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

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 highsensitivity 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.

Minimizing isotropic and deviatoric membrane energy – An unifying formation mechanism of different cellular membrane nanovesicle types

“…The composition of vesicles shed from the membrane is determined by the local composition of the budding membrane and the enclosed part of the cell interior, where the key property is the mismatch between the local membrane curvature and the intrinsic curvature of the constituents. As there is a huge number of membrane constituents yet discovered, wide variety of the composition of enclosed adjacent cell interior [62] and different conditions/processing of samples applied [63][64][65][66], the composition and the size of EVs were found to be largely heterogeneous. Our results indicate that the intrinsic curvatures of the constituents are key factors in formation of EVs, therefore the mechanism would be non-specific primarily with respect to the chemical composition of molecules, but specific as regards their shape within the types shown in…”

The Significance of Phenotyping and Quantification of Plasma Extracellular Vesicles Levels Using High-Sensitivity Flow Cytometry during COVID-19 Treatment

“…We performed a previously described and experimentally approved method of extracellular vesicles isolation from the whole blood by differential centrifugation [ 10 ]. Briefly, whole blood samples were centrifuged at 1500 g for 10 min, followed by transfer of plasma to a new tube that was centrifuged twice—first at 1500 g for 10 min and then the obtained supernatant was centrifuged at 3000 g for 10 min.…”

“…Instrument calibration setup was performed using the Cytometry Sub-Micron Particle Size Reference Kit, Molecular probes by Life Technologies), and Megamix-Plus FSC and Megamix-Plus SSC (Biocytex, Marseille, France) containing FITC-labeled reference beads of various diameters. All control samples, detergent effectiveness treatments, and possible false-positive or false-negative controls, were performed in accordance with the previously published requirements [ 7 , 11 ] and as previously described by our group [ 10 , 12 ]. Stained sample dilution was performed in accordance of prevention of coincidence effects, as previously described [ 10 , 12 , 13 ].…”

Regulatory Action of Plasma from Patients with Obesity and Diabetes towards Muscle Cells Differentiation and Bioenergetics Revealed by the C2C12 Cell Model and MicroRNA Analysis

“…MicroRNA quantification was performed as described previously [ 31 ]. Briefly, for the amplification of microRNAs, TaqMan Universal Master Mix II no UNG and the corresponding TaqMan Assay (Thermo Fisher Scientific, Waltham, MA, USA, ) were used as recommended by the manufacturer.…”