Figure 6

Gap junctional coupling with CMs promotes the cardiogenic differentiation of MSCs. (a,b) Immunofluorescence labeling of cardiac specific markers revealed the transdifferentiation of MSCs into a cardiac-like phenotype. MSCs co-cultured with CMs showed expression of GATA-4 and NKX2.5, two early cardiac specific transcription factors. (c) MSCs were found to express α-actinin, indicating the formation of pre-sarcomeric structures. (d) Quantitative assessment of NKX2.5 and GATA-4 expression by fluorescence microscopy demonstrated a marked increase when MSCs were co-cultivated with CMs, n = 3, 175 cells, note the logarithmic scale. (e) qRT-PCR confirmed the pronounced up-regulation of NKX2.5 and GATA-4 induced by co-culture with CMs, compared to MSC mono-culture, n = 3. (f) The establishment of cell-cell contacts between MSCs and CMs resulted in a significant higher protein level of GATA-4 and NKX2.5 compared to MSCs that share the same medium but lack direct cell-cell interaction with CMs, n = 3, 148 cells, note the logarithmic scale. (g) Downregulation of Cx43 in co-cultured MSCs reduced the expression levels of both transcription factors, indicating a promoting role of GJs in the transdifferentiation process of MSCs induced by adjacent CMs, n = 5, 409 cells. Scale Bars 20 µm, Graphs are shown as mean ± SEM. Statistical significance was analyzed using two-tailed Student’s t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).