Behaviour of iPSC-Derived and Donor-Derived Hepatic Stellate Cells in Organoid Cultures Is Highly Variable and Contributes to Fibrotic Outcome.

Hepatic stellate cells (HSCs) are central drivers of liver fibrosis, yet their heterogeneity is often overlooked in human liver organoid models. Here, we investigated how HSC-intrinsic variability influences fibrotic phenotypes in multicellular liver organoids using induced pluripotent stem cell-derived HSCs (iHSCs) and primary HSCs (pHSCs). iHSCs derived from two different iPSC lines exhibited distinct differentiation phenotypes towards iHSCs. Consistently, co-culture of the hepatocyte-like cell line HepaRG with these two iHSC populations generated organoids with distinct fibrotic and hepatic features. BJ1-iHSC/HepaRG organoids contained an approximately 2.9-fold higher proportion of CYP3A4-positive cells, whereas Sigma-iHSC/HepaRG organoids showed around a 4-fold higher percentage of Sirius red-positive area. Single-cell RNA analysis further showed that the two iHSC populations differed in cell cycle distribution, HSC-related signatures, extracellular matrix-associated pathways, and matrisome enrichment. Similarly, human pHSCs cultured as organoids with HepaRG showed marked donor-dependent differences at baseline and after single acetaminophen, repeated thioacetamide or transforming growth factor beta treatment. Together, our findings suggest that HSC heterogeneity, whether iPSC-derived or donor-derived, is an important factor in fibrotic output and response variability in human liver organoid models and should be considered in model design, interpretation and comparison between studies.