Fig 2: Podoplanin-expressing MSCs can protrude through endothelium. Cells cultured on Transwell filters (A–F) or in umbilical cord tissue sections (G–L) were stained for podoplanin, along with markers of ECs (CD31) and MSCs (CD90). (A) Monolayer of CD31-positive ECs in green on the apical surface of the filter for an EC:MSC co-culture filter. Arrows indicate red podoplanin staining on the apical surface of the filter. (B) Monolayer of MSCs expressing CD90 (in blue) and podoplanin (in red), where dual-positive cells are purple, on the basal surface of the filter for an EC:MSC co-culture filter. (C) Image obtained mid-way through the filter for an EC:MSC co-culture. Arrows indicate podoplanin from the basal MSC layer protruding up through the filter. Monolayer of CD31-positive ECs in green on the (D) apical and (E) basal surface of the filter for an EC mono-culture. (F) Image obtained mid-way through the filter for an EC mono-culture. (G) CD90 expression in red around vessels in the umbilical cord. (H) Podoplanin expression in blue around vessels in the umbilical cord. (I) CD31 expression in green around vessels in the umbilical cord. (J) CD31, podoplanin and CD90 expression around vessels in the umbilical cord. The white box represents the area magnified in panel K. (K) Zoomed image of CD31, podoplanin and CD90 expression. White arrows indicate cells positive for CD31 and CD90, but not podoplanin. Yellow arrows indicate cells appearing positive for all three markers. (L) CD31, podoplanin and CD90 expression across the entire umbilical cord section. The white box represents the zoomed in areas for G–J. Representative confocal images, obtained of using Zen Black software, are shown. Scale bars: 20 µm (A–F,K), 50 µm (G–J), 1 mm (L).

Fig 3: Verification of OOM-SCs Characteristics. (A) Microscopic images depict the CFU of OOM-SCs that were visualized with 0.15% crystal violet solution after two weeks of culture. Images of the stained colonies were captured using phase-contrast microscopy. (B) RT-PCR results of the expression of the stemness markers Nanog, Sox2, and Rex1 in comparison with those of WJ-MSCs. GAPDH was used as a housekeeping gene. (C) RT-PCR results of specific markers, including ITGA6, CD146, and TM4SF1, distinguish between multipotent MSCs and NHDFs. These genes are only expressed in multipotent MSCs. (D) RT-PCR data for the expression levels of the surface markers of the isolated OOM-SCs compared with those of WJ-MSCs. These markers included the negatively (CD19 and CD45) and positively (CD73, CD105, and CD166) expressed markers. (E) FACS analysis for confirming the expression of the negative markers CD34 and CD45 and positive markers CD73, CD90, and CD105 for OOM-SCs, compared with those of WJ-MSCs. FACS results are summarized in the table (right panel), and all results are presented in percentages. (F) Microscopic images of adipogenic, chondrogenic, and osteogenic differentiation were evaluated by Oil Red, Alcian Blue, and Alizarin Red S staining, respectively. The upper and lower panels represent undifferentiated and differentiated cells, respectively. Scale bar = 200 µm.

Fig 4: Characterization of 3,2'-DHF-treated WJ-MSCs and EVs. (A) The proliferation rate of WJ-MSC treated with various concentrations of 3,2'-DHF. WJ-MSCs have the best proliferation rate with 1–2 µM 3,2'-DHF. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ** p < 0.05, **** p < 0.0001. (B) Protein expression of MEK/ERK signaling proteins in WJ-MSCs with and without 3,2'-DHF treatment. The bar graph shows the protein expression of AKT and MEK in 3,2'-DHF-treated and untreated WJ-MSCs. (C) Flow cytometry analysis to confirm the characteristics of WJ-MSCs with or without 3,2'-DHF. The expression of the positive markers CD73, CD90, and CD105, and the negative markers CD34 and CD45, was assessed. (D) The relative EV production amounts of Cont-EV and Fla-EV were measured using nanoparticle tracking analysis (NTA). Statistical analysis was determined using two-way ANOVA: **** p < 0.0001. (E) Transmission electron microscopy (TEM) images of Cont-EV and Fla-EV. Scale bar: 100 nm. The size of the EVs was determined with NTA. (F) Characterization of EV-positive markers CD9 and CD63, and negative markers calnexin and GM130, in Cont-EV and Fla-EV using Western blotting. (G) Representative plot of EV-surface markers CD63 and CD81 in Cont-EV and Fla-EV via flow cytometry analysis.