The prevalence of bone and articular cartilage injuries is expected to increase with the aging population. As a possible therapeutic option, stem cell-based therapies are being investigated. It has previously been reported that muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle can undergo osteogenic and chondrogenic differentiation in vitro and in vivo when stimulated with bone morphogenetic protein 4 (BMP4). One goal of this project was to determine whether cell sex influences this differentiation potential. Using in vitro osteogenic assays, it was determined that male MDSCs (M-MDSCs) stimulated with BMP4 expressed osteogenic markers and displayed more mineralization than female MDSCs (F-MDSCs). In vivo, M-MDSCs expressing BMP4 and implanted into the hind limb of mice induced a more robust ectopic bone formation when compared to F-MDSCs. These results suggest that cell sex influences the osteogenic differentiation potential of MDSCs. In the second study, the signaling pathways involved during BMP4 stimulation were investigated to further characterize the osteogenic differentiation process. The phosphatidyl inositol 3-kinase and p38 MAPK pathways played a positive role in MDSC osteogenesis, while the extracellular signal-regulated kinase pathway was identified as a negative regulator of osteogenesis. These results suggest that the osteogenic differentiation of MDSCs could be manipulated by regulating these pathways. In the third study, the effect of BMP4 and transforming growth factor-b1 (TGF-b1) on the chondrogenic differentiation of F- and M-MDSCs in vitro was investigated. All MDSCs tested underwent chondrogenic differentiation, with no significant sex-related differences observed. However, addition of TGF-b1 synergistically enhanced BMP4-induced chondrogenic differentiation. In the final study, the effect of mechanical stimulation on the proliferation and osteogenic differentiation of MDSCs was investigated by using both biaxial and uniaxial strain. Mechanical stimulation affected cell orientation, but did not significantly affect the proliferation or osteogenic differentiation of MDSCs. In conclusion, the BMP4-induced osteogenic and chondrogenic differentiation of MDSCs can be influenced by several factors including cell sex and growth factors and can be guided through the manipulation of cell signaling pathways. The results from this project support the continued investigation of MDSCs as a potential cell source for bone and articular cartilage tissue engineering
