Figure 7

GJ-dependent delivery of small RNA from CM influences the transdifferentiation of MSCs. (a) EGFP-labeled MSCs were co-cultured with CMs transfected with DY-547 labeled miRNA. 24 h after co-cultivation, miRNA was also found in MSCs (asterisk), indicating the exchange of small RNAs between these two cell types, Scale bar 20 µm. (b) CM-derived small RNAs induce gene silencing in MSCs. MSCs were transfected with a plasmid coding for EGFP and co-cultured with CMs transfected with anti-EGFP siRNA. Following co-cultivation for 2 days, EGFP expression in MSCs was decreased by 20% when co-cultured with CMs containing anti-EGFP-siRNA. Downregulation of Cx43 in MSCs diminishes the EGFP-reducing effect of CMs. Efficiency of the EGFP/siRNA reporter construct was evaluated by double transfection of MSCs with EGFP and siRNA, resulting in a reduced EGFP intensity of ~50%. n = 3. (c) To verify the impact of CM-derived small RNAs on the transdifferentiation of MSCs, Dicer was down-regulated in CMs. Resulting disruption of the miRNA machinery in CMs impaired the transdifferentiation process in co-cultured MSCs. While NKX2.5 was significantly decreased in MSCs, no effect was observed on the expression level of GATA-4 upon co-cultivation with dicer knockdown CMs, n = 3, 255 cells. Graphs are shown as mean ± SEM. Statistical significance between EGFP fluorescence intensities was analyzed using one-way ANOVA, followed by Dunn’s post hoc test (**P < 0.01, ***P < 0.001). Statistical significance for GATA-4 and NKX2.5 levels was analyzed using two-tailed Student’s t-test (*P ≤ 0.05, ***P ≤ 0.001).