Project Summary HBV and HCV infections are among the leading causes of chronic liver disease worldwide. In the United States, it is estimated that over 850 thousand people are currently infected with HBV and more than 2.4 million for HCV. Despite the availability of highly effective HBV vaccine and HCV treatment, the mortality and the burden associated with HBV and HCV are nevertheless increasing as individuals with existing infections advance into more advanced stages including fibrosis, cirrhosis, and HCC. Moreover, HCV-infected patients, even though cured of viremia, remain at a significantly elevated risk for advanced liver diseases. Due to their shared modes of transmission and epidemiological features, HBV and HCV frequently coexist in patients in endemic areas or among subjects at high risk of infection. Coinfection is usually more complex than monoinfection, leading to an accelerated disease progression and complicated viral interaction for their treatment. Despite the severity of HBV and HCV infections, the mechanisms by which they lead to liver disease, of how coinfection causes an increased severity and risk for complications, and of how HBV and HCV interact in the liver of coinfected patients that may lead to reactivation of HBV upon the cure of HCV remains largely unclear. This stems, in part, from the lack of relevant human models that recapitulate key disease phenotypes and permit detailed mechanistic studies that could answer these questions. The primary research goal of this proposal is to establish a novel, high fidelity multicellular in vitro liver model to address the aforementioned gaps. In this system, we plan to coculture human pluripotent stem cells (hPSCs)-derived hepatocytes, hepatic stellate cells, macrophages, and endothelial cells. Unlike existing traditional cell culture models, we propose to create an innovative three dimensional platform that mimics the liver’s multicellularity and microenvironment, by culturing these four cell types in a configuration that resembles the liver’s in vivo spatial organization, its hemodynamics, and its cell-cell interaction. To distinguish cell type- specific responses, we will develop methods to purify individual cell types for downstream analysis. With this multicellular culture platform, we will pursue our long-range objectives, across which we capitalize on the unique features of this novel platform to address questions that cannot be adequately answered with any existing in vitro human-relevant system. These include the mechanisms of continued disease progression following the cure of HCV infection, an accelerated disease progression with HBV/HCV coinfection, and the interaction between HBV/HCV that leads to HBV reactivation following the cure of HCV infection (Aim 1). In Aim 2, we will combine hPSCs with gene editing to understand how genetic variants affect the course of disease and treatment following HBV and HCV infection. These studies will not only provide new insights ...