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inaccessible to manipulation, especially in ova, may now be accessible by EV technology. Will the
Weismann Barrier be breached? How does virus spread in the body and are EVs being hijacked by virus as
some reports suggest, to facilitate spread? Is Long COVID due, in part, to the distribution of virus across
biological barriers? Lastly, will EV applications flourish? EV technology may change the practice of
medicine through diagnostics and therapeutics. EV applications in agriculture are still in a nascent stage,
and undoubtedly, more will come. The long and short of this narrative is that starting from a key basic
science discovery, a new era of cell communication has emerged that will reveal more of what is novel about
the human condition.

FUTURE PERSPECTIVES AND ROADMAP FOR THE NEXT GENERATION
Finding the evolutionary origins of EV biogenesis and secretion
The mechanisms of vesicle formation and secretion in prokaryotes are undoubtedly the forerunners of EV
secretion in eukaryotes. Comparative analyses of EVs from the three kingdoms will be revealing.

Elucidating the biogenesis and secretion of EVs in eukaryotic cells
Understanding the biogenesis of multivesicular bodies harboring exosomes and the assembly of nascent
EVs at the plasma membrane will be essential to the goal of transferring EV technology to diagnostics and
therapeutics. There are likely numerous junctions and inputs along the various trafficking pathways,
including different modes of packaging: at the cell surface; within endosomes; in autophagosomes; and even
in nuclear outer membranes. How would the assembly process be regulated at the transcriptional level? Do
EV assembly and discharge pathways operate as escalators that constantly move along the secretory
pathway with or without cargo or would the assembly process be tied to the production of cargo or cargo
carriers?

Cognition and reception
Specificity is key to EV-dependent communication. Signal transmission by EVs will require some form of
molecular recognition of EVs by target cells. How recognition molecules are packaged into EVs that also
contain informational content will remain a central but challenging question. Recognition of EVs by target
cells might be achieved by low affinity interactions enhanced by vesicle clustering akin to a quorum effect
found in bacteria. Understanding the modes of access to target cells will be essential to the development of
EV-based therapeutics.

Methods, models and the microenvironment
Tracking EV discharge and delivery at the single-cell level in the microenvironment will reveal the role of
EVs in the creation of so-called niche or pre-metastatic environments. Methods to study EVs at the single-
vesicle level, including improved imaging technologies and innovative in vitro reconstitution techniques,
will be required to examine all aspects of EV biogenesis. Exploitation of organoid technologies will be
needed to create novel microenvironmental simulations. Lastly, animal models will be needed to monitor
EV traffic and exchange in real time, opening a new level of understanding of the interplay between EV
signaling and the endocrine system.

Diagnostic and therapeutic applications
A major challenge in the coming decades is the application of EV-based technology to overall human
health. Advances in EV-based diagnostics will depend on the discovery of unique EV markers that would
serve as molecular signatures that report on both disease and physiological states. Finally, reconstitution or
packaging of targeting molecules and informational content into deliverable EVs will open up a new era in
therapeutics.