Comprehensive characterization of human brain‐derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications

Abstract

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain‐derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size‐exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG‐UC). Our results showed that SG‐UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass‐tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG‐UC showing the best enrichment of common EV proteins with less contamination of non‐EV proteins. In addition, SG‐UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell‐substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single‐particle analysis using the super‐resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Single‐extracellular vesicle (EV) analyses validate the use of L1 Cell Adhesion Molecule (L1CAM) as a reliable biomarker of neuron‐derived EVs

“…Additional results obtained by fluorescent confocal microscopy and FCA confirmed the sorting of L1CAM extraluminal epitopes to bona fide single NDEVs carrying the canonical EV markers Alix and tetraspanins CD9, CD63 and CD81 (Figure 1d–o ). Particularly, FCA results showed that a vast majority of L1CAM+ EVs from iNeurons co‐express the synaptic protein VAMP2 (Figure 1o ), previously reported to be enriched in EVs secreted by mice, rat and human neurons (Vilcaes et al., 2021 ; You et al., 2022 ), and differentiating them from glia‐derived EVs (You et al., 2022 ; Zhang et al., 2023 ). Our findings at the single EV level validate previous studies detecting L1CAM in EVs from neuronal culture media via immunoblotting, L1CAM Simoa ® of SEC EV fractions and mass spectrometry (Faure et al., 2006 ; Lachenal et al., 2011 ; Norman et al., 2021 ; Vilcaes et al., 2021 ).…”

Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation

“…We found a strong enrichment of known EV markers and a reduction of plasma, nuclear, cytosolic, and most mitochondrial proteins. A recent study comparing three CNS tissue EV separation protocols—SEC, sucrose gradient ultracentrifugation, and phosphatidylserine affinity capture—highlighted the speed and robustness of SEC, but also reported contamination with soluble proteins using this method [ 42 ]. However, our protocol differs from this publication in several steps: we carried out filtration with a smaller cut-off (0.22 µM vs 0.45 µM) and used higher centrifugation speeds to eliminate larger EVs—which reduces contamination and refines the studied EV subset.…”

Alterations of plasma neuron-derived exosomal microRNAs in patients with bipolar disorder

“…This variance in miRNA uctuations might root from the diversity in exosomes origins. Considering exosomes can emerge from a variety of organs, with those stemming from neurons forming just a minute segment, this distinction is not entirely surprising [15]. Compounding the complexity, prior research often hinges on smaller participant groups and tends to focus on a con ned set of miRNAs.…”

“…While prior studies con rm altered miRNAs in blood exosomes of patients with mental disorders [12][13][14], it's important to note that the origin of these exosomes in peripheral blood is multifaceted. Only a minimal fraction is sourced directly from brain tissue [15]. However, a breakthrough is the capability to concentrate neuron-derived exosomes from blood samples.…”