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. 2018 Jun 21;8:9448. doi: 10.1038/s41598-018-27881-5

Figure 2.

Figure 2

Elimination of the epicardium after FG-aided epicardial placement of MSCs. (a) Immunohistostaining for cleaved caspase 3 (c-caspase 3) showed a reduction of apoptosis of donor MSCs (white arrowheads) in the FG-MSC group compared to the IM-MSC group at day 3 after cell therapy. Orange signals in the c-caspase 3 panels were leaked fluorescence from the highly bright CM-DiI (red). Scale bars = 50 µm. *p < 0.05, n = 6 in each group. (b) Immunostaining for Ki67 showed rare proliferation of donor MSCs (white arrowheads) at day 3 after cell therapy in either group. Orange signals in the Ki67 panels were leaked fluorescent signals from the bright CM-DiI (red). Scale bars = 40 µm. n = 6 in each group. (c) Immunohistostaining analysis detected an ICAM-1+ monolayer of the epicardium in the normal (no-MI) heart and the heart of the Sham group (sham treatment in ICM rats). This epicardium disappeared by day 1 after FG-aided, instant epicardial placement of MSCs (labeled with CM-DiI; yellow). CM-DiI is originally red/orange, but CM-DiI-labeled MSCs exhibit yellow color in the figure. This is because CM-DiI-labeling of MSCs were extremely intense, and the strong red fluorescence leaked into the green channel. This was particularly the case when the labelled MSCs densely aggregated. Scale bar = 100 µm. (d) Immunohistostaining analysis detected PECAM1+ (green) and CM-DiI+ (yellow) vessels, suggesting trans-differentiation of donor MSCs to endothelial cells in both FG-MSC and IM-MSC groups (white arrows). As explained above, CM-DiI-labeled MSCs exhibited yellow color, but these yellow cells were distinguishable from the PECAM1+CM-DiI+ cells, which showed a different yellow color. In addition, PECAM1+ (green) but CM-DiI− vessels were observed, indicating host-derived endothelial cells (blue arrows). Scale bar = 100 µm.