Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers

Abstract

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

Proteomics analysis of extracellular vesicles for biomarkers of autism spectrum disorder

“…Human peripheral blood samples were drawn from the HCs and ASD individuals into EDTA tubes. In the processing of blood samples, blood components were separated using density gradient centrifugation as previously described 11 . The plasma supernatant was further centrifuged for 15 minutes to remove platelets and blood cells to obtain platelet-free plasma, which was aliquoted and stored at −80°C until further use for EV isolation.…”

Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome

“…We used the known exosome marker proteins CD9, CD63, PDCD6IP (aka Alix), FLOT1, FLOT2, TSG101, SDCBP (aka Syntenin-1) and NCAM1 [38][39][40][41][42] . NCAM has been used in the past for the immuno-enrichment and detection of neuronal derived exosomes from plasma or serum [43][44][45][46] . We used the proteins CD40, SEPTIN2, and ATP5F1A as markers of microvesicles 39,40,42,47,48 .…”

Extracellular Vesicles: Techniques and Biomedical Applications Related to Single Vesicle Analysis

“…It was suggested that plasma NCAM-positive neuronal EVs could be used as a promising liquid biopsy tool for the diagnosis of neurological disorders. 201 The vitally important role of EVs in neurodegenerative diseases holds potential for the development of diagnostic and therapeutic approaches. 202,203 Moreover, EVs show promise as drug carriers for neurodegenerative diseases due to their excellent biocompatibility, stability, target specificity, as well as blood−brain barrier (BBB) permeability.…”

“…Ali Moussa et al employed a flow cytometry method to identify single neuronal EVs released from cortical neurons and brain organoids derived from induced pluripotent stem cells (iPSCs) and found that neural cell adhesion molecule (NCAM) was highly expressed and enriched. It was suggested that plasma NCAM-positive neuronal EVs could be used as a promising liquid biopsy tool for the diagnosis of neurological disorders …”