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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: PPARβ/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia–reperfusion injury

Fig. 4

Increased cardioprotection in hearts treated by PPARβ/δ-primed MSC. a C57Bl6 mouse hearts were mounted on a Langendorff system and subjected to IR injury. The ex vivo protocol comprises a 15 min-period of stabilization, followed by 30 min of global ischemia achieved by stopping the flow through the aorta (no-flow). Reperfusion was achieved by restoring the Tyrode perfusion during 60 min (IR group). For the PostC group, a postconditioning stimulus comprising 3 cycles of 1 min ischemia-1 min reperfusion was applied at the onset of reperfusion. In the MSC group, reperfusion was achieved with a solution of MSC cells prepared in a Tyrode buffer at various concentrations (2500; 5000 or 10,000 cells/mL). At the end of the protocol, hearts were dedicated to infarct size (TTC-staining method; b–e) or immunochemistry (f) analysis. b Scatter plot and bars (mean ± SD) were represented for Infarct size in IR (n = 11), PostC (n = 7), MSC 2500 cells/mL (n = 11), MSC 5000 cells/mL (n = 10), and MSC 10,000 cells/mL (n = 10); Statistical analysis was performed using Kruskal–Wallis with the Dunn’s post hoc test for multiple comparison. Statistical significance compared to IR is noted *** for p = 0.0004 (PostC vs IR), ns for p = 0.8021 (MSC 2500 vs IR), *** for p = 0.0004 (MSC 5000 vs IR), ns for p > 0.99 (MSC 10,000 vs IR) and for comparisons vs PostC: ns for p = 0.097 (MSC 2500), ns for p > 0.9999 (MSC 5000), ** for p = 0.011 (MSC 10,000). c Scatter plot and bars (mean ± SD) were represented for Infarct size in IR (n = 11), MSC 2500 cells/mL (n = 12), MSC (2500 cells/mL) + GW0742 0.1 µM (n = 8), MSC (2500 cells/mL) + GW0742 1 µM (n = 7) and MSC (2500 cells/mL). Statistical analysis was performed by Kruskal–Wallis followed by the Dunn’s post-test. Statistical significance versus IR was noted ns for p = 0.0881 (MSC), * for p = 0.0309 (MSC/GW0742 0.1 µM) and **** for p < 0.0001 (MSC/GW0742 1 µM) and versus MSC: ns for p > 0.999 (MSC/GW0742 0.1 µM), * for p = 0.0429 (MSC/GW0742 1 µM). d Scatter plot and bars (mean ± SD) were represented for Infarct size in IR (n = 11), MSC 5000 cells/mL (n = 12) and MSC (2500 cells/mL) /1 µM GW0742 (n = 7). Statistical analysis was performed by Kruskal–Wallis followed by the Dunn’s post test. Statistical significance was noted *** for p = 0.001 (MSC 5000 vs IR), *** for p = 0.0003 (MSC 2500/GW0742 vs IR) and ns for p > 0.9999). e Scatter plot and bars (mean ± SD) were represented for Infarct size in IR (n = 11), MSC 5000 cells/mL (n = 12), MSC (5000 cells/mL) + GSK0660 (n = 11). Statistical analysis was performed by ANOVA (since the normality test passed) followed by the Tukey’s multiple comparisons test. Statistical significance was noted **** for p < 0.0001 (MSC vs IR), ns for p = 0.3467 (GSK0660 vs IR) and * for p = 0.0220 (MSC vs GSK0660). f Left panel: Representative pictures of microscopic observations for an MSC-treated heart section with corresponding enlarged immunostaining images (Original magnification: × 40 oil immersion) showing DI-I labelled (a) MSC and (b) MSC pre-treated with GW0742 1 µM in left ventricle collected 60 min after reperfusion (same time than for infarct size evaluation). Right panel: Scatter plots for the % of fluorescence measured in each slice of MSC versus MSC + GW0742 1 µM injected hearts (n = 3 for each group). g Representative pictures of microscopic observations of immunostaining from MSC-treated LV sections showing MSC (DiI, red labeling), vessels (IsoB4, green), cell nuclei (DAPI, blue) and cardiac actinin staining (anti-actinin antibody, cyan). The last picture represents the merge of the co-immunostaining showing that MSC are detected in microvessels at one hour of reperfusion. Original magnification: × 40 oil immersion; Scale bars are indicated on each picture

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