FIG. 1.

Incorporation of MSC-EVs and RNA transfer in proximal tubular epithelial cells (PTECs). (A) MSCs were double-stained in red (with Vybrant Dil, 15-min incubation) and green (with Syto-RNA, 30-min incubation). Original magnification: ×200. Labeled MSCs released double-labeled EVs (see “Materials and Methods” section). (B) Double-labeled MSC-EVs were incubated for 6, 12, and 24 h with PTECs in normal condition and after ATP depletion injury. The first column of panels from the left shows the internalization of MSC-EV membranes. The second column of panels is the nuclei of PTECs stained with DAPI (blue). The third column of panels shows the distribution of Syto-RNA carried by MSC-EVs inside PTECs. The fourth column of panels shows a merge between the two previous panels. These experiments were realized in normal culture condition. (C) MSC-EV incorporation in PTECs after 24 h of incubation in normal culture condition and after ATP depletion injury. The panel description is the same as indicated above. Three experiments were performed with similar results using MSC-EVs derived from different MSCs. Original magnification: ×630. (D) FACS analysis of Vybrant Dil-labeled MSC-EV incorporation rate by PTECs. White bars represent the experiments realized in normal control conditions and black bars represent incorporation rate by PTECs after ATP depletion injury. Statistical analysis was performed by ANOVA with Newman-Keuls multicomparison test: *,**statistical difference between the 6- and 24-h experimental conditions; ^#statistical difference between normal and injury conditions, in the same incubation time (P<0.05; n=4). EV, extracellular vesicle; MSCs, mesenchymal stromal cells; FACS, fluorescence activated cell sorting.