Figure 5

Survival and engraftment of mesoangioblasts in a dystrophic mouse model. Shown are different time-point samples (1, 3, and 5 weeks, respectively) of the dystrophic tibialis anterior (TA) muscles from 12-month-old α-sarcoglycan null mice treated (n = 18 per group) with intra-muscular injections of nuclear (n)lacZ mesoangioblasts in PBS (A-C) or polyethylene glycol-fibrinogen (PF) (E-G). X-Gal staining is shown in blue and laminin immunostaining in red. Histological analysis showed a higher number of lacZ+ cells in the TA muscle treated with the PF mesoangioblasts (E-G) compared with the PBS mesoangioblasts (A-C). High magnification of X-Gal and laminin staining reveals an amelioration of the muscle morphology, showing the localization of lacZ-positive nuclei at the periphery of the host’s regenerating muscle fibers (arrow) for the TA injected with the PF mesoangioblasts (H), whereas donor nuclei are mainly located in the extracellular matrix (arrow) in the TA treated with PBS mesoangioblasts (D). Quantitative analysis of the total number of nlacZ+ nuclei on X-Gal/laminin-stained TA sections reveals higher mesoangioblast engraftment at each time point in the TA muscles treated with PF mesoangioblasts, (I) and ameliorated integration of PF mesoangioblasts into host regenerated myofibers (J). The number of mesoangioblasts in PBS-injected TA (black bars) and PF-injected TA (white bars) was documented at 1, 3, and 5 weeks after treatment (*P<0.05 by ANOVA test). Counting analysis was performed by scoring lacZ-positive labeled cells under a phase-contrast microscope (× 40) in five randomly selected fields of different non-adjacent sections for three mice per group. (K) The representative western blots for lacZ in total protein extracts from three different treated dystrophic TA muscles (n = 5, one representative shown in the figure) show the progressive increase of lacZ expression in the TA muscle treated with PF mesoangioblasts compared with the samples treated with PBS mesoangioblasts. (L) Densitometric analysis of the lacZ/GAPDH ratio from five different western blots confirms the histological data analysis, and documents a steady increase in lacZ protein as a function of engraftment time; the influence of the PF carrier on survival and integration of nlacZ-mesoangioblasts is also evident (*P<0.05 by ANOVA test). Scale bar: (A, B, C, E, F, G) 500 μm, (D, H) 20 μm.