Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

Figures & data

Text Box 1. Roundtable topics, moderators, and descriptions.

Roundtable #1: Biogenesis and Release Moderators: Matias Ostrowski, Hang Yin, Roosmarijn Vandenbroucke

EV subpopulations are commonly defined by site or mechanism of biogenesis. Roundtable 1 aimed to identify outstanding questions about how various molecules and pathways influence formation and release of EVs.

Roundtable #2: Uptake and Fusion

Moderators: Pieter Vader, Jeanne Sisk, David Carter

After release, EVs may exert effects through autocrine, paracrine, or endocrine processes, all of which require interaction of EV membranes with target cells. This roundtable focused on current knowledge of EV-cell interactions, including uptake and fusion, and experimental approaches needed to dissect mechanisms.

Roundtable #3: Technologies and Strategies

Moderators: Marca Wauben, Paolo Bergese

Since unique technologies and approaches may be needed to study EVs and their membranes, this roundtable discussion sought to identify technologies, experimental methods, and models that have not yet been well applied to EV studies or should be further developed to enable more sophisticated analysis of EVs and membranes.

*Roundtable #4: Transfer and Functional Assays

Moderators: Jan Lötvall, Daniel Anthony

How EVs transfer cargo to recipient cells and how to assess effects of transfer were the considerations for this roundtable discussion. Also discussed were best practices for conducting and reporting EV studies (especially visualization), use of in vitro generated EVs for in vivo uptake studies, and the future of EV-based therapeutics.

*Because of substantial content overlap of Roundtable 4 with Roundtables 1–3, information from this roundtable has been integrated into other sections below.

Table 1. Workshop pre-survey questions.

Pre-Workshop Survey Questions
What, in your opinion, are the top publications (up to 3) in the last five years that have addressed important questions of EV/membrane biology?
What are the most pressing current questions in EV biogenesis? (Up to 4)
What are the most pressing questions surrounding EV uptake and/or fusion? (Up to 4)
What important questions remain about EV component loading (natural or artificial) – this includes lipids, proteins, internal cargo? (Up to 4)
What are the most important unanswered questions about EV function as related to target cell interaction/uptake/fusion? (Up to 4)
To help answer outstanding EV/membrane questions, are there any technologies, methods, or models that have not yet been developed or fully applied? If so, what are they? Or what are the capabilities you would want? (Open response)
Do you have any position or opinion related to this workshop that you suspect some of your colleagues would disagree with? If so, what? (Up to 3)
Other comments or suggestions? (Open response)

Table 2. Survey questions regarding EV biogenesis.

EV Biogenesis Survey Questions
Figure 1	Budding into the multivesicular body (MVB) as intraluminal vesicles (ILVs) and budding from the plasma membrane are the two major EV biogenesis pathways, with at least partially independent molecular machinery of biogenesis.
	It is possible that what we refer to as MVBs are actually physical extensions of the plasma membrane and not late endosomes.
	It is currently possible to distinguish, using protein, lipid, or other markers, an “exosome” (former ILV in the MVB) from a “microvesicle” (from the plasma membrane) after the respective vesicle has left the cell.
	Size can be used to separate EVs by biogenesis pathway.
	EVs from the endosomal system are smaller, on average, than EVs that bud from the plasma membrane.
	We know the basic size distribution of EVs from biofluids and cell culture.
Figure 2	Excluding apoptotic bodies and other “macrovesicles”, the average diameter of most EV populations is:	Significantly smaller than 100 nm
		Roughly in the 100–150 nm range
		Significantly larger than 150 nm
Figure 3	Asymmetric distribution of lipids (inner, outer leaflet) is the same in EVs as in the cell membrane of origin and remains stable over time.
	The inner and outer sides of the EV membrane are revealed by inner and outer domains of proteins in the expected orientation relative to the cell. That is, the cellular membrane topology is maintained by EVs.
Figure 4	The weight of the evidence supports preferential packaging of certain miRNAs or other RNA cargo into specific subsets of EVs.
	The RNA Cargo of larger EVs correlates with cellular expression, but that of small EVs does not.
Figure 5	It is possible to create cells or organisms that do not produce EVs.
	EV biogenesis is essential for life, as evidenced by lethality of TSG101 knockouts and knockouts of multiple biogenesis-linked proteins.
Figure 6	Lipid-raft domains (endosome-like domains, rich in cholesterol, etc.) play a role in EV biogenesis; without them, many EVs would not form.
	nSMase2 is not involved in biogenesis of all EV subtypes in all cells, hence discrepant results of nSMase2 blocking.
	Energetic requirements of EV biogenesis are largely unknown.

