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.

Keywords: NCAM; biomarker; exosome; extracellular vesicle; neurological disorder.

FIGURE 1

Enrichment of EVs from plasma using SEC. (A) Schematic overview of the procedure for isolating EVs from plasma using SEC. EVs elute first, followed by smaller protein complexes. (B) Elution profiles of EVs and plasma protein. EV particle numbers and protein concentration in each fraction from 1 to 30 were determined by NTA and the absorbance at a wavelength of 280 nm, respectively. Fractions 1–5 were subsequently pooled together as EV samples (blue dotted line).

FIGURE 2

Physical and biochemical characterization of EVs isolated from plasma. (A) Size distribution of plasma EVs determined by NTA. Median diameter (nm), 90.2 ± 4.58 SD. (B) AFM image (scale bar, 400 nm) and (C) line scan profiles of isolated EVs. (D) Size distribution of plasma EVs determined by AFM. (E) Negative-stain TEM images of EVs. Scale bar, 200 nm. (F) Purity of EV samples tested by Western blot. Plasma proteins, albumin, and ApoA-I are removed from plasma EV samples. Seven micrograms of protein from whole plasma, plasma EVs isolated using SEC, and HEK293 cell lysate were subject to SDS–PAGE and immunoblotted with antibodies against albumin and ApoA-I.

FIGURE 3

Characterization of EVs using flow cytometry. (A) Plasma EVs labeled with DiI were compared with different sizes of beads, i.e., 1, 2, and 3 μm. Light scatter signals correspond to the size of beads, and DiI labeling of EV samples (black circle) increases fluorescence signal. Plasma EVs labeled with DiI (black circle) were mixed with 1 μm beads in the absence (B) and presence (C) of 0.1% Triton X-100. Light scatter and fluorescence signals of DiI-labeled EVs were absent after detergent lysis. Dot plot of forward scatter (FSC) signal for plasma EVs (black square) and 1 μm beads (black circle) in the absence (D) and presence (E) of 0.1% Triton X-100.

FIGURE 4

Analyzing individual EVs using flow cytometry. Shown are dot plots of fluorescent intensity for plasma EVs labeled with DiD. Background acquisition with buffer-only control (A) and DiD-only control (B). Plasma EV samples were unstained (C) and stained with DiD (black circle) (D). (E) 0.1% Triton X-100 treatment disrupts light scatter and fluorescent signals of DiD-labeled EVs.

FIGURE 5

NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C). (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G_i) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G_ii). (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10⁸) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I), single stained with DiI (J), and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.