TY - JOUR

T1 - Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting

AU - Kondratov, Kirill

AU - Nikitin, Yuri

AU - Fedorov, Anton

AU - Kostareva, Anna

AU - Mikhailovskii, Vladimir

AU - Isakov, Dmitry

AU - Ivanov, Andrey

AU - Golovkin, Alexey

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - erythrocyte-derived extracellular vesicles

KW - high-sensitivity flow cytometry (hs-FCM)

KW - high-sensitivity fluorescence-activated vesicles sorting (hs-FAVS)

KW - microRNA

KW - nucleic acid repertoire

KW - platelet-derived extracellular vesicles

KW - Subpopulations of extracellular vesicles

KW - STORAGE

KW - CELLS

KW - MITOCHONDRIAL

KW - SIZE

KW - MICROVESICLES

KW - RELEASE

KW - MICROSCOPY

KW - PLATELETS

KW - EXOSOMES

UR - http://www.scopus.com/inward/record.url?scp=85082751651&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/8cee59d1-1da7-32a3-84be-16098777c22d/

U2 - 10.1080/20013078.2020.1743139

DO - 10.1080/20013078.2020.1743139

M3 - Article

AN - SCOPUS:85082751651

VL - 9

JO - Journal of Extracellular Vesicles

JF - Journal of Extracellular Vesicles

SN - 2001-3078

IS - 1

M1 - 1743139

ER -