Efficient Generation of Fetal Hepatic Stellate Cells From hiPSC

Human induced pluripotent stem cell (hiPSC)-derived liver organoids have emerged as valuable models for studying human liver development. However, existing organoid systems often lack developmentally matched cell populations, particularly fetal hepatic stellate cells (HSCs), limiting their ability to recapitulate key developmental processes. Current approaches for generating HSCs rely on primary cells, immortalized cell lines, or hiPSC differentiation methods that frequently produce activated HSC-like cells and often require cell sorting. Here, we describe an efficient protocol for generating expandable fetal-like HSCs from hiPSCs through a stepwise differentiation strategy that mimics embryonic HSC development. The resulting cells can be robustly expanded while maintaining characteristic molecular and functional features of fetal HSCs. This protocol provides a reproducible and scalable source of fetal-like HSCs without cell sorting and supports the generation of multicellular liver organoids containing developmentally relevant stromal components. Beyond the validation of the protocol in studies of liver maturation and vascularization, it can be applied to investigations of HSC biology and congenital liver diseases. Key features • Efficient generation of fetal-like HSCs from hiPSCs without cell sorting. • Robust expansion of hiPSC-derived HSCs in vitro while maintaining fetal HSC characteristics. • Generation of developmentally relevant HSCs for multicellular liver organoid construction. • Applicable to studies of HSC biology, liver development, vascularization, and cell-cell interactions; successful implementation requires experience in hiPSC maintenance and differentiation.