SUMMARY

The Wnt signaling pathway plays an important role in the development of many tissues in the body, notably cardiac tissue, from the very earliest stage of the process. However, the precise mechanisms of the Wnt pathway and the specific roles it has in development in the context of different tissue types remain poorly understood. This is in part due to the complexity of embryonic development and in part due to the hydrophobicity of Wnt ligands which renders them expensive and difficult to purify in a usable form. To overcome issues associated with the use of natural Wnt ligands, we have developed a heterodimer of Fabs which bind to the Wnt co-receptors LRP6 and Frizzled. We have demonstrated that this dimer can activate Wnt signaling with an efficacy comparable to that of the natural ligand. To elucidate the mechanisms of downstream events in the Wnt pathway, we constructed a kinetic model consisting of a system of ordinary differential equations. We fit this model to empirical time course data derived from Western blots of HEK293T cells treated with Wnt. From this fit we were able to gain insights into how the intracellular levels of the Wnt pathway component β-catenin are regulated. To better characterize the downstream effects of Wnt signaling during the manufacturing of therapeutic cells, we are also generating CRISPR/Cas9 edited reporter iPSC lines which we hope will be able to detect the expression of Wnt-regulated marker genes such as Brachyury and COUP-TFII with high specificity. Luminescent signals secreted by these cell lines during directed differentiation into cardiomyocytes will permit continuous non-destructive monitoring of the manufacturing process. These cell lines could potentially guide process optimization and enable production of cardiomyocytes with a more mature phenotype. Due to the short notice of this announcement, a recording of the presentation will be made available upon request.