Isolation and Characterization of Mesenchymal Stem Cell-Derived Extracellular Vesicles

MSCs were isolated from bone-marrow aspirates of four healthy individuals after informed consent as described before [6 (link)]. Approval of the Ethical Committee of the University Hospital Essen was obtained (12-5295-BO, 17.01.2013 and 25.06.2018). Cell culture supernatants (conditioned media; CM) were collected for EV purification by differential centrifugation and PEG precipitation as recently described [15 (link)]. For details, see supplementary information. MSC-EV fractions were studied (i) by nanoparticle tracking analysis on the ZetaView (Particle Metrix, Meerbusch, Germany) to define size and particle concentration (Table S1) [16 (link)], (ii) by protein assay (Thermo Scientific, Darmstadt, Germany) to define the protein concentration (Table S1) and (iii) by SDS PAGE and western blot to verify the expression of components associated with EVs including TSG101 (Sigma-Aldrich, St. Lois, MO, USA), Syntenin (clone EPR8102; Abcam, Cambridge, UK), CD81 (clone 5A6; Biolegend, San Diego, CA, USA), CD9 (VJ1, kindly provided by Francisco Sánchez-Madrid) and the absence of cytochrome C (clone 6H2.B4; Biolegend) to exclude cellular protein contamination as previously recommended [17 (link),18 (link)] as minimal requirement for definition of EVs (Figure S1).