Project Summary Fibrotic liver disease is a growing public health concern significantly impacting the global population. Fibrotic liver disease is often the result of sustained insults; chronic viral hepatitis B and C as well as NAFLD are the most prevalent causes. While antiviral regimens for hepatitis B and C have decreased the viral cirrhosis burden, other causes like NAFLD are increasingly prevalent, now affecting 14-27% of individuals in developed countries. Despite this high disease burden, there are currently no approved therapies. Rather, the lack of consensus on optimal drug targets or strategies reflects a gap in our mechanistic understanding of disease drivers. Similarly, the utility and clinical relevance of animal models is equally controversial. A multitude of models are currently used, but each recapitulates only isolated aspects of human pathophysiology. There is an unmet need for human-relevant systems that recapitulate key elements of fibrotic liver disease to enable mechanistic dissection. The measurable improvement in liver fibrosis in some but not all patients following successful antiviral treatment reinforces the need for better understanding of fibrogenesis at a molecular level to aid development of new treatments to prevent fibrosis or encourage regression. For NAFLD, despite identification of increasing numbers of susceptibility-associated genetic variants and lifestyle-dependent risk factors, the mechanisms by which they individually or synergistically contribute to disease progression remain largely unclear. The primary research goal of this proposal is to exploit a unique renewable and genetically manipulatable human pluripotent stem cell (hPSC)-derived multicellular culture system to address the aforementioned gaps. In this multicellular system, we coculture hPSC-derived hepatocytes, hepatic stellate cells (HSCs), and macrophages in a manner that recapitulates the complexity of liver physiology in both health and disease. Modeling hepatitis virus infection and NAFLD in the multicellular cultures, we found that both HCV infection and a lipotoxic milieu induced inflammatory signals and stellate cell activation. A lipotoxic milieu also triggered other features of NAFLD clinical phenotypes. Eliminating HCV reversed fibrosis-like phenotypes and treating with obeticholic acid showed improvement in NAFLD-like features, as observed in the clinic. Across three aims, using cell and molecular approaches, we capitalize on the unique features of this novel platform to address questions that cannot be adequately answered with any existing ex vivo human-relevant system, including the roles of cytokines in HSC activation (Aim 1), the mechanisms of reversion after activation (Aim 2) and the role of genetics and lifestyle-associated risk factors in NAFLD (Aim 3). Understanding the molecular mechanisms of stellate cell activation and fibrosis development are critical for developing diagnostics and designing new treatments to block ...