Project Summary The bipotential differentiation of liver progenitor cells to hepatocytes and biliary epithelial cells (cholangiocytes) is integral to liver development, regeneration, and diseases including bile duct paucity and liver cancer. In particular, the most common congenital liver diseases are associated with bile duct dysfunction. In addition to the developmental processes during embryogenesis, duct morphogenesis also occurs in the adult liver in response to severe and chronic injury. These so-called ductular reactions exhibit highly variable differentiation patterns, and although these reactions significantly contribute to the proliferative responses in the liver, they remain poorly characterized. Notably, ductular proliferations in the adult liver are concentrated near the portal vein region, similar to the formation of bile ducts during development. Despite substantial research efforts, the structural complexity and dynamic nature of liver development and regeneration has limited the comprehensive understanding of disease mechanisms as well as the advancement of new therapeutic options. The long-term goal of this project is to develop complementary two-dimensional and three-dimensional engineered tissue platforms that can be applied towards the investigation of liver progenitor cell differentiation mechanisms that are presently inaccessible with current cell culture systems and animal models. Towards this end, we will pursue the following research objectives, which are specifically targeted towards deconstructing the combined influence of biochemical and biomechanical signals in liver progenitor cell fate specification. In Aim 1, we will utilize a cell microarray platform to investigate the influence of spatial gradients of Notch signaling and cell mechanical stresses in progenitor cell differentiation. Our approach will enable the independent control of cell- cell interactions, defined by multicellular geometry, and specific exogenous microenvironmental signals presented within the array platform. In Aim 2, we will develop and utilize a three-dimensional microtissue culture platform to systematically investigate the effects of three-dimensional geometry and determine how distinct multicellular geometries regulate differentiation patterns. These research efforts will establish microscale tissue engineering tools that enable the controlled presentation and systematic perturbation of a range of microenvironmental signals. In Aim 3, we will extend our studies towards the direct analysis of human liver differentiation mechanisms through the integration of human induced pluripotent stem (iPS) cell-derived liver progenitor cells. Collectively, our approach will allow for novel studies into the mechanisms of liver progenitor cell differentiation, including the unique examination of the combinatorial influence of cell mechanical stress gradients and Notch signaling. Further, we envision that these platforms are generalizable, and could be imp...