Fig. 2.
Full characterization of passage 15 (P15) selected clones. A Differentiation assessment of cMSCs and hMSCs using alizarin red S, oil red O, and Alcian blue, respectively, for osteogenic, adipogenic, and chondrogenic differentiation (bar: 100 µm). B Expressions of mesenchymal specific differentiation genes evaluated by RT-qPCR. Data are compared to the undifferentiated state. Each bar represents three individual replicates. Mean ± SD, n = 6. P-values less than 0.05 were considered significant (*P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001). C In vitro immunomodulation of P15 cMSCs and P5 hMSCs on healthy donor PBMCs under allogeneic conditions. Co-culture effect of MSCs on the suppression percentage of PBMCs. PBMCs were stimulated with PHA. Data were normalized with the stimulated PBMCs in the absence of MSCs. Each bar shows proliferation suppression of 1 × 105 CFSE-labeled PBMCs at ratios of 1:2 and 1:10 (MSCs:PBMCs). Mean ± SD, n = 2. P-values less than 0.05 were considered significant (***p < 0.001). D Scratch wound migration assay induced by 48-h secretome of cMSCs (P15) and hMSCs (P3) in resting and primed conditions. Migration was assessed by measuring the scratched area at 0, 12, 24, and 48 h after starting the test on mitomycin C-treated HUVECs. The bar chart shows the quantitative assessment of the wound area. P-values lower than 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001; N.S.: Non-significant). E In vitro angiogenesis stimulated by 48-h secretome of cMSCs (P15) and hMSCs (P3) (Scale bar: 100 µm). The bar chart shows the quantitative assessment of angiogenesis by ImageJ software. Each bar represents three replicates. F Histogram and bar charts for flow cytometry analysis of immunogenicity markers (HLA-DR, CD80, and CD86) in cMSCs, hMSCs, and moDCs (positive control). Data are from three replicates. P-values lower than 0.05 were considered significant (**p < 0.01; ***p < 0.001). G Karyotype of cMSCs (P15) through the G-banding method. cMSCs: Clonal mesenchymal stromal cells, hMSCs: Heterogeneous mesenchymal stromal cells, PBMCs: Peripheral blood mononuclear cells, PHP: Phytohemagglutinin PHA-P, HUVECs: Human umbilical vein endothelial cells, moDCs: Monocyte-derived dendritic cells