Figure 5.

In vivo studies: histological analysis. Panels a through d are related to the confocal microscopy analysis and immunohistochemistry performed on adductor muscles harvested at 14 days from induction of ischemia and the local injection of 2 × 10⁴ hVPCs (left panels) or control culture medium (right panels) (original magnification, ×400). In a, endothelial cells were detected by fluorescence lectin (green) and transplanted human cells by their positivity to human nuclear antigen (hNAg⁺ in red). Nuclei were counterstained by Hoechst (blue). Arrow in left panel indicates an area containing a hNAg⁺ cells belonging to a blood capillary vessel. The same area is shown at ×1000 in the upper right corner. A microvessel negative to hNAg is shown for reference in the right panel. b: Sections stained for α-SMA (red) and hNAg (green). In the left panel, hNAg⁺ cells are incorporated into the arteriolar wall of mouse adductor muscles injected with hVPCs. As expected, the CM-injected muscle (negative control) does not show the presence of human cells. Nuclei are counterstained by Hoechst (blue). c: Sections stained for laminin (a basal lamina marker, red) and hNAg (green). Transplanted hNAg⁺ cells are incorporated in myofibers (left panel). The CM-injected muscle does not show hNAg⁺ cells. Nuclei are counterstained by Hoechst (blue). These figures are representative of three independent experiments. d: Immunohistochemical staining on 3-μm sections using an anti-CD31 antibody (brown, peroxidase). The arrow in the left panel points to a cell associated with muscle microvessels within skeletal muscle, which stained positively to human-specific anti-CD31. As expected, the CM-injected muscle (negative control) does not show the presence of human cells. e: Images of the hindlimb region of mice that had been submitted to left limb ischemia and injected with hVPCs or CM 14 days in advance. f: Real-time PCR was performed with primers specific for MyoD and MYF5 using total RNA from adductor muscles injected with hVPCs, differentiated hVPCs, or control. MYF5 is approximately 80-fold up-regulated in the hVPC-transplanted muscles with respect to differentiated hVPC-injected muscles. Error bars represent SDs calculated on three different replicas. g: Distribution of necrotic toes in the left foot of mice that were exposed to ipsilateral ischemia. Zero means absence of necrotic toes, and 5 indicates all toes being necrotic or absent. One point was scored for every necrotic toe. Each circle is representative of a single mouse. Colored transverse lines represent the median for each group. Blue, CM-injected mice; green, EPCs; yellow, differentiated hVPCs; red, hVPCs.