PHD

Autologous adult human skeletal muscle cells have numerous potential cell transplantation applications, including regenerating heart and skeletal muscle, and for therapeutic protein delivery when genetically engineered to express a recombinant protein. Human myoblasts offer a number of advantages over other cell types for implantation therapies, including a readily available cell source, ease of growth in vitro, and strong safety profile when implanted in humans. In addition, postmitotic muscle fibers formed from myoblasts are long lived (decades), have a high protein synthetic capacity, and the postmitotic muscle fibers (myofibers) are nonmigratory, making treatment reversibility possible (an attractive safety feature). But when implanted as either proliferating myoblasts or differentiated muscle fibers, loss of cell viability and/or function occurs. Our project examines the survival and functionality of human muscle cells transplanted as either proliferating myoblasts or differentiated myofibers into an immunodeficient murine model. Primary human myoblasts isolated by needle biopsy from the vastus lateralis muscle of volunteers are bioengineered into either human 'bioartificial muscles' (HBAMs) containing organized postmitotic muscle fibers in a collagen gel or as a disorganized but tensioned muscle cell layer on bioresorbable polymer scaffolds (MBPS). Proliferating myoblasts, HBAM and MBPS are implanted subcutaneously into immunodeficient NOD-SCID mice. Muscle cell survival as well as transcriptional/translational activity is determined over a two month period and correlated with implant vascularization. Development of a functional interface between cell implants and host blood vessels is assessed by immunohistochemistry, laser doppler perfusion imaging, fluorescent microsphere perfusion, and secretion of recombinant proteins into the systemic circulation. Survival and performance of the implanted cells is studied following the local delivery of angiogenic/vasculogenic/anabolic proteins either short term by their release from the scaffold polymer, or long term by genetically engineering the muscle cells with replication deficient viral vectors. It is hypothesized that ex vivo differentiated and genetically 'enhanced' skeletal muscle fibers that are rapidly vascularized will provide the best functional skeletal muscle implant technology. Successful completion of this project will be a step toward the development of improved methods for skeletal muscle cell transplantation for the treatment of numerous endocrine, neuromuscular, and cardiovascular disorders.