Fig. 5

SHED-EVs enhanced Tert-associated telomerase activity and osteogenic functions of human BMMSCs (hBMMSCs). hBMMSCs were incubated with SHED-EVs (EV; 20 μg) or PBS (MOCK) for 3 days. a, b hBMMSCs were incubated with SHED-EVs loaded with SHED-EVs labeled with CFSE and PBS, CSFE-EVs, and MOCK-EVs, respectively, for 3 days. Representative fluorescent images of hBMMSCs are shown. Nuclei were stained with DAPI (a). Representative histograms of hBMMSCs loaded with CFSE-EV and MOCK-EV by FCM analysis. Numbers indicate the means of the positive cells (b). c Expression of human TERT in hBMMSCs by RT-qPCR. d Expression of human TERT in hBMMSCs by Western blot analysis. H3 histone 3. e Telomerase activity in hBMMSCs as detected by RQ-TRAP. 293T, HEK 293T cells; 293T H.I., heat-inactivated HEK 293T cells. f Expression of CD146 and proportion of BrdU⁺ cells in hBMMSCs by FCM and BrdU incorporation analyses. g Representative images of mineralized nodules after Alizarin Red staining 4 weeks after osteogenesis. Percentage of the Alizarin Red⁺ area in the dish area. h Ratio of the expression of RUNX2, BGLAP, and SEMA3A by RT-qPCR 1 week after the induction of osteogenesis. i The expression of osteoblast markers, including RUNX2 and BGLAP by Western blot analysis 1 week after the induction of osteogenesis. ACTB actin, beta. j The expression of SEMA3A in the conditioned medium by ELISA 1 week after the induction of osteogenesis. a–j CFSE-EV, CFSE-pretreated SHED-EV-loaded group; MOCK-EV, PBS-pretreated SHED-EV-loaded group. a, g Bars = 20 μm (a), 10 mm (g). b, c, e–h, jn = 7 for all groups. Comparisons between two groups were analyzed by an independent two-tailed Student’s t test. Multiple group comparisons were performed by a one-way analysis of variance followed by Tukey’s post hoc test.*P < 0.05, **P < 0.01, ***P < 0.005. ns no significance. Graph bars represent mean ± SEM. c, h The results are shown as a ratio to the expression in the MOCK-EV-treated group (MOCK = 1)