Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting

Kirill Kondratov; Yuri Nikitin; Anton Fedorov; Anna Kostareva; Vladimir Mikhailovskii; Dmitry Isakov; Andrey Ivanov; Alexey Golovkin

doi:10.1080/20013078.2020.1743139

Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting

J Extracell Vesicles. 2020 Mar 30;9(1):1743139. doi: 10.1080/20013078.2020.1743139. eCollection 2020.

Authors

Kirill Kondratov¹, Yuri Nikitin², Anton Fedorov¹, Anna Kostareva^{1

3}, Vladimir Mikhailovskii⁴, Dmitry Isakov^{5

6}, Andrey Ivanov², Alexey Golovkin¹

Affiliations

¹ Institute of Molecular Biology and Genetics, Almazov National Medical Research Centre, St. Petersburg, Russia.
² Department of Clinical Biochemistry and Laboratory Diagnostics, Military Medical Academy N.a. S.M. Kirov, St. Petersburg, Russia.
³ Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden.
⁴ Interdisciplinary Resource Center for Nanotechnology, Saint-Petersburg State University, St. Petersburg, Russia.
⁵ Department of Immunology, Institution of Experimental Medicine, St. Petersburg, Russia.
⁶ Department of Immunology, Pavlov First Saint Petersburg State Medical University, St. Petersburg, Russia.

Abstract

The aim of this study was to investigate cell source-dependent nucleic acids repertoire of diverse subpopulations of plasma extracellular vesicles (EVs). Blood plasma from nine healthy volunteers was used for the analysis. Samples of EVs were obtained by differential centrifugation of plasma. The application of high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS) using fluorophore-conjugated anti-CD41-FITC (Fluorescein isothiocyanate) and anti-CD235a-PE antibodies allowed the isolation of three subpopulations of EVs, namely CD41+ CD235a-, CD41-CD235a+ and CD41-CD235a dim. The high purity (>97%) of the sorted subpopulations was verified by high-sensitivity flow cytometry. Presence of nanosized objects in sorted samples was confirmed by combination of low-voltage scanning electron microscopy and dynamic light scattering. The amount of material in sorted samples was enough to perform Quantitative polymerase chain reaction (qPCR)-based nucleic acid quantification. The most prominent differences in the nucleic acid repertoire were noted between CD41+ CD235- vs. CD41-CD235a+ vesicles: the former contained significantly (p = 0.004) higher amount of mitochondrial DNA, and platelet enriched miR-21-5p (4-fold), miR-223-3p (38-fold) and miR-199a-3p (187-fold), but lower amount of erythrocyte enriched miR-451a (90-fold). CD41-CD235a+ and CD41-CD235a dim vesicles differed in levels of miR-451a (p = 0.016) and miR-21-5p (p = 0.031). Nuclear DNA was below the limit of detection in all EV subpopulations. The hs-FCM-based determination of the number of sorted EVs allowed the calculation of per single-event miRNA concentrations. It was demonstrated that the most abundant marker in CD41+ CD235a- subpopulation was miR-223-3p, reaching 38.2 molecules per event. In the CD41-CD235+ subpopulation, the most abundant marker was miR-451a, reaching 24.7 molecules per event. Taken together, our findings indicate that erythrocyte- and platelet-derived EVs carry different repertoires of nucleic acids, which were similar to the composition of their cellular sources.

Keywords: Subpopulations of extracellular vesicles; erythrocyte-derived extracellular vesicles; high-sensitivity flow cytometry (hs-FCM); high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS); microRNA; nucleic acid repertoire; platelet-derived extracellular vesicles.