Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of the extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analysis of HSCs isolated from mice defined the murine ortholog of TILAM. We then generated Tilam-deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl4) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM. Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in murine HSCs during the development of fibrosis in vivo. In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease.

Chronic liver injury promotes inflammation, which can progress to fibrosis and cirrhosis, a major cause of mortality. Resolving the transcriptional changes orchestrating this transformation remains challenging. Here, we derive single-cell transcriptomic maps of progressive liver injury in human pluripotent stem cell (hPSC) - derived liver organoids (HLOs), modeling steatohepatitis with palmitic acid and fibrosis through TGF-β1 treatment. We observe that palmitic acid drives inflammation and non-alcoholic fatty liver disease (NAFLD) expression signatures, while TGF-β1 expands hepatic stellate-like populations, remodels cell cycle patterning, and induces extracellular matrix pathways. Analysis of receptor-ligand expression defines the induction of genes regulating Notch and fatty acid signaling with palmitic acid treatment, while the TGF-β1 response is shaped by the co-expression of COL1A1 and integrins to promote crosstalk between hepatocytes, cholangiocytes, and stellate cells. Finally, inflamed and fibrotic HLOs sequentially induce genes predicting disease progression in NAFLD. Our findings highlight HLOs as dynamic human in vitro systems to study evolving liver injury, providing a single-cell transcriptomic reference that will facilitate benchmarking future organoid-based liver injury models.