Shh and Dll-4 together with hMAPCs additively increase formation of arterial-like vascular structures in vivo. (A-F) Histologic analysis on cross-sections through Matrigel plugs containing hMAPCs and VEFG₁₆₅ (A,D) or hMAPCs and VEGF₁₆₅ + Shh + Dll-4 (B,E). (A-B) Double immunofluorescent staining of 3 μm paraffin cross-sections through Matrigel plugs stained with SMC α-actin (red) and BS-I lectin (staining ECs in green) showing more SMC-coated (indicated by arrowheads) vessels when the arterial media were used (B) in comparison with standard media (A). (C) Diagram comparing the fraction of SMC-coated vessels (expressed as percentage ± SEM versus the total number of vessels) for the conditions outlined in the box; *P < .05; **P < .01. Note the additive effect of hMAPCs. (D-E) Sirius red staining (visualized by polarized light microscopy) indicating abundant and thick (orange-red birefringent) fibrillar collagen around vessels in Matrigels containing hMAPCs combined with the arterial mix (E) as compared with the less abundant and thinner collagen in Matrigels containing hMAPCs combined with VEGF₁₆₅ alone (D); dashed lines indicate the edge of the Matrigel (M). (F) Diagram comparing the collagen fractional area (expressed as percentage ± SEM versus the total area) for the conditions outlined in the legend; *P < .05; **P < .01. Note the additive effect of hMAPCs. (G,H) ELISA for PDGF-BB (G) and active TGF-β1 (H) on cell supernatants of undifferentiated hMAPCs (“baseline”) or differentiated for 7 or 14 days to ECs either in standard media (gray bars) or arterial media (green bars). While PDGF-BB production was only slightly and temporarily higher, active TGF-β1 production was significantly higher in arterial media versus standard media. *P < .05; **P < .01. Data are expressed as picogram per 10⁵ cells and represent the mean (± SEM) of 3 experiments performed in triplicate.