Table 3. Survey questions regarding EV uptake.

EV Uptake Survey Questions
Figure 7	Most cell types, sooner or later, internalize at least a proportion of stained EVs, seemingly regardless of the cells of origin.
	EV-cell fusion is most likely to occur through endosomal uptake and acidification.
	Proteins on the EV surface are required for most fusion events between EVs and cellular membranes.
	EV-cell fusion events are actually quite rare in vivo, and may involve minority subpopulations of EVs and specific uptake pathways.
	Current in vitro studies of uptake (anything involving 2D tissue culture plastic substrates) are not worthwhile as unrepresentative of in vivo biology.
Figure 8	Rank the following from 1 (most likely) to 5 (least likely). Interacting with target cells, EVs exert effects by:		Signalling through proteins displayed on the target cell surface or in the endosomal lumen
			Transferring functional proteins
			Transferring functional lipids
			Transferring functional RNA molecules
			Serving as a form of nutrition/molecular recycling for the recipient cell
Figure 9	Current technologies are adequate to measure both functional and physical stability of EVs.
	Physical stability of EVs (defined here as the tendency to maintain vesicular form) is related to size.
Figure 10	Regarding freeze-thaw of EVs:	All EVs are generally resistant to freeze-thaw damage
		Small EVs are generally resistant to freeze-thaw damage
		EVs are damaged both physically and functionally
		EVs are damaged functionally, but may show the same physical characteristics
		We still don’t know enough to answer this question
Figure 11	In vitro EV transfer experiments are highly time-dependent, and the relevance to timing/EV stability in vivo is often unclear.
	Dose-response studies are essential in establishing any effect of EVs.
	Most EVs in vivo are bioactive.
	EVs in circulation (blood) are less likely to be bioactive and are cleared rapidly.
	EVs are most likely to have a signalling function in tissue, i.e. locally.
	Tumour-bearing mice accumulate more EVs in cancer tissue mostly because of vascular leakiness.
	The apparently low rates of EV:cell fusion indicated by systems such as the Cre/lox stoplight system may reflect sensitivity or idiosyncrasies of the assay and not imply that fusion is really so rare.

Table 4. Survey questions regarding current technologies for studying EVs.

EV-Technology Survey Questions
Figure 12	Different measurement technologies are biased to certain EV size ranges.
	Optical scattering methods of EV measurement such as nanoparticle tracking are not specific to EVs.
	Lipid dyes form artefactual particles on their own and with non-EV materials; results of lipid dye experiments are unreliable unless one can effectively separate EVs and artefacts by flotation gradients.
Figure 13	With which statement do you agree more?	High-resolution single EV analysis by flow cytometry is now possible for labs with access to a standard flow cytometer.
		It remains necessary to have specialized equipment, reagents, and expertise to perform single EV flow analysis for EVs below about 500 nm in diameter.
Figure 14	Fluorescence triggering in EV flow cytometry allows better resolution than scatter.
	Better generic dyes of EVs are needed for flow cytometry and other investigations.
	Development of reagents such as single chain antibodies, aptamers, and less bulky fluorophores is needed to improve sensitivity of EV flow.
Figure 15	It is currently possible to make artificial EVs that faithfully mimic genuine EVs
	It is currently possible to affect EV distribution to tissues by manipulating EV surface features.
	New animal models and more relevant in vitro systems are needed to address questions about production and function of subsets of EVs.

Table 5. Summary of topics on which there is largely agreement, relative consensus, or clear lack of consensus; a set of specific recommendations are included.

