Engineering Tools to Promote and Characterize Wnt-Mediated Stem Cell Differentiation

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Stathos, Mark
Kane, Ravi S.
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The Wnt signaling pathway plays an important role in the development of many tissues in the body from the earliest stage of the process. However, the mechanisms of the Wnt pathway and the roles it plays in development remain incompletely understood. This is in part due to the complexity of embryonic development and in part due to the hydrophobicity of Wnt ligands. 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 shown 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 also generated CRISPR/Cas9 edited reporter iPSC lines which are designed to detect the expression of Wnt-regulated marker genes with high specificity. Luminescent signals produced with the help of luciferase secreted by these cell lines during directed differentiation into cardiomyocytes 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.
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