Multiple Insights from Myogenic Cell Transplants

This single image (Fig. 1) illustrates several issues of myoblast transplantation that are important to interpretation of cell transfer therapy for primary myopathies but are not discernible in standard histological preparations.

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FIG. 1.

This muscle fiber with multiple branches was extracted from the tibialis anterior muscle of a 43-day-old mdx nude mouse whose hind limbs had been irradiated at 21 days of age with 18 Gy to disable the endogenous satellite cells and then transplanted 3 days later with 5×10⁵ wild-type myogenic cells that had been transduced with a retrovirus expressing nuclear-localizing β-galactosidase (pMFG NL LacZ). Three weeks after grafting, the mouse was euthanized, and the tibialis anterior muscle was removed and digested with type 1 collagenase to release the individual muscle fibers. These were fixed briefly with 2% paraformaldehyde in phosphate-buffered saline and stained for β-galactosidase (monoclonal anti-β-galactosidase and fluorescein isothiocyanate–conjugated goat anti-mouse secondary antibody) and costained for dystrophin (P6 polyclonal followed by biotinylated sheep anti-rabbit and Texas red–streptavidin) with 4′,6-diamidino-2-phenylindole counterstain.

First, it provides evidence on the debate as to the origin of muscle “splitting” or “branching” (Hall-Craggs, 1972), showing that this is generated in this instance by misalignment of newly formed myotubes to the stump of the damaged muscle fiber. Immunostaining for β-galactosidase (in green) shows that the branches that had formed over the 3-week period are derived from donor cells, that is, they had formed as a result of regeneration. This was true of all branches in fibers extracted from these muscles (Blaveri et al., 1999), supporting the idea that branching is a result of inaccurate regeneration rather than pulling apart of fiber tips by physical forces into separate projections. So complex a pattern of repair implies, in accord with other studies (Morgan et al., 1990), that the donor satellite cells had fused to form several individual myotubes and that each had fused independently to the end of the surviving segment of the dystrophic muscle fiber.

Second, the myonuclei in these new-formed fibers lie predominantly in peripheral positions, not in the centrally placed strings that are the norm in regenerated mouse muscle. This peripheral localization of newly formed muscle nuclei does not occur in the mouse unless the endogenous satellite cell population is inactivated by high-dose preirradiation. Thus resident satellite cells impose a dominant effect on the positioning of nuclei of transplanted cells, reinforcing the idea that the minority of myoblasts that survive transplantation constitute a stem cell-like subpopulation that differs in a number of respects from the resident satellite cell (Beauchamp et al., 1999; Boldrin et al., 2012).

Third, the red immunostaining shows that the dystrophin produced by myonuclei within the branches undergoes only limited longitudinal diffusion from the myonuclei that encode it, penetrating the surviving stump of the repaired muscle fiber by only a few tens of microns. Thus, transplants of genetically normal or corrected myogenic cells are likely to have limited benefit outside of the immediate region of their integration into each dystrophic muscle fiber, emphasizing the goal of diffuse integration of therapeutic myogenic cells for inherited myopathies.

To gather such a detailed picture from serial histological sections of such a region would be difficult and arduous.