	Consensus	Most agree	No consensus	Recommendations
Biogenesis, size and content	Fusion of MVBs and budding from the PM are the main routes of EV biogenesis	It is not possible to distinguish microvesicles from exosomes using size, proteins, lipids or other markers, though most agree that MVs are on average larger	Most EV populations are <100 or 100-150nm	Researchers should consider the entire trafficking landscape of intracellular vesicular organelles which can directly, or indirectly affect EV biogenesis and secretion
		EV biogenesis is essential for life	MVBs may actually be physical extensions of the plasma membrane and not late endosomes	Substantial additional work is required to identify specific markers of EV subtypes released across or within cell types
		It is not possible to generate cells or animals that don’t produce any vesicles	RNA cargo of larger EVs shows better correlation with cellular expression compared to that of smaller EVs	Improved separation and characterization technologies are required for unbiased and accurate counting and sizing of EVs
		The energetics of EV biogenesis are unknown	Asymmetry of lipids (in inner/outer leaflet) and proteins (orientation of proteins) is maintained relative to the cell	Reproducible in vitro assay systems which closely mimic the physiological context are needed to study EV cargo loading
		Lipid rafts are important in EV biogenesis, and nSMase2 is not involved in the biogenesis of ALL EV subtypes		Unbiased genetic screens and small molecule modulator screens may be needed to resolve unappreciated and combinatorial contributions to EV biogenesis
		There is some specific loading of cargo into specific subsets of EVs		The roles of various sphingomyelinases, ceramides, and lipid rafts in EV biogenesis requires further investigation
Transfer, uptake and biodistribution	The apparent low rates of EV:cell fusion indicated by systems such as the Cre/lox stoplight system reflect the sensitivity and idiosyncrasies of the assay and do not imply that fusion is really so rare	Fusion is most likely to occur through endosomal uptake and acidification	EV:cell fusion events are rare, involving a minority subpopulation of EVs and specific uptake pathway	Further experiments are required to decipher the rules of cellular targeting and uptake by EVs
	EV transfer experiments are time-dependent	Most cells eventually internalize at least some stained EVs		Greater understanding of how EVs induce their most important effects (direct interactions, transfer of different cargo etc) is needed
	EVs accumulate in cancer tissue in xenografts because of vascular leakiness	EVs in vivo are bioactive; there is less consensus on whether EVs in circulation are bioactive, with most believing that EVs are most likely to have signalling functions locally within tissues		Serial or differential dosing may be necessary for in vivo studies aimed at understanding the function or biodistribution of EVs
		Proteins on the EV are required for fusion		Improved methodology, including imaging and staining, is required for the study of EV biodistribution
		The most significant interaction of EVs with cells is via signalling that occurs through proteins displayed on the target cell surface or in the endosomal lumen		There is a need for advanced animal models to study the physiological importance of EV-mediated cargo transfer between cells and tissue
		It is possible to affect EV distribution to tissues by manipulating EV surface features		The field needs to establish guidelines for defining and/or concluding which EV subpopulations and associated cargo are involved in homeostatic maintenance and pathological responses
		Even studies in 2D culture systems are worthwhile as a representation of at least some aspects of in vivo uptake
Methodology	Dose-response studies are essential for establishing functions for EVs	Lipid dyes can form artefactual particles making results of experiments less reliable unless one can effectively separate these by flotation	It is possible to make EVs that faithfully mimic genuine EVs	Dose-dependency is a key factor to include in experimental design
	Fluorescence triggering in EV flow cytometry allows better resolution than scatter			Free dye controls are needed for experiments using stained EVs
	High-resolution single EV analysis by flow cytometry requires specialized equipment, reagents and expertise			Better generic EV dyes and tags are needed for flow cytometry and other studies
	Optical scattering methods of EV measurement are not specific to EVs			There is a need for reagents such as single chain antibodies, aptamers and less bulky fluorophores to improve the sensitivity of EV detection in flow cytometry
	Different measurement technologies are biased to certain EV size ranges			New animals and more relevant in vitro systems are needed to address questions about production and function of subsets of EVs
				Improved and novel methodology is needed for single EV analysis
				Improved imaging techniques are needed for EV analysis at different levels of resolution
Storage and stability		Current technologies are not adequate to measure both functional and physical stability of EVs	Stability of EVs is related to their size	More work is needed to understand the effects of storage on the stability and function of EVs
			Freeze-thawing affects the structure and/or functionality of EVs

Figure 1. EV Identification and size. Six questions regarding EV identification and sizing were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Most responders believe that there are multiple distinct pathways for vesicle biogenesis that result in heterogeneity in terms of size. Identifying vesicles from these pathways based on size, protein or lipid markers remains difficult.

