# 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.

## Key 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).