ABSTRACT / PROJECT SUMMARY Non-alcoholic steatohepatitis (NASH) represents a rapidly growing epidemic of liver disease that can predispose affected individuals to advanced fibrosis and hepatocellular carcinoma. Strategies for targeting cellular mediators of liver fibrosis, such as activated hepatic stellate cells (HSCs), remain limited due to an incomplete understanding of the mechanisms governing myofibroblastic differentiation and the signaling between liver parenchymal and non-parenchymal cell (NPC) types. The importance of microenvironmental signal crosstalk, including interactions between extracellular matrix (ECM) protein composition and mechanical stiffness in cell phenotypic alterations, is increasingly appreciated. Although animal models have provided insights into NASH, significant differences across species in drug metabolism and disease pathways exist. Thus, there is a need for human-relevant in vitro approaches that enable the investigation of hepatocellular phenotypes within physiological and NASH-like microenvironments and could facilitate the high-throughput discovery of novel therapeutics. The goal of this project is to implement engineered culture platforms for selectively modulating microenvironmental signals and utilize these systems to reveal key phenotypic programming pathways and interaction mechanisms within the context of a NASH-like microenvironment. Our approach will enable hypothesis-driven studies incorporating controlled perturbations of extracellular signals. In Aim 1, we will investigate the microenvironmental regulation of myofibroblastic phenotype. Utilizing defined ECM compositions and mechanical stiffness regimes in engineered cultures, we will examine the role of epigenetic gene regulatory mechanisms and test the hypothesis that combinatorial microenvironmental cues regulate epigenetic changes that are critical for the myofibroblastic programming of human hepatic stellate cells within the context of normal and NASH-like soluble triggers. In Aim 2, we will examine the influence of ECM composition and stiffness on Kupffer cell (KC) and primary human hepatocyte (PHH) functions including reciprocal intercellular interactions and cooperative effects of NASH-like soluble stimuli. Our approach will facilitate modular control and deconvolution of the effects of ECM composition, substrate stiffness, and soluble signals. In Aim 3, we will develop and implement multicellular 3D liver microtissues for the systematic analysis of multicellular phenotype regulation in the context of disease-like ECM alterations. We will investigate heterotypic cellular crosstalk mechanisms between non-parenchymal cell types (HSC, KC, and liver sinusoidal endothelial cells), and their collective influence on primary human hepatocyte functions. We will further establish capabilities for assessing NASH-relevant therapeutics within the multicellular liver platform. The proposed studies will provide a) fundamental insights into the microenvironme...