For years, exosomes were regarded as relatively homogeneous extracellular vesicles (EVs), simple carriers of biomolecules. However, advances in single-vesicle analysis have revealed a remarkable heterogeneity, not only in size or composition but also in biological function. This diversity raises new key questions:
What functionally defines an exosome subpopulation? How can they be separated with greater resolution? And what is their actual role in pathophysiology?
What functionally defines an exosome subpopulation?
Specific combinations of surface biomarkers, molecular cargo, and cellular origin define functional subpopulations of exosomes and determine their interaction with target cells and biological impact. Beyond the classical markers (CD9, CD63, CD81), researchers have discovered that distinct profiles of proteins, lipids, and nucleic acids confer specialized functions.
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Immunomodulatory exosomes, carrying MHC-associated proteins or costimulatory molecules.
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Proangiogenic exosomes, enriched in VEGF, miR-210, or specific integrins.
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Oncogenic exosomes, with unique proteomic signatures that facilitate invasion or immune evasion.
Ultimately, functionality is not defined by a single marker but by a multimodal profile, whose integrated interpretation requires advanced characterization technologies.
How can they be separated with greater resolution?
The current challenge is to discriminate exosome subpopulations with enough resolution to correlate phenotype and function. Classical techniques—such as ultracentrifugation or precipitation—do not offer this capability.
The most promising strategies include:
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Multiparametric immunocapture combined with high-sensitivity flow cytometry (such as single-EV flow cytometry), capable of detecting co-expression of markers at the single-particle level.
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Affinity-based microfluidics, enabling isolation of specific subpopulations based on ligands or antibodies.
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Nanoflow cytometry or Raman spectroscopy on individual EVs, providing a more comprehensive view of biochemical composition.
These tools achieve unprecedented functional resolution, enabling the interpretation of each exosome as a distinct “informational unit.
The role in pathophysiology: the importance of diversity
Understanding exosome heterogeneity is not merely an analytical matter. Each subpopulation may play divergent—even opposing—roles in pathophysiology.
For example:
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In cancer, some vesicles promote metastasis while others may induce antitumor immune responses.
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In neurodegenerative diseases, certain EVs facilitate the spread of misfolded proteins, whereas others contribute to repair mechanisms.
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In immunology, the coexistence of exosomes with pro- and anti-inflammatory signals suggests a fine-tuned regulation of homeostasis mediated by their relative balance.
Thus, heterogeneity is not biological noise but an essential dimension of vesicular function.
Looking ahead
The future of exosome research lies in mapping this functional diversity with precision. Technologies such as high-resolution flow cytometry and multiparametric characterization will redefine the very concept of an “exosome population.”
Understanding what makes each subpopulation unique will be key not only for basic biology but also for developing more specific liquid biomarkers and more targeted therapeutic vehicles.