Fluorescence-based Sorting Found to Alter miRNA Signature of Extracellular Vesicles

A recent proof-of-concept study, published in Extracellular Vesicles and Circulating Nucleic Acids, evaluated how fluorescence-based sorting influences the microRNA (miRNA) molecular profile of extracellular vesicles (EVs) from adipose-derived mesenchymal stromal cells (ASCs). ASCs from five human donors were characterized by flow cytometry and cultured to obtain conditioned media. EVs were isolated either by standard ultracentrifugation (ASC-EVs) or via high-sensitivity fluorescence sorting targeting particles ≥100 nm (sASC-EVs).

Both ASC-EVs and sASC-EVs showed similar size distributions, as measured by nanoparticle tracking analysis, and comparable surface marker profiles for EVs and mesenchymal stromal cells via flow cytometry. Electron microscopy further confirmed consistent morphology across groups.

Small RNA sequencing highlighted notable differences. Sorted sASC-EVs contained 285 detectable miRNAs, far fewer than the 749 in unsorted ASC-EVs. The two groups shared 271 miRNAs, which displayed strong correlations in relative abundance. These common miRNAs enriched pathways involved in angiogenesis, inflammation modulation, and gene silencing.

Differential expression analysis identified 32 miRNAs upregulated and 6 downregulated in sASC-EVs, alongside 14 miRNAs unique to the sorted fraction. Despite these variations, both EV preparations maintained a positive overall balance between protective and detrimental OA-related miRNAs.

The team from IRCCS Ospedale Galeazzie attributes the reduced miRNA count in sASC-EVs to factors like lower EV recovery, exclusion of miRNA-bound non-vesicular particles, or inability to detect small EVs with fluorescence sorting. This approach increases EV purity but sacrifices miRNA diversity, revealing a clear trade-off.

The findings stress the need to optimize EV isolation protocols for clinical applications. Sorting preserves key functional signals, yet the loss of complexity warrants refined methods to retain fuller miRNA profiles without compromising precision.