Figure 3.

MSCs act as reservoirs of MV130 and are able to transfer it to DCs. MSCs were treated for 24 h with MV130 labeled with CFSE (CFSE-MV130; green) for monitoring. After washing cells to remove the drug, its uptake, processing and transference to DCs were studied. (A) Uptake and maintenance of CFSE-MV130. Histograms show the percentage of positive cells and MFI in brackets (n = 6–8 independent experiments). (B, C) Spatiotemporal monitoring of MV130 (green) in MSCs. Immunostaining in red for CD63 (B) or LAMP2 (C) in MV130-MSCs at different times. Inset indicated by arrows in B, scale bar: 10μm (24 h) or 1μm (120 h). Right: Higher magnification of the white square in (C) (120 h); examples of LAMP2-positive compartments with CFSE-MV130. Hoechst was used for nucleus staining (blue). Images are representative of 5 independent experiments. (D–G) DCs differentiated from monocytes were co-cultured with MSCs, previously treated with MV130 or CFSE-MV130 as described in Material & Methods section, and transfer of MV130 from MSCs to DCs was studied. A representative dot plot (D) and the mean ± SEM of five independent experiments (E) are shown. DCs and MSCs were gated according to CD1a or CD90 expression, respectively. In (E) control MSCs and DCs directly treated with CFSE-MV130 are also shown. (F) MV130 transfer from MSCs to DCs studied by immunofluorescence. Hoechst was used for nucleus staining in all cases (blue). Co-cultures were labeled with phalloidin (red) and anti-HLA-DR (magenta). The absence of HLA-DR expression on MSCs allow to distinguish it from DCs (HLA-DR⁺). White arrows in (F) indicate area of insert image magnification. (G) MSCs were labeled with anti-CD63 (red) and Hoechst was used for nucleus staining (blue). Co-localization of CD63⁺ extracellular microvesicles with CFSE-MV130 was observed free in the medium. White arrows indicate area of insert image magnification. Representative images of 3 independent experiments (*p < 0.05 significance by Wilcoxon test).