Project Summary Alcohol contributes to nearly half the liver related deaths in the United States, mostly from cirrhosis and hepatocellular cancer. Even though large numbers of people misuse alcohol, only a small fraction develops liver disease. A better understanding of the mechanisms that contribute to hepatocyte damage from alcohol is clearly warranted. Mouse models are one tool to further our understanding of the role of alcohol on hepatocytes. However, in part due to species differences and because human genetic predispositions cannot be modeled in mice, ethanol studies in mice have been of limited translational use. An alternative approach to study the effects of alcohol on human hepatocytes are liver chimeric mice. These models are based on the transplantation of primary human hepatocytes into immunodeficient mice with liver injury, after which the human cells proliferate and repopulate the mouse liver parenchyma. There are many limitations with these models, one of them being that only very healthy hepatocytes engraft. Hepatocytes from patients with inflamed livers or cirrhosis generally fail to engraft, which has limited our ability to create disease-specific chimeric models. Possible solutions to this problem would be to genetically manipulate primary hepatocytes or to create chimeric mice with pluripotent stem cells. However, manipulating primary human cells has remained challenging and pluripotent stem cells have largely failed to reconstitute liver chimeric models. We have recently created protocols to efficiently engraft a liver chimeric model with induced pluripotent stem cells or with primary cells that were genetically altered in culture These protocol leads to chimeric animals in which the majority of the liver has been humanized. In addition we can isolate large numbers of human hepatocytes from these mice to create primary human hepatocyte cultures that are stable for months. These advances, combined with a new organoid system, provides us with unique tools to study various hepatocyte responses to alcohol. We here propose to combine these advances to test the effects of alcohol on human hepatocytes. We will use both primary human hepatocytes and pluripotent stem cell-derived hepatocytes to establish systems of alcohol toxicity in hepatocytes, and test a genetic variant that predisposes humans to develop alcoholic hepatitis. In addition the new organoid technology will allow us to test the effects of alcohol on hepatocyte regeneration. At the completion of these studies we will have established systems with which the effects of long- term alcohol toxicity on human hepatocytes can be modeled. These systems will further advance the creation of patient-specific chimeric models for alcoholic liver disease, and may advance the use of stem cell therapies for therapeutic use in patients.