Abstract
German Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing cause of morbidity with limited treatment options. Thus, accurate in vitro systems to test new therapies are indispensable. While recently, human liver organoid models have emerged to assess steatotic liver disease, a systematic evaluation of their translational potential is still missing. Here, we evaluated human liver organoid models of MASLD, comparatively testing disease induction in three conditions: oleic acid, palmitic acid, and TGF-β1. Through single-cell analyses, we find that all three models induce inflammatory signatures, but only TGF-β1 promotes collagen production, fibrosis, and hepatic stellate cell expansion. In striking contrast, oleic acid ameliorates fibrotic signatures and reduces the hepatic stellate cell population. Linking data from each model to gene expression signatures associated with MASLD disease progression further demonstrates that palmitic acid and TGF-β1 more robustly model inflammation and fibrosis. Our findings highlight the importance of stratifying MASLD organoid models by signatures of clinical disease progression, provide a single-cell reference to benchmark future organoid injury models, and allow us to study evolving steatohepatitis, fibrosis, and HSC susceptibility to injury in a dynamic, multi-lineage human in vitro system. Metabolic dysfunction‐associated steatotic liver disease (MASLD, formerly NAFLD) is a major cause of chronic hepatic injury and failure, however the details of its induction remain unclear. This benchmark resource reports in vitro dissection of fatty liver disease in human organoids uncovering distinct disease phenotypes triggered by specific lipids. Human pluripotent stem cell‐derived liver organoids serve as models for injury related to MASLD.Palmitic acid induces inflammatory signatures while TGF‐β1 signaling induces inflammatory and fibrotic signatures.Oleic acid reduces hepatic stellate cell numbers and suppresses collagen production.Gene expression predicting MASLD severity increases from palmitic acid to TGF‐β1 treatment.