LS-10-P-1903 Experimental stem cell therapy of skeletal muscle diseases: role of transplanted bone marrow cells in regenerative myogenesis

Recently discovered ability of the adult bone marrow cells (BMCs), incl. hematopoietic and mesenchymal stem cells, to contribute to injury–induced skeletal muscle regeneration has raised new possibilities in treatment of skeletal muscle diseases. However, mechanisms by which BMCs participate in regenerative myogenesis have still remained to be fully elucidated. To extend our knowledge of experimental stem cell therapy of skeletal muscle diseases and investigate the role of exogenous adult BMCs in the skeletal muscle regeneration, we intravenously transplanted mouse lacZ⁺ or GFP⁺ freshly isolated BMCs into whole-body lethally irradiated immunocompetent mice 7 hours after or 4 weeks before the cardiotoxin-induced injury of the recipients’ tibialis anterior muscles. Seven to 33 days after the toxin injection, injured muscles were excised and fixed in 4% paraformaldehyde, processed for X-gal histochemistry to detect lacZ⁺ cells, embedded into GMA resin (to obtain 1 µm thin sections) or paraffin or fixed in 2% paraformaldehyde, frozen in methylbutane, chilled in liquid nitrogen, cryosectioned and examined for GFP fluorescence. The presence of lacZ gene in injured muscles was determined by qPCR. The skeletal muscles of recipients injured 7 h before the transplantation did not regenerate, nevertheless, X-gal positivity was predominantly identified in desmin^- and nestin^- multinucleated cells resembling foreign body giant cells located in the injured areas, 14 and 33 days after grafting. On the contrary, the recipients’ muscles injured 4 weeks after the transplantation fully regenerated. X-gal or GFP positivity was observed in the regenerating muscles excised 7 days after the injury in numerous inflammatory cells, in some newly formed myoblasts and myotubes and in some cells of the endomysium and in the regenerated muscles examined 28 days after the toxin injection in some endomysial cells and rarely in newly formed myofibers. qPCR verified presence of transplanted lacZ⁺ BMCs in injured recipients’ muscles. Our results confirmed ability of intravenously transplanted exogenous BMCs to settle in the injured skeletal muscle and participate in the skeletal muscle regeneration. They generated blood cells that infiltrated endomysium and took part in the cleaning reaction, the first step of the regeneration process, and moreover, they contributed to new myofibers formation.