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Eitan et al. Extracell Vesicles Circ Nucleic Acids 2023;4:133-150 https://dx.doi.org/10.20517/evcna.2023.13 Page 139

Data availability: De-identified data are available upon request from a qualified investigator.

RESULTS
ExoSORT captures extracellular vesicles
ExoSORT is a novel immunoaffinity-based method for EV isolation and was therefore evaluated for
[19]
compliance with MISEV guidelines [Figure 1]. Spherical morphology was shown by electron microscopy
[Figure 1A], and average particle diameter in the 50-250 nm range was confirmed by nano-tracking analysis
(NTA; Figure 1B), even though it seems that NDEVs display a non-normal size distribution. The presence
of typical EV markers, CD9 and flotillin (FLOT1), as well as reduced levels of the non-EV protein albumin,
were shown by Western blot/WES [Figure 1C]. The depletion of contaminating plasma proteins was
assessed using ELISAs for Apolipoprotein A1 (ApoA1) and albumin, with depletion of 95.95 ± 0.94% and
99.8 ± 0.2%, respectively (N = 6, P < 0.001; Figure 1D and E). The relatively higher level of ApoA1
contamination compared to albumin is consistent with previous reports demonstrating EV interactions
with HDL/LDL . Lipidomic analysis showed high concentrations of characteristic EV lipids, such as
[26]
cholesterol, phosphatidylserine, and sphingomyelin [Supplementary Figure 1A and Supplementary Table
1A], and unbiased proteomic analysis revealed multiple EV-specific proteins in the NDEV preparations
[Supplementary Figure 1A and Supplementary Table 1B].

NDEVs captured by ExoSORT are enriched for neuronal markers
NDEVs captured by ExoSORT showed higher levels of neuron-specific proteins and 24-hydroxycholesterol,
[27]
a lipid enriched in the brain , compared to the procedural control, material immunocaptured using IgG
isotype antibody [Table 4]. The enrichment of neuronal proteins was also demonstrated by WES with
antibodies against neuron-specific proteins, GluR2 and NeuN [Figure 1F]. Unbiased proteomic analysis also
revealed significant enrichment for neuronal proteins, as was shown by gene ontology analysis
[Supplementary Table 2]. Since EVs are the major carriers of circulating mRNAs and RNA measurements
[28]
are less sensitive to matrix effects, neuron-specific mRNA content was compared between NDEVs and
unprocessed plasma [Table 4 and Figure 1G]. The NDEV levels of mRNA encoding for hemoglobin beta
chain (HBB, erythrocyte marker), as well as platelet factor 4 (PF4, platelet marker), and albumin (ALB, liver
marker), were less than 6% of those in unprocessed plasma, suggesting successful depletion of non-specific
material; in contrast, the levels of mRNA encoding for the neuronal marker NRGN were similar in NDEVs
and plasma, pointing to efficient recovery of neuron-specific EVs [Figure 1G]. The levels of neuronal
markers in NDEVs were 35 times higher than in the material isolated with control IgG (procedural control,
P < 0.0001; Table 4). The diminished levels of non-neuronal mRNAs and the preservation of neuronal
mRNAs and proteins corroborate the specificity of the NDEV isolation.

ExoSORT isolates endogenous and spiked NDEVs with high efficiency and precision
To estimate the NDEV isolation efficiency, we compared NRGN mRNA levels in NDEVs isolated by
ExoSORT and in unprocessed plasma and found that NRGN levels in NDEVs comprised 86 ± 7% of those
in the parent plasma [Figure 1G]. Further estimates of NDEV isolation efficiency were based on conducting
sequential rounds of ExoSORT, wherein the supernatant remaining after capture bead removal in each
round was retained and subjected to a new round of ExoSORT [Supplementary Figure 2]. With NRGN
mRNA level used as a measure of NDEVs extraction, the difference between two sequential yields was
87 ± 9% providing a measure of isolation efficiency [Figure 2A and B]. Next, we used a classical spiking
approach using EVs isolated from human iPSC-derived cortical neurons. Culture-derived EVs, which were
characterized according to MISEV guidelines [Supplementary Figure 3A-C], contain exceptionally high
[19]
Tau levels [Supplementary Figure 3D], at least ten times higher than those expressed in endogenous plasma
NDEVs. This difference enabled the use of Tau measurements to estimate spiked iPSC EV recovery from
plasma since their contribution far exceeds that of endogenous NDEVs. iPSC EV in amounts containing