UC Riverside

Because of their pluripotent nature, as defined by their self-renewal and differentiation capacity, mouse embryonic stem cells (mESCs) have become an invaluable resource for unraveling the underlying mechanisms that control developmental programs. Understanding the formation and maintenance of bone tissue will undoubtedly facilitate the creation of new therapeutic approaches that address bone-related pathologies. Numerous reports have demonstrated the importance of the Wnt signaling pathway in regulating osteogenesis, however, given the revealed complexity of this regulation further elucidation of the time- and cell-specific response is needed. The in vitro culture of mESCs allows investigators to examine multiple stages of differentiation while simultaneously manipulating this signaling pathway in a time-dependent manner. Through the use of known modulators and activity reporters, studying beta-catenin (CTNNB1) (i.e. the central effector of canonical Wnt signaling) will provide invaluable insights towards a greater understanding of osteogenic processes. For instance, we have found that supplementation of the non-canonical Wnt ligand Wnt5a, during a narrow time window of osteoblast precursor formation, was able to improve osteogenic differentiation at later stages. This supplementation resulted in increased activation of multiple downstream effectors and was also correlated with a decrease in CTNNB1-dependent LEF/TCF transcriptional activity. Interestingly, when studying the impact of high concentrations of glucose within culture media, we found a decrease in osteogenic differentiation, which corresponded to an upregulation of CTNNB1-dependent LEF/TCF reporter activity within the same time period. This change in transcriptional activity was linked to differential activation of the AKT/FOXO signaling pathway. Thus, our work continues to reinforce the dynamic role of CTNNB1 in regulating in vitro osteogenesis. With this knowledge we employed multi-stage supplementation of either canonical or non-canonical Wnt ligands to direct osteogenic differentiation of mESCs in serum-free cultures. We found that these ligands, or corresponding chemical modulators, could yield measures of osteogenic output that was comparable to cultures containing fetal bovine serum. With these advancements in our understanding of the role of CTNNB1 in both developmental osteogenesis and the dysregulated processes underlying bone-related pathologies, novel strategies can be developed that improve differentiation protocols and therapeutic applications.