Large-scale heparin-based bind-and-elute chromatography identifies two biologically distinct populations of extracellular vesicles

Abstract

Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.

Keywords: exomere; exosome; extracellular particles; extracellular vesicles; fibronectin; heparin.

FIGURE 1

Heparin chromatography separates EVs into two fractions. (a) Purified (post Capto‐Core‐700) EVs were loaded on a 1 mL Hitrap Heparin‐Sepharose column using PBS (pH 7) as the running and washing buffer. The NHB were pooled from the flow‐through fractions. HB were eluted with NaCl in PBS at a maximum concentration of 1 mol/mL in a gradient manner. A representative chromatography curve out of three biological repeats is shown. The horizontal axis shows buffer volume. The vertical axis on the left shows UV and the vertical axis on the right shows conductivity. (b) ZETA potential and (c) particle concentrations were measured by NTA. (d) Protein concentrations were determined by BCA assay. Five biological repeats were performed for ZETA potential and the p‐value was calculated by One‐way ANOVA. Three biological repeats were performed for particle concentrations and protein concentrations.

FIGURE 2

NTA, TEM, and dSTORM report EV sizes with variations. (a) Representative TEM images of EV in the Input, NHB, and HB EV fractions. (b) Size distribution of Input, NHB, and HB EV samples determined by TEM, dSTORM, and NTA. ‘R. Gyr.’ stands for ‘radius of gyration,’ which measures the radial distance to where the inertia equaled the mass, hence roughly half the ‘true’ particle diameter. Here the R.Gyr. numbers were multiplied by two to correspond to EV diameter as reported by other methods. n: number of particles measured. d: diameter. Five biological repeats were performed for TEM and NTA. Three biological repeats were performed for dSTORM.

FIGURE 3

Proteomics analysis revealed conventional EV markers in NHB. (a) Venn diagram comparison of proteins identified in the Input, NHB, and HB samples. (b) Volcano plot of the quantitative differences of proteins in NHB and HB. Red dots indicate proteins enriched either in NHB or HB (p‐value < 0.05, |Log2| > 0.58). Blue dots annotate the Top 100 EV proteins. (c) Waterfall plot of the relative abundance of proteins in NHB versus HB. Three biological replicate samples were used for proteomics analysis. Raw data is available for download from ProteomeXchange PXD039845. (d) Western blot confirmation of validated EV protein markers, specifically CD81, CD63, Alix, Flotillin‐2, Syntenin, Fibronectin, Histone H3. Representative images of three biological repeats were shown (complete blots are provided as supplemental material).

FIGURE 4

Cellular component analysis of proteins identified in NHB and HB. Proteins enriched in either NHB or HB were analyzed by DAVID Bioinformatics Resources (https://david.ncifcrf.gov/home.jsp) and the top 10 hits are shown.

FIGURE 5

Quantitative EV surface marker profiling by dSTORM super‐resolution microscopy. Representative dSTORM images of (a) Input, (b) NHB, (c) HB EV samples labeled by CM‐DeepRed (magenta), CD81 (cyan), and fibronectin (yellow). Percentages of Fibronectin+ (Red), CD81+ (Green), and double positive (Blue) EVs in Input (d, n = 2025), NHB (e, n = 1545), HB (f, n = 1837) samples were quantified by CODI analysis (ONI Inc.).

FIGURE 6

Heparin fractionates EVs into two biologically active but functionally distinctive populations. (a) Representative immunofluorescent images of hTERT‐HUVEC cells treated with CM‐Red dye labeled Input, NHB, and HB EV for 24 h. Nuclei were labeled by DAPI (blue). Presence of CM‐Red dye (yellow) indicates EV taken in by cells. (b) WB of phosphorylated ERK (top) and total ERK (bottom) upon Input, NHB, and HB EV treatment for 1, 4, 8, and 24 h. (c) Quantification of phosphorylated ERK intensity on WB upon 24 h of EV treatment from three independent experiments. (d) Representative immunofluorescent images of hTERT‐HUVEC cells treated with PBS, Input, NHB, and HB EVs for 24 h. Ki67, nuclei (DAPI), and composite images were shown. (e) Percentage of Ki67 positive cells upon different treatments. ****p < 0.0001, ***p < 0.0005 by T‐Test.

FIGURE 7

Principal component analysis (PCA) and expression fold change of transcriptomics in hTERT‐HUVEC cells upon EV treatment. hTERT‐HUVEC cells were treated with PBS, Input, NHB, and HB EVs and subjected to transcriptomics analysis by RNAseq. Each treatment included four replicates. After 1 h (a) and 24 h (b) of treatment, data were subjected to PCA. Expression fold change between NHB and HB treated cells were represented in (c). Red and blue dots indicate mRNA up‐regulated in HB and NHB, respectively (p‐value < 0.05, |Log2| > 0.5). For comparison, Interferon‐stimulated genes (ISG) were annotated by gene names. Fold changes between 1 and 24 h were represented in (d). Red and blue dots indicate mRNA up or down‐regulated after 24 h of treatment (FDR < 0.05, |Log2| > 0.58). Data and the R codes used for analysis are available on Bitbucket (see Section 2).