Studio in vitro e in vivo di cellule mesenchimali da sangue cordonale e cellule satellite da fibre muscolari scheletriche nella rigenerazione muscolare

Previous works showed that muscle substitutes composed by acellular matrix and myoblasts may represent a promising approach for the treatment of diseases characterized by congenital absence or loss of large areas of skeletal muscle tissue. Here, the regeneration process occurring in vivo within the prosthetic material has been studied verifying the expression, as mRNA and protein, of some skeletal muscle and nerve tissue markers during three month after reconstructive surgery. The experimental evidences indicate that a progressive muscle remodelling within the implants exists and it is not completed after three months. Indeed, the simultaneous presence of markers belonging to both early and late differentiative stages indicates the presence of activated progenitors towards the myogenic line and cellular elements of advanced stages. Moreover, the involving of circulating precursors, probably of marrow origin, in the regeneration process it is suggested by the following observations: i) persistence of poorly differentiated cells at 3 months after surgery, ii) important cell migration from vessels without inflammatory phenomena, and iii) implanted myoblasts, that are cells already committed toward a specific fate, presumably form multinucleated elements in a short time. Finally, there is a rapid appearence of both vascular network and the nervous component. Although these promising results, in view of medical application a problem arise when the function of muscle satellite cells is impaired or their number inside the muscle fibers is low. To overcome this problem, this research has been addressed to the identification of alternative cell sources. For this reason, it has been evaluated both in vitro and in vivo the myogenic potential of mesenchymal cells (MSCs) obtained from umbilical cord blood (UCB). There is the possibility to withdraw the umbilical cord blood at the birth time and to store it in tissue banks, and having an autologous MSC reserve to use for congenital muscular diseases. Evidence for myogenic differentiation was found when MSCs were seeded on Matrigel® coated plates and cultured with myogenic media. After 4 days Myf-5, detected by immunofluorescence, was expressed by 13% of MSCs increasing to 30% at 8 days. We used quantitative PCR for human MyoD and Myogenin mRNAs to further demonstrate myogenic differentiation of MSCs. To evaluate the regenerative capacity of MSCs we used a skeletal muscle chemical injury model (bupivacaine hydrochloride), resulting in necrosis of muscle fibers in Lewis rat Tibialis Anterior. Undifferentiated green fluorescent protein (GFP)-labeled MSCs were injected into the injured muscle, without immunosuppression. The cells engrafted after 1 week and stained positive for Myf-5 and MyoD. After 2 weeks, we noted striations in fibers containing UCB-MSCs suggesting an organization of cytoskeletal proteins into sarcomeres. The skeletal muscle appeared intact by histological analysis, without signs of significant immunologic response, and the presence of MSCs was detected by immunostaining with a monoclonal antibody against human-Nuclei. In addition, these cells also expressed myosin and sarcomeric tropomyosin, a motor-protein and an actin-binding filament protein, respectively. At both time points we observed fibers with centrally located nuclei, indicating regenerated fibers. Finally, the number of GFP-positive fibers/total fibers counted in several fields was 40% for both time points and the area covered by these fibers was 20% of all fibers at 7 days increasing to 26% at 14 days, indicating a maturation of these myofibers. Our in vitro and in vivo data indicate that human MSCs are able to differentiate towards the myogenic lineage and may be efficiently incorporated into injured skeletal muscle, supporting the muscle regenerative process.