Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

Microglia-derived small extracellular vesicles (MGEVs) are key mediators of neuroimmune communication, yet their cross-species comparability and translational relevance remain poorly defined. Here, we establish a harmonized framework to compare the molecular and biochemical signatures of sEVs derived from immortalized mouse (BV2) and human (HMC3) microglial cells as well as assess their bioactivity on a human Schwann cell (HuSC) line. MGEVs were isolated via MISEV-aligned size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and immunoblotting for canonical EV markers CD9, CD63, CD81, TSG101. Human and mouse MGEVs exhibited similar morphology but displayed distinct membrane tetraspanin protein enrichment patterns. Functionally, mouse and human MGEVs attenuated HuSC migration while enhancing HuSC proliferation and their resistance to H₂O₂-induced oxidative stress, with human MGEVs providing stronger protective effects, suggesting they retain similar core functional properties. Short, non-coding-miRNA sequencing analysis identified 196 shared miRNAs (Spearman ρ = 0.72) with species-specific enrichment: human MGEVs-derived miRNAs favored regenerative and metabolic pathways, whereas mouse MGEVs-derived miRNAs aligned more so with inflammatory signaling. This study delivers the first integrated cross-species blueprint of MGEVs, revealing conserved neuroprotective actions alongside species-biased miRNA cargo that define translational boundaries and highlight human-relevant MGEV signatures for therapeutic innovation, therefore contributing to the importance of considering these differences in translational research.