Engineered microtissue systems for identifying the roles of Wnt and YAP signaling in hepatoblast differentiation and organization.

Liver development requires precise coordination of biochemical and biophysical cues to establish proper zonation and localized cell fate specification. However, there are limited models for studying the interactions between signaling pathways in defined microenvironmental contexts. Here, we employed complementary 2D microarray and 3D microwell platforms to systematically investigate how Wnt and YAP signaling pathways regulate hepatoblast differentiation and influence spatial patterning. siRNA-mediated knockdown of APC enhanced biliary marker expression and activated Notch signaling targets Hey1 and Hes1, while disrupting spatial organization patterns. YAP inhibition predominantly affected hepatocyte specification in 2D but dramatically inhibited biliary differentiation in 3D microtissues, revealing platform-dependent effects. Array culture analyses revealed that decreased cytoplasmic YAP levels, facilitated by YAP knockdown, were associated with a concomitant change in adherens junction protein expression. Collectively, the differential responses between 2D and 3D microtissue platforms are indicative of the context-dependence of intercellular interaction signals, with geometry-dependent effects influencing spatial distribution of differentiated cell types. Combinatorial pathway modulation demonstrated that Wnt and Notch signaling cooperatively regulate biliary fate, while YAP functions as a critical determinant through geometry-specific mechanisms. These findings highlight the application of engineered culture models for investigating the pathways that coordinate biochemical and biophysical signals during liver progenitor cell fate determination.