Therapeutic Potential of Dermal Cells Following Transplantation and In Vivo Myogenic Conversion in Dystrophic Muscle

Thesis (Ph.D.)--University of Washington, 2012Cell-based therapies have the potential to contribute to functional muscle regeneration in Duchenne muscular dystrophy (DMD) and other muscle disorders. Various cell types have been studied for their ability to fulfill this requirement, with reports of high engraftment after transplantation into muscle. However, studies rarely demonstrate improvements in whole muscle function, and very few have characterized cell populations that are amenable to autologous transplantation. To this end, we have characterized the in vitro and in vivo potential of dermal fibroblasts in the mdx4cv mouse model of DMD. Fibroblasts were isolated from transgenic mice carrying a minidystrophin gene, transduced with a lentiviral vector carrying tamoxifen-inducible MyoD, and transplanted into muscles of mdx4cv mice to model ex vivo gene therapy for DMD. Treatment of host mice with tamoxifen drove conversion of transduced fibroblasts into myogenic cells that fused into muscle fibers that subsequently expressed high levels of minidystrophin. Transplantation of various cell doses revealed a limit for reliable engraftment of up to 1 x 106 cells in single injections. In vivo converted dermal fibroblasts engrafted similarly to primary myoblasts, with up to 30% of the host muscle expressing minidystrophin. We found no evidence of fibrosis or structural abnormalities following in vivo conversion of dermal fibroblasts. However, these muscles showed no improvement in force development or protection from contraction-induced injury. We hypothesized that engraftment was below a threshold for improvement of contractile properties in dystrophic muscle, and therefore tested whether a cocktail of pro-survival factors could enhance engraftment. A quantitative PCR-based method allowing rapid screening for these cells in host tissue revealed significant early cell death, and a nearly 3-fold increase in engraftment when cells were injected intramuscularly with the pro-survival cocktail. Histologic analysis showed increased dystrophin-positive fiber number, area engrafted, and spread of engrafted cells with the pro-survival cocktail. Furthermore, engraftment of cells with pro-survival cocktail led to modest but statistically significant protection from contraction-induced injury over mdx4cv controls. The clinical relevance of this strategy lies in the transplantation of autologous cells that can survive and form functional muscle fibers without prior muscle irradiation or toxin treatment in immunocompetent hosts