Using Molecular, Cellular and Bioengineering Approaches Towards Understanding Muscle Stem Cell Biology

Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function.