PROJECT SUMMARY Primary sclerosing cholangitis (PSC) is a chronic inflammatory condition of the biliary epithelium characterized by periductal fibrosis, a heightened risk for cholangiocarcinoma, and frequent comorbidity with inflammatory bowel disease (IBD). The periductal fibrosis observed in PSC narrows the lumen of affected bile ducts and causes extrahepatic cholestasis, which mediates further liver injury. PSC patients typically develop end-stage liver disease within 10-20 years of diagnosis, making PSC a clinically significant cause of liver transplant in the United States. There are currently no disease modifying drugs for PSC, underscoring the unmet need for novel research that characterizes the pathologic mechanisms that drive periductal fibrosis in PSC patients. Aim 1 seeks to dissect the cellular heterogeneity of the periductal niche and characterize cell subpopulations mediating periductal fibrosis in PSC patients using single-cell multiomics. In this aim, the 10X Genomics Next GEM single-cell RNA-sequencing platform will be used to perform integrative analysis of gene expression and chromatin availability in single cells. The gene expression signatures of PSC patient tissue and PBMCs will be compared to patients with metabolic dysfunction-associated steatohepatitis (MASH) and non-cirrhotic controls. Aim 2 seeks to map the spatial organization of cell subpopulations within PSC periductal lesions and reveal cellular interactions within intact tissue. In this aim, the 10X Genomics Xenium in situ Gene Expression Analyzer will be employed to measure the expression of 400 genes in 10µm thick, 12mm x 22mm tissue sections produced from explanted liver tissue. This work will shed new light on the cellular landscape that drives periductal fibrosis, biliary-type cirrhosis, which is often present in patients with advanced PSC, and MASH cirrhosis, which is included as a control. Aim 3 seeks to characterize the effects of a leaky biliary epithelium on macrophage function and collagen production in the periductal niche using an in vitro model. Bile-stimulated macrophages will be characterized at the RNA and protein level and differences in the activating potential of PSC patient bile versus control bile will be assessed. Macrophage-fibroblast co-cultures will also be treated with bile and collagen production will be measured using the Sircol assay. This work utilizes patient-derived bile samples and an in vitro myeloid- stromal co-culture model to resolve luminal, myeloid, and stromal contributions to periductal fibrosis. Mount Sinai Hospital cares for one of the largest and most diverse PSC patient populations in the United States, and the Icahn School of Medicine home to pioneers in the field of hepatic fibrosis research. The training plan for this fellowship takes full advantage of the academic environment at Mount Sinai, and the research strategy combines a single-cell approach with the development of a novel cell culture model to assess cellula...