Importance of Transplantation Variables in Marrow to Muscle Conversion.

We have previously shown that conversion of marrow to skeletal muscle is increased by cardiotoxin injury and we have demonstrated levels of up to 12% conversion. We have now used the same model of cardiotoxin injury to determine variables that affects this conversion. Muscle fibers were defined by their morphology and presence of desmin and dystrophin staining and absence of CD45 staining. In a GFP to B6 transplant model, there was a significant increase in the incidence of muscle chimersim with increasing the dose of marrow donor cells unrelated to the level of hematopoietic chimersim. Radiation was a critical factor and a combination of 500cGy of whole body and 500cGy of lower extremity radiation was found as the optimal model. However radiation impact was mostly due to its effect on hematopoietic chimerism and there was very little local effect on muscle. Higher doses of radiation were detrimental and resulted in significant muscle atrophy. Transplantation of marrow cells to NOD/Scid mice without radiation resulted in muscle engraftment although this was a rare event. Furthermore, timing of the cardiotoxin injury relative to the transplantation was an important factor since injection of cardiotoxin before or right after transplant resulted in minimal conversion while injury seven days or more after transplant resulted in significant conversion events. When sublethal doses of cardiotoxin were injected systemically after marrow transplant GFP positive muscle fibers were seen but the incidence was much lower than direct intramuscular injections of cardiotoxin. Multiple cycles of mobilization and injury also improved the incidence of GFP positive fibers in the regenerated muscle. We conclude that in addition to injury, cell mobilization and cell selection, improvements in the efficiency of this process can be achieved by applying specific doses and distribution of radiation (i.e. local and systemic), by giving the appropriate doses of marrow cells and by selecting the appropriate time for injury. Combining these models gives quantitatively significant fiber conversions approaching levels that would be therapeutically effective in the treatment of muscular dystrophies.