Single-cell analysis of heterogeneity in reverted hiPSC-derived human hepatic stellate cells

TL;DR

Liver cells called HSCs can partly return to a healthy state after injury is removed, but remain sensitive to reactivation, with immune signals like IL-10 playing a key role.

Contribution

Demonstrates human HSC plasticity and identifies IL-10's role in reversion through vitamin A metabolism using a hiPSC-derived liver model.

Findings

Activated HSCs revert to a less activated state after injury resolution, regaining lipid droplets and vitamin A storage.

Single-cell RNA sequencing reveals heterogeneity in reverted HSCs, including a subset resembling naïve quiescent HSCs.

Macrophage-derived IL-10 promotes reversion by inducing vitamin A metabolism genes like LRAT and RBP1.

Abstract

Activated HSCs are known to drive fibrogenesis, but their fate following injury resolution remains unclear. We aimed to investigate whether human activated HSCs revert to a less activated state, and to characterize features of such reversion using a human induced pluripotent stem cell (hiPSC)-derived multicellular liver model.

We used a hiPSC-derived liver culture containing hepatocytes, HSCs, and macrophages. HSCs were activated by HCV infection or a lipotoxic milieu modeling metabolic dysfunction-associated steatotic liver disease (MASLD) and subjected to injury resolution through antiviral treatment or replacement with a healthy medium. Reverted HSCs were characterized via gene expression profiling, functional assays, and single-cell RNA sequencing (scRNA-seq). The role of macrophage-derived IL-10 in HSC reversion was investigated through receptor knockdown and cytokine treatment experiments.

Following either HCV clearance or withdrawal of lipotoxic stress, activated HSCs reverted to a less activated state, regaining lipid droplets and vitamin A storage while re-expressing quiescent HSC markers. scRNA-seq revealed heterogeneity among reverted HSCs, identifying subpopulations expressing apoptotic, senescent, or quiescent-like signatures. A distinct lipid-high, PTK2-low population closely resembled naïve quiescent HSCs. Functional assays demonstrated that rHSCs retained partial quiescence but exhibited heightened sensitivity to fibrogenic re-stimulation (n = 4, p <0.05). Mechanistically, macrophage-derived IL-10 promoted HSC reversion by inducing vitamin A metabolism-related genes, including LRAT and RBP1 (n = 4, p <0.01).

Activated human HSCs demonstrate plasticity, reverting to a quiescent-like state following resolution of viral or metabolic injury, although they remain primed for reactivation. Macrophage-derived IL-10 plays a critical role in driving this reversion by regulating vitamin A metabolism. These findings provide insights into HSC dynamics and suggest potential therapeutic avenues for liver fibrosis by targeting HSC reversion.

Removing the cause of liver injury—curing hepatitis C or withdrawing lipotoxic stress—allows scar-forming liver cells (hepatic stellate cells) to partly revert to a healthier, vitamin-A-storing state; single-cell profiling reveals its heterogeneity and identify a subset nearing true quiescence. This rebound depends on intercellular interaction, in part on the immune signal IL-10 from macrophages, yet reverted cells remain easier to re-activate. These findings provide insights into dynamics of hepatic stellate cells and suggest potential therapeutic avenues for liver fibrosis by targeting stellate cell reversion.

Image 1

•HSCs revert toward quiescence after HCV cure or lipotoxic withdrawal.•Reverted HSCs regain vitamin A and quiescent markers but remain primed.•ScRNA-seq reveals heterogeneity and a lipid-high, PTK2-low rHSC subset.•Macrophage IL-10 drives reversion via vitamin A metabolism genes (LRAT and RBP1).

HSCs revert toward quiescence after HCV cure or lipotoxic withdrawal.

Reverted HSCs regain vitamin A and quiescent markers but remain primed.

ScRNA-seq reveals heterogeneity and a lipid-high, PTK2-low rHSC subset.

Macrophage IL-10 drives reversion via vitamin A metabolism genes (LRAT and RBP1).

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