Figure 3.
A lentivirus ENoMi vector labels extracellular vesicles secreted by brain-implanted human neural progenitor cells
(A) The ENoMi construct encodes for a CD63 TSN membrane scaffold that exposes an FLAG tag peptide and NanoLuc reporter on the EV surface and an mCherry reporter in the EV lumen. The ENoMi transgene is in-frame with two other separate transgenes encoding a fluorescent copGFP protein and antibiotic-selectable puromycin resistance gene. Transcription of this lentiviral construct is initiated by an EF-1alpha promoter, and three separate proteins (ENoMi, copGFP, and puromycin) are produced due to T2A and P2A self-cleavage sites (top) separating them. A schematic of how EVs incorporate the ENoMi construct by their donor hNPC. In hNPCs expressing copGFP in the cytosol, mCherry faces the cytoplasm, while FLAG tag and NanoLuc are located toward the extracellular milieu or the lumen of the multivesicular body (MVB). Next to ENoMi labeling of EVs, ENoMi-EVs incorporate exRNAs from their donor hNPCs. These exRNAs include pathology-related transcripts, such as the XDP hallmark TAF32i or other ENoMi-related transcripts, such as copGFP (bottom). (B) Representative fluorescent microscopy image of hNPCs in culture expressing the ENoMi construct. Upon ENoMi expression, hNPCs express a dotted mCherry pattern in the cytosol, while the copGFP expression is diffuse in the cytosol. Bottom right, DAPI staining of nuclei; bottom left, merged images. Scale bar, 100 μm. (C) Size exclusion chromatography (SEC) of conditioned media from ENoMi-transduced control hNPCs and XDP hNPCs in culture. In the secrete of hNPCs, ENoMi bioluminescence derived from its NanoLuc reporter is detectable in early SEC fractions (F7–F11) where the EVs reside and not so much in the late SEC fractions (F13–F27) that consist of free proteins. The profile is generated with data from four replicates per genotype. (D) Nanoparticle tracking analysis (NTA) of EVs isolated with SEC from conditioned media of ENoMi-transduced control hNPCs or XDP hNPCs in culture. The upper histogram shows the profile of control ENoMi-EVs (mean particle size = 221 nm, and particle number = 2.26 × 109 particles/mL, while the lower histogram illustrates the XDP ENoMi-EVs (mean particle size = 238 nm and particle number = 7.9 × 108 particles/mL). (E) SEC-isolated EVs were verified with an immunoblot against hCD81. SEC fractions 7 to 15 were resolved on a western blot. CD81 appears only in the EV fractions 7 to 11 and not in the protein fractions 12 to 15. (F) Comparison of ENoMi collection of EVs (anti-FLAG tag pulldown) with other magnetic bead-based immune-capture methods using antibodies to TSNs. Bars represent percent NanoLuc luminescence in conditioned media of ENoMi-transduced XDP hNPCs after exposure to anti-FLAG, anti-CD63, anti-CD81, and anti-CD9 coated magnetic beads. Luminescence was measured on beads (immuno-captured EVs - red bars) and in suspension (non-captured EVs - gray bars) after affinity pulldown. Data represent mean ± SEM of n = 3 for each hNPC genotype. (G) Representative electron microscopy image of ENoMi-EVs after isolation from conditioned medium of hNPCs. ENoMi-EVs were isolated from conditioned media mixed with magnetic anti-FLAG-tag immune-capture beads and recovered from the beads with a competing recombinant FLAG tag peptide. Black arrowheads represent eluted ENoMi-EVs after negative stain. Scale bar, 100 nm. (H) Detection of XDP-related exRNA in ENoMi-EVs. Bars represent TAF1-32i RNA levels in conditioned media of ENoMi-transduced hNPCs of XDP patients after exposure to anti-FLAG-tag, anti-CD63, anti-CD81, and anti-CD9 coated magnetic beads. Data representing qRT-PCR analysis of TAF1-32i levels (ratio of immunocaptured Ct values over non-captured Ct values). TAF1-32i was undetectable in control hNPCs. Data represent mean ± SEM of n = 3 for each hNPC genotype.