Comparison of Rapidly Proliferating, Multipotent Aortic Valve-Derived Stromal Cells and Valve Interstitial Cells in the Human Aortic Valve

Figure 1. Cell morphology comparison between valve-derived stromal cells (VDSCs) and valve interstitial cells (VICs). (a) Phase-contrast images of VDSCs and VICs (passages 1 to 3: P1 to P3), (b) VDSCs and VICs with crystal violet staining, and (c) immunofluorescence staining of cellular actin stress fibers with rhodamine phalloidin and nuclei were stained with DAPI. (d) Quantitation of cell area (μm2) and cell perimeter (μm) comparison between VDSCs and VICs, ∗,#p < 0.05 are accepted as have significant difference, n = 12.

Figure 2. Cell proliferation ability comparison between VICs and VDSCs. (a) Cell proliferation curves show the difference between VDSCs and VICs; ∗p < 0.05 (vs. VICs) are accepted as having significant difference, n = 3. (b) MKI67 immunofluorescent staining, and nuclei are stained with DAPI. (c) The cartogram (based on (b)) shows the differences between passage 2 and passage 3 of VDSCs and VICs; ∗p < 0.05 are accepted as having significant difference, n = 4. (d) FACS analysis for the cell cycle of VDSCs and VICs; (e) S, G0/G1, and G2/M phases were counted and statistically compared. ∗p < 0.05 (vs. VICs) are accepted as having significant difference, n = 4.

Figure 3. Comparative analysis of immunophenotype and differentiation abilities on VDSCs and VICs. (a) Immunofluorescent staining for α-SMA, vimentin, SOX-10, and CD146 of VDSCs and VICs. Nuclei are stained with DAPI; scale bars: 50 μm. (b) Flow cytometric analysis of mesenchymal markers of CD90, CD44, CD29, CD117, CD163, CD133, and CD146 and endothelial markers of CD34, CD31, and CD106 and also hematologic markers of CD11b and CD68 in VDSCs and VICs. (c) Osteogenic, adipogenic, and chondrogenic comparison of the VDSCs and VICs (Alizarin Red S staining for osteogenesis, Oil Red O staining for adipogenesis, hematoxylin staining for the nuclei, and Alcian blue for chondrogenesis); scale bars: 100 μm.

Figure 4. Comparison of global gene expression profiles between VICs and VDSCs. (a) Box plot shows the distribution of gene expression levels in each sample, and the dispersion of the data distribution can be observed; adipose mesenchymal stem cells (AdMSCs) were used for the reference. (b) Heat map for the Pearson correlation coefficient of all gene expression levels between samples; the higher the correlation coefficient indicates, the more similar the gene expression level. (c) Heat map for the global gene expression with group clusters (n = 2). (d) Scatter plot of differentially expressed genes (DEGs) in VDSCs versus VICs (upregulation: 301 and downregulation: 1294); FC (fold change) > 1 was accepted as positive DEGs. (e) GO enrichment of those selected DEGs including the biological process (red), cellular component (blue), and molecular function (yellow); broken line indicates p value (-log10). (f) KEGG pathways enrichment bubble map; a larger p value (-log10) indicates a higher degree of enrichment.

Figure 5. Significant pathways (ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, and cell cycle) were selected for further significant DEG analysis. (a) Heat maps for typical selected functional DEGs based on previous selected pathways, (b) Venn interaction of DEGs, and (c) selected ECM-related DEG fold change in VICs and VDSCs.