secretion rate, thus, confounds EV data analyses. To mitigate these challenges, animal models have been developed, and the MISEV2023 includes a new section on experimental invertebrate and vertebrate models. This section covers model organisms, such as zebrafish and rodent transgene models, that use reporter proteins to label endogenously expressed EVs. The reporter proteins create a robust link between cell type and its EVs, enabling cell-specific EV cargo analyses, determination of biodistribution, and demonstration of molecular cargo transfer between organs by endogenously produced EVs.9 The animal models will be essential to decipher the EVs' contribution to health and diseases. Yet, to establish how EVs are causally involved in physiological regulation, good experimental tools to stimulate and inhibit EV secretion are needed. Pharmacological approaches for stimulation and inhibiting EV secretion have been identified; however, different cell types often share signaling pathways for EV secretion, and pharmacological interventions, therefore, lack cell type specificity. Genetic manipulation of EV secretion, on the other hand, could provide cell type-specific interventions. However, the knowledge about genes controlling EV secretion is still limited, and the identified genes often affect other critical cellular processes. Thus, new experimental tools are needed to cell type-specifically interfere with EV secretion and determine whether EVs are involved in, for example, feedback loops for homeostatic control of vital parameters. The research on EV biology is still filled with unanswered questions, but the newly updated and revised MISEV2023 provides a strong foothold for exploring how EVs contribute to physiology. Crucially, physiology is a quantitative science, and the MISEV2023's updated information on, for example, in vivo approaches to explore EV biology offers an excellent opportunity to gain new physiological knowledge. The best practice recommendations in the MISEV2023, thus, refine the experimental approaches to facilitate a robust interpretation of EV data and gain an accurate understanding of EVs in health and diseases. Didde R. Hansen: Writing – review and editing; writing – original draft. Per Svenningsen: Conceptualization; writing – original draft; writing – review and editing; visualization. None. The data that support the findings of this study are available from the corresponding author upon reasonable request.