Single-Cell Analysis of Heterogeneity in Reverted Human iPSC-Derived Hepatic Stellate Cells

We employed a single-cell RNA sequencing (scRNA-seq) approach using the 10x Genomics platform to investigate the heterogeneity of reverted hepatic stellate cells (HSCs) derived from human induced pluripotent stem cells (hiPSCs). HSCs are key mediators of liver fibrosis; although their activation is well known to drive fibrogenesis, their fate following removal of the fibrogenic insult remains poorly understood. To address this, we utilized a hiPSC-derived liver culture system comprising hepatocytes, HSCs, and macrophages. HSCs were activated by hepatitis C virus (HCV) infection and subsequently treated with antivirals to achieve viral clearance. Reverted HSCs were then characterized through gene expression profiling, vitamin A quantification, functional assays, and scRNA-seq. Our data reveal that activated HSCs exhibit plasticity and can revert to a quiescent-like state upon removal of the activating stimulus, although they retain heightened sensitivity to re-stimulation. These findings enhance our understanding of HSC dynamics and highlight potential therapeutic strategies targeting HSC reversion to treat liver fibrosis. Overall design: HSCs were activated through hepatitis C virus (HCV) infection and subsequently treated with antiviral agents to achieve viral clearance. To enable single-cell RNA sequencing (scRNA-seq), reverted HSCs were isolated by depleting co-cultured hepatocytes.