A small and complex new world

Originally described as "platelet dust" (Wolf, 1967), followed by the discovery of transferrin shedding by Peter Stahl and Rose Johnstone in the early 1980s (Harding et al., 1984; Ban et al., 1983), extracellular vesicles (EVs) have become a fascinating subject of research, especially over the last decade. These small lipid nanoparticles with a size of 40-5,000 nm in diameter are secreted by all living cells and transport bioactive molecules to recipient cells. They have been ascribed various functions in health and disease: next to the rather trivial possibility of a disposal pathway, these comprise the regulation of gene expression, immune responses, cell survival, proliferation and migration. Nowadays, it is clear that cells secrete not only large particles, such as the plasma membrane-derived large oncosomes or ectosomes (Di Vizio et al. 2009, Muradlidharan-Chari et al. 2009), but also endosomal-derived small particles, the so-called exosomes (Raposo et al. 1996), or even smaller extracellular particles, such as exomeres or suprameres (Zhang et al. 2018; Zhang et al. 2021). EV heterogeneity is further expanded by the finding that specific cellular states can induce the release of specific EV subpopulations. Examples include apoptosis, which results in the massive shedding of apoptotic bodies from the dying cell (Kerr et al. 1972), or cell migration, which is associated with the release of migrasomes from the retraction fibers of moving cells (Ma et al. 2015). Furthermore, cells have been shown to release distinct populations of EVs from the apical and basolateral sides, resulting from different biogenesis pathways (Matsui et al. 2021). Several studies have demonstrated that the EV subpopulations differ in their functionality (Menck et al. 2013; Menck et al. 2015; Tkach et al. 2017; Collino et al. 2017; Zhang et al. 2018). Thus, the discussion about EV heterogeneity is not only a question of nomenclature but also important for further advancing our understanding of EV biology and their use as therapeutics in clinics.

Methodological limitations when studying EV heterogeneity

The presence of specific subpopulations in EV preparations is not only critically influenced by the state of the secreting cell, but also by the method chosen for EV isolation. To study the biophysical properties of these small particles, special methods were and are required to isolate and characterize EVs from various sources (e.g. different biofluids, cell culture supernatants). Due to their overlapping biophysical characteristics in terms of size or density, the different EV subpopulations often co-fractionate in isolation methods such as size exclusion chromatography or density gradient ultracentrifugation which fail to reliably separate large and small EVs (Menck et al, 2015, Kowal et al. 2016, Saludas et al. 2022). Differential ultracentrifugation, which is still the most commonly used method for isolating EVs, at least allows to collect EV pellets at different centrifugal forces which differ in size, morphology and cargo (Crescitelli et al. 2013; Kowal et al. 2016, Cvjetkovic et al. 2017). However, small EVs from distinct cell lines have been shown to differ in their sedimentation characteristics and centrifugal parameters (e.g. time, rotor type, speed) have a significant impact on EV yield (Jeppesen et al. 2014; Cvjetkovic et al. 2014). Moreover, in particular high-speed ultracentrifugation (>100,000xg) pellets not only exosomes, but co-isolates small ectosomes, extracellular particles, protein aggregates and/or lipoprotein particles. It is thus clear that also ultracentrifugation-based EV preparations are far from being pure and fail to reliably separate the distinct EV subpopulations. Combining orthogonal methods might improve the purity of EV preparations but at the cost of very low yields that limit downstream analyses.

One size fits all?

As a result of mostly heterogeneous EV samples, it is not surprising that the classical tetraspanins formerly suggested as ‘general’ EV markers (e.g. CD9, CD81, CD63), in fact, seem to represent distinct subpopulations: While CD9- and/or CD81-positive sEVs arise from the cellular plasma membrane, endosomal-derived sEVs are rather enriched for CD63 and negative for the other two tetraspanins (Kowal et al. 2016; Mathieu et al, 2021, Fan et al. 2023). Some cell types express the three tetraspanins also on apoptotic bodies and large EVs, while others do not express CD9 or CD81 at all on the EV surface (Bobrie et al. 2012; Crescitelli et al. 2013; Koliha et al. 2016). Likewise, the endosomal sorting complex required for transport (ESCRT) system mediates cargo sorting at endosomal membranes as well as the cell surface. Consequently, ESCRT (-associated) proteins such as Tsg101, or Alix, have been detected on several EV subpopulations (Nabhan et al. 2012; Saludas et al. 2022) and thus are no reliable markers to describe the subcellular origin of an EV population. Another challenge is that the distribution of markers into the distinct centrifugation-based EV pellets seems to be highly heterogeneous among different cell lines (Kowal et al. 2016; Schöne et al. 2024). Due to the divergence in EV isolation and characterization protocols, wide variations in results between different laboratories have been observed and multicenter studies are largely lacking. Consequently, experts from neighboring fields have raised concerns about the reliability of EV research.

