Wnt signaling pathway inhibitor promotes mesenchymal stem cells differentiation into cardiac progenitor cells in vitro and improves cardiomyopathy in vivo

Figure 1 Isolation, propagation, and characterization of human umbilical cord-derived mesenchymal stem cells. A: Explant processing and isolation of human umbilical cord-derived mesenchymal stem cells (MSCs). Phase contrast images showing homogenous fibroblast like morphology and growth; B: Characterization of MSCs by immunocytochemistry representing the positive expression of CD117, CD29, Lin28, CD105, Vimentin, and negative expression of CD45; C: Immunophenotyping of MSCs by flow cytometry showing positive expression of CD105, Vimentin, and CD73, and negative expression of CD45; D: Tri-lineages differentiation of MSCs into osteogenic, adipogenic, and chondrogenic lineages.

Figure 2 Cytotoxicity analysis of mesenchymal stem cells treated with different concentrations of inhibitor Wnt production-4 by JC-1 assay. A: Mesenchymal stem cells (MSCs) treated with 5 μM concentration of inhibitor Wnt production-4 showed non-significant number of apoptotic cells, while 10 μM concentration or above showed significant cytotoxic effect on MSCs; B: Quantification of apoptotic and non-apoptotic cells presented in bar graphs. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Values are expressed as means ± SEM; level of significance is P < 0.05 (^aP < 0.05, ^cP < 0.001).

Figure 3 Cardiac markers gene expression analysis by quantitative real-time polymerase chain reaction. A: Seven days treatment of mesenchymal stem cells (MSCs) with inhibitor Wnt production-4 (IWP-4); B: Fourteen days treatment of MSCs with IWP-4 in comparison with untreated control showing significant increase in the expression of early cardiac markers, GATA-4, Nkx2.5, and late cardiac markers, MHC-β, MLC-2v, Mef-2D, cTnT, cTnC, cTnI, α-actinin, Ca-channel and Na-channel. Quantitative two fold (2^-ΔΔCT) difference of mean is represented by ΔΔCt method. Statistical analysis was performed using an Independent sample t-test. Values are presented as mean ± SEM from three independent biological triplicates; level of significance is P < 0.05 (^aP < 0.05, ^bP < 0.01, and ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; NS: No significant.

Figure 4 Wnt pathway gene expression analysis: Wnt pathway gene expression analysis by quantitative real-time polymerase chain reaction. Seven days, and fourteen days treatment of mesenchymal stem cells with inhibitor Wnt production-4, in comparison with untreated control showing significant decrease in the expression of Wnt pathway genes, DVL, Wnt, TCF, Axin, and β-catenin, and downstream transcription factors C-jun, C-myc, and Cyc-D. A: Seven days; B: Fourteen days. GSK increased in the fourteen days treatment. Quantitative two fold (2^-ΔΔCT) difference of mean is represented by ΔΔCt method. Statistical analysis was performed using an Independent sample t-test. Values are presented as mean ± SEM from three independent biological triplicates; level of significance is P < 0.05 (^aP < 0.05, ^bP < 0.01, and ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; NS: No significant.

Figure 5 Analysis of fourteen days inhibitor Wnt production-4 treated mesenchymal stem cells. A: Morphology of untreated mesenchymal stem cells (MSCs), seven days inhibitor Wnt production-4 (IWP-4) treated MSCs and fourteen days IWP-4 treated MSCs; B: Immunocytochemical analysis of untreated MSCs, seven days and fourteen days IWP-4 treated MSCs showing positive expression of cardiac-specific proteins α-actinin, connexin-43, cTnI, Desmin, GATA-4, Nkx2.5, secondary control, and DAPI control. Alexa fluor 488 secondary antibody was used for detection, then counterstained with DAPI to stain the nuclei; C: Quantification of fluorescence intensities in untreated MSCs, seven days, and fourteen days IWP-4 treated MSCs. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Data are presented as mean ± SEM with significance level P < 0.05 (where ^aP < 0.05, ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; NS: No significant.

Figure 6 Cardiac functional analysis by echocardiography. A: Ultrasound images taken at parasternal long axis showing B and M mode scans of left ventricle in sham control, myocardial infarction (MI+) group, mesenchymal stem cells (MI+MSC) treated group, and inhibitor Wnt production-4 (IWP-4) (MI+IWP-4) treated groups; B: Bar graphs representing cardiac functional analysis in terms of left ventricular systolic internal dimensions, left ventricular diastolic internal dimensions, end-systolic volume and end-diastolic volume, ejection fraction and fractional shortening, after 2 and 4 wk of MI model development. Both untreated and IWP-4 treated MSC groups showed cardiac functional improvement. However, IWP-4 treated group exhibited more significant results. Statistical analysis was performed using One-way ANOVA followed by Bonferroni post-hoc test. Data are presented as mean ± SEM with significance level P < 0.05 (where ^bP < 0.01; ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; MI: Myocardial infarction; LVIDs: Left ventricular systolic internal dimensions; LVIDd: Left ventricular diastolic internal dimensions.

Figure 7 Histological analysis of heart sections. A: Large scan images showing Masson’s trichrome stained transverse section of the whole heart; B: Magnified images showing Masson’s trichrome stained sections in sham control, myocardial infarction (MI+), mesenchymal stem cells (MSCs) (MI+MSCs), and inhibitor Wnt production-4 (IWP-4) treated MSCs (MI+IWP-4) groups after 4 wk of MI; C: Images showing isolated rat hearts of sham control, MI+ group, MI+MSC group, and MI+IWP-4 groups; D: Bar graph showing percent infarcted area as compared to MI+ group. Percent infarcted area was significantly decreased in MI+MSC and MI+IWP-4 groups after 4 wk of MI; E: Bar graph showing left ventricular wall thickness as compared to MI+ group; left ventricular wall thickness was non-significant in MI+MSC group and significantly increased in the MI+IWP-4 group after 4 wk of MI. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Data is presented as mean ± SEM with significance level P < 0.05 where (^bP < 0.01, ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; MI: Myocardial infarction; NS: No significant.

Figure 8 Hematoxylin-Eosin staining of heart section. Large scan images showing Hematoxylin-Eosin (H&E) stained transverse section of the whole heart. Magnified images showing H&E stained sections in sham control, myocardial infarction (MI) group, mesenchymal stem cells (MSCs) (MI+MSC) group, and inhibitor Wnt production-4 (IWP-4) treated MSCs (MI+IWP-4) group after 4 wk of MI. IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; MI: Myocardial infarction.

Figure 9 Immunohistochemistry of heart tissue section. A: Immunohistochemical images of heart sections showing the transplanted untreated mesenchymal stem cells (MSCs) and fourteen days inhibitor Wnt production-4 (IWP-4) treated MSCs labeled with red fluorescent DiI dye. Cardiac specific proteins α-actinin, cTnI, and GATA-4 were immunostained for the expression of cardiac proteins. Alexa fluor 488 secondary antibody was used for detection; B: Quantification of the fluorescence intensities of the DiI labeled cells presented in bar graphs. As compared to normal MSCs, fluorescence intensity was significantly increased in case of alpha actinin, GATA-4, and cTnI in the fourteen days IWP-4 treated MSCs group in the infarcted myocardium. For statistical analysis, One-way ANOVA was used followed by the Bonferroni post-hoc test. Data are presented as mean ± SEM with significance level P < 0.05 where (^bP < 0.01, ^cP < 0.001). IWP-4: Inhibitor Wnt production-4; MSC: Mesenchymal stem cell; MI: Myocardial infarction.