Figure 2. The average diameter of EVs (Excluding apoptotic bodies and other “macrovesicles”). In the post-workshop survey, participants were asked to choose from the three listed options. Responders believe that most EV populations are less than 150 nm in size. Those vesicles less than 100 nm in size are difficult to detect using techniques based on light scattering.

Figure 3. EV membrane topology. Two questions regarding EV membrane topology were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders are uncertain as to whether the lipid distribution of EV membranes is the same as the original cell membrane.

Figure 4. Membrane Involvement in EV cargo packaging. Two questions regarding the involvement of membranes in EV cargo packaging were administered in the Post-Workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders are not sure whether miRNA or RNA cargo is specific to certain subtypes of EVs.

Figure 5. Importance of EVs for Life. Two questions regarding the importance of EVs for life were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. The majority of responders believe that EV production is necessary for cell and organism survival.

Figure 6. Requirements for EV Biogenesis. Three questions regarding requirements for EV biogenesis were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders believe that the roles of lipid-raft domains, nSMase2, and the energetic requirements of EV biogenesis need to be further explored.

Figure 7. EV-cell fusion. Five questions regarding EV-cell fusion were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders agree that recipient cells internalize EVs from different cell types through endosomal uptake and acidification, and that proteins on the EV surface are responsible for fusion events. Survey participants are not sure how frequent EV-cell fusion events are in vivo.

Figure 8. EV-cell interactions. In the post-workshop survey, participants were asked to rank order the most to least likely ways in which EVs interact with target cells. Answers are depicted in a heat map, with pink shades indicating a higher number of responders, and blue indicating a lower number of responders. Responders believe that EVs primarily interact with target cells by signalling through proteins displayed on the target-cell surface or endosomal lumen. Transferring functional RNA, proteins and lipids is seen as a secondary effect. Most believe that EVs are indirectly a form of nutrition or molecular recycling for recipient cells.

Figure 9. EV stability. Two questions regarding EV stability were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. While EVs are physically stable, most survey participants believe that current technologies need to be improved to simultaneously measure the functional and physical stability of EVs.

Figure 10. Storage of EVs. In the post-workshop survey, participants were asked to choose from five options whether or not they believe freeze-thawing causes damage to EVs. Responders agree that we do not know enough about how freeze-thawing affects EV stability, uptake, and functionality.

Figure 11. Bioactivity of EVs. Seven questions regarding the bioactivity of EVs were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders believe that the use of EVs for in vitro transfer experiments is time-dependent, that dose–response studies are important, and that EVs have a greater functional impact in the local tissue environment. Survey participants are undecided on how to determine and identify bioactive EVs. The survey reveals the need for improved technology for the study of EV-cell fusion.

Figure 12. Current particle tracking technologies. Three questions regarding current particle tracking technologies were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Survey participants require improved, non-biased technologies for determining EV size. The use of lipid dye can cause experimental artefacts.

Figure 13. Current opinion on EV-flow cytometry. In the post-workshop survey, participants were asked to choose between two options regarding the current status of applying flow cytometry to the study of EVs. Almost all responders to this question call for specialized equipment, reagents and expertise to characterize single EVs through flow cytometry.

Figure 14. Fluorescent labelling of EVs. Three questions regarding fluorescent labelling of EVs were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Survey question participants acknowledge that better dyes and reagents are needed to study EVs using flow cytometry. Still, using fluorescent flow cytometry to study EVs provides better resolution than scatter.

Figure 15. Current EV Technologies. Three questions regarding current EV technologies were administered in the post-workshop survey. For each question, participants’ answers are depicted horizontally on a Likert-scale from 0 to 10, with bubble size reflecting of the number of responders at each point on the scale. Responders agree that new animal and in vitro models are needed to address questions concerning EV production and function. Survey participants are not sure whether artificial EVs can mimic genuine EVs, or that manipulation of EV surface features will affect biodistribution.