MISEV - defining commonly accepted standards

In the early 2010s, an international interest group was formed to discuss the aspects of EV biology, and their purification and subsequent analysis, resulting in the foundation of the International Society for Extracellular Vesicles (ISEV) followed by a growing number of national societies, including the German Society for Extracellular Vesicles (GSEV). A major goal of the ISEV has been to develop standards in order to improve the rigor, reproducibility, and transparency of EV research by providing recommendations and tools to help researchers, reviewers, and editors evaluate EV-related work. A notable milestone of these endeavors was the publication of the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines in 2014 (Lötvall et al. 2014). They provide a framework to support innovative EV research across various disciplines across multiple applications, ensuring standardized practices and broad consensus in the field, relevant to both basic and clinical research. Meanwhile, these guidelines were updated in 2018 (MISEV2018) (Théry et al. 2018) and 2023 (MISEV2023) (Welsh et al. 2024). More than 1051 authors from 52 countries, including 50 authors from Germany, contributed to the latest edition. Thus, the MISEV guidelines provide a solid foundation to gain an overview of the opportunities and approaches of EV research and to identify current, broad consensus opinions as well as uncertainties and growth areas in the EV field. However, such guidelines may not cover all aspects of a research field, especially given the heterogeneous nature of EVs in terms of size, biogenesis, composition, and function. Indeed, the MISEV guidelines clearly emphasize that they are not a "one size fits all" blueprint, but aim to summarize the most critical elements and prevailing perspectives in the field of EV research.

EV heterogeneity in the MISEV 2023 guidelines

Critical considerations about the diverse nature of an EV preparation isolated with a given method are often neglected in experimental planning. As a consequence, EV heterogeneity has been discussed as one of the major challenges in the field (Giebel et al. 2017; Willms et al. 2018). In particular the lack of standardized EV isolation protocols limits our advances in understanding EV biology and function. Therefore, we highly appreciate the attempt to standardize EV nomenclature and characterization in the recent MISEV guidelines (Welsh et al. 2024). Since it is extremely difficult, or almost impossible, to separate and distinguish between different EV subpopulations based on the currently available methods, the use of the generic term ‘EV’ is encouraged and distinguished from non-vesicular extracellular particles (NVEPs) and some special nanoparticles such as artificial EVs (Welsh et al. 2024). The use of biogenesis-related terms (e.g. ‘ectosomes’ or ‘exosomes’) is discouraged unless the cellular origin of the EV population can be proven. Likewise, the previously popular operational terms “small” or “large” EVs introduced in the MISEV 2018 guidelines (Théry et al. 2018) are recommended to be used only with caution as no common consensus on upper or lower size limits has been reached yet. The approach of reducing EV heterogeneity to the umbrella term “EV” sounds logical, but for newcomers in particular it harbors the risk of underestimating the heterogeneity that actually exists.
In order to improve comparability of results and advance our understanding of EVs, a basic characterization of the investigated EV population isolated with a given method is key. The recommendation of MISEV 2023 to use orthogonal methods for EV characterization will surely help to achieve this goal. Unfortunately, no general EV marker has been identified yet. To help EV researchers in selecting markers to assess the purity and quality of their EV preparation, MISEV 2023 provides an overview of frequently used EV positive and negative markers (Table 3 in Welsh et al. 2024). It should be noted that this selection mainly contains proteins that have been associated with small EVs, in particular exosomes, whereas known markers for other EV subpopulations (e.g. CK18 for large oncosomes (Minciacchi et al. 2015) or Rgap1, Mitofilin, EMMPRIN/Basigin, SLC3A2 or Actinin-4 for ectosomes (Kowal et al. 2016; Menck et al. 2015; Lischnig et al. 2022; Mathieu et al. 2021) are missing among this list. Although their universal applicability for the indicated subpopulation has not been addressed in detail yet, these markers might help EV researchers to assess the heterogeneity of a given EV preparation and attribute specific effects to the distinct subpopulations.

Conclusion

Due to the lack of methods that allow the isolation of specific EV populations based on their different biogenesis pathways, heterogeneity in EV samples remains a major challenge. Although immunocapture methods might allow the specific selection of a desired EV subpopulation, how to define specific capture antigens when the currently available methods fail to reliably separate the distinct EVs? Cell biology studies in combination with novel methodological advances in the isolation of distinct subtypes, such as free-flow electrophoresis to achieve charge-based separation (Preußer et al. 2022), as well as the study of EVs at the single-particle level, including nano-flow cytometry or imaging approaches, might help to overcome this problem. Being at the crossroads of biology, physics, chemistry and medicine, EV research will surely benefit from the interdisciplinary development of novel methods to study the diverse EV subpopulations and advance our understanding of EV heterogeneity.