Abstract Liver fibrosis is a pathological condition that results from extracellular matrix (ECM) accumulation in response to chronic liver injury and is a major global cause of death in adults (~1M per year) due to inadequate therapeutic options. To address this limitation, we have developed human hepatic organoid models that enable hypothesis- driven, mechanistic evaluation of novel drug candidates for treatment of liver fibrosis. One model is produced by engineering iPSC to express a common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). ARPKD organoids develop the key hallmarks of hepatic fibrosis: they accumulate thick collagen fibers; and have a marked increase in collagen-producing myofibroblasts whose transcriptomic profile is like those present in liver tissues obtained from patients with commonly occurring (acquired) forms of liver fibrosis (viral- induced cirrhosis and advanced non-alcoholic steatohepatitis, NASH). We also developed a NASH organoid fibrosis model; along with two live cell imaging methods for monitoring for the appearance of collagen fibers and collagen producing cells. We hypothesize that since the fibrosis that develops in this human, multi-lineage, hepatic organoid resembles that in patients with congenital and acquired forms of liver fibrosis, it can be used to advance liver fibrosis research and to discover and characterize anti-fibrotic therapies. In Aim 1, ARPKD and NASH organoids are used to develop a novel platform for assessing the anti-fibrotic efficacy of 10 agents whose mechanism of action is relevant to liver fibrosis, and to identify drug combinations with increased anti-fibrotic efficacy. Since nine are FDA-approved drugs, but none are currently used to treat liver fibrosis, these studies could have significant translational importance. In Aim 2, these models evaluate the fibrogenic effect of ECM cues using a novel, fully chemically defined, biosynthetic matrix. ECM changes are widely thought to promote fibrotic remodeling. A novel, synthetic chemistry scheme enables tuning of the key mechanical (stiffness, viscoelasticity) and biochemical (cell-adhesive ligand identity) matrix properties. ARPKD and NASH organoids grown in synthetic matrices will enable us to examine the effects that matrix cues have on fibrosis, and this analysis includes single cell RNA sequencing (scRNA-Seq). In Aim 3, to identify common pathogenetic drivers that are shared among congenital and acquired forms of liver fibrosis, we extend our modeling approach to generate and characterize organoid models for Joubert Syndrome Related Disorder (JSRD), which is a multi- system genetic disease that causes liver fibrosis in some cases, and we characterize a unique NASH organoid model. JSRD liver disease cannot be modeled in animals. JSRD and NASH organoids and isogenic controls will be analyzed using scRNA-Seq, high-dimensional time of flight mass cytometry (CyTOF) and two semi-targeted metabolomic methods. JSRD...