Heterogeneity of the nucleic acid repertoire of plasma extracellular vesicles demonstrated using high-sensitivity fluorescence-activated sorting. / Kondratov, Kirill; Nikitin, Yuri; Fedorov, Anton; Kostareva, Anna; Mikhailovskii, Vladimir; Isakov, Dmitry; Ivanov, Andrey; Golovkin, Alexey.
In: Journal of Extracellular Vesicles, Vol. 9, No. 1, 1743139, 2020.Research output: Contribution to journal › Article › peer-review
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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 -
ID: 62766071