Affiliation: School of Medicine, Department of Pathology and Laboratory Medicine
Duchenne muscular dystrophy (DMD) is a debilitating disease that principally affects striated muscles (skeletal and cardiac) and is the most severe form of muscular dystrophy. Disruption of the dystrophin gene is the primary cause of disease leading to excessive muscle damage. Regenerative processes counterbalance damage but individuals with DMD eventually succumb to immobilizing loss of strength and death from cardiac and pulmonary complications in their late teens and twenties. Golden retriever muscular dystrophy (GRMD) is a large animal model with better mimicry of the human disease than mouse models. Its development and characterization are critical to developing therapies for DMD. The cells primarily responsible for the regenerative response in skeletal muscle are satellite cells. These cells have been characterized at the protein level previously with only minor differences found between normal and dystrophic cultures. However, satellite cells have not been characterized at the transcriptional level. Pax7, MyoD, Myogenin and Utrophin act as critical members in the path to myogenesis. In this work, we have looked at the mRNA variation in cells collected from normal and GRMD animals and found substantial differences in mRNA expression profiles. These finding are also reflected in cell fusion experiments done on the same cultures. Studying these proteins and mRNAs in vitro under growth and differentiating conditions can help characterize satellite cells in the GRMD model. To sort through the heterogeneity of satellite cell populations, clonal cultures are needed to better characterize protein and mRNA patterns in these cells. Methods such as limiting dilution or flow cytometry require considerable time and resources to clone and verify large numbers of colonies for analysis. Micropallet array technology is a cell sorting method that permits clonal culture of large numbers of cells in very small spaces. Employing its flexible nature, micropallet array technology has been adapted to culture primary satellite cells from the GRMD model. Using these adaptations, clonal colonies have been cultured and shown to proliferate on tri-partite micropallet arrays. This forms two sister colonies where one sister colony can be analyzed and the other reserved for continued culture and downstream experiments.