Single-Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell-Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice

Abstract

Background and aims: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL₄ -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing.

Approach and results: We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL₄ -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential gene-expression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL₄ -treated livers but showed no therapeutic efficacy in bile duct ligation livers.

Conclusions: This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies.

FIG. 1

ScRNA‐seq of nonparenchymal liver cells. (A) Schematic overview depicting the approach for the experiments. (B) UMAP visualization of isolated hepatic cells, based on 47,752 single‐cell transcriptomes pooled from control (10,636), CCL₄‐treated (18,185), and bile duct‐ligated mice (18,931). Inset: Three‐dimensional UMAP showing the spatial distribution of HSCs and portal fibroblasts. (C) Heatmap depicting the top 25 marker genes (ordered by adjusted P‐values) for each cell cluster. (D) Distinct expression of the cell‐type‐specific genes overlaid on the UMAP of (B). (E) The expression patterns for Gucy1a1 and Gucy1b1. (F) The EGFP expression pattern in the livers of Gucy1a1‐EGFP mice, showing typical HSC morphology. Liver sections were co‐stained with the HSC‐specific marker Desmin and the endothelial cell‐specific marker CD31.

FIG. 2

Transcriptomic roadmap of HSC activation. (A) UMAP visualization of the qHSCs and aHSCs from pooled HSCs of control, CCL₄‐treated, and bile duct‐ligated mice. (B) The expression of the qHSC marker genes Lrat and Ecm1 and aHSC marker genes Col1a1 and Acta2 overlaid on the UMAP of (A). (C) Heatmap depicting the top 50 representative genes (ordered by adjusted P‐values) of qHSC and aHSC. (D) Pseudotime trajectory indicating the activation of HSC during liver fibrosis. (E) Heatmap representing four gene clusters based on the dynamic gene expression pattern over pseudotime from the qHSC state to the aHSC state. Cluster 1: 202 genes; cluster 2: 82 genes; cluster 3: 288 genes, and cluster 4: 401 genes. Plot showing average expression of the four gene clusters along pseudotime. GO biological pathway analysis of the four gene clusters. Expression kinetics of representative genes of four gene clusters.

FIG. 3

Subclusters of activated HSCs. (A) UMAP displaying the qHSC and the three subclusters of aHSCs (stage‐1 aHSCs, stage‐2 aHSCs, stage‐3 aHSCs). (B) Heatmap depicting the top 50 differentially expressed genes for qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, and stage‐3 aHSCs (sort by average logFC in descending order). (C) GO biological analysis of the top 50 differentially expressed genes of (B). (D) UMAP visualization of the expression pattern of the cell proliferation markers Mki67 and Mcm6. (E) UMAP displaying the qHSC and the three subclusters of aHSCs in the individual livers of control, CCL₄, and BDL mice (left panel). Proportion of qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, and stage‐3 aHSCs in the individual livers of control, CCL₄, and BDL mice (right panel).

FIG. 4

Morphological analysis of HSCs in liver fibrosis. (A) The liver sections of sham‐operated, CCL₄‐treated, or bile duct‐ligated Gucy1a1‐EGFP mice were stained with collagen‐1 and CD31 antibodies. The morphology of EGFP+ HSCs were skeletonized; cell bodies are in blue and cellular processes are in red. (B) Morphological analysis of EGFP+ HSCs in the liver sections of sham‐operated, CCL₄‐treated, or bile duct‐ligated Gucy1a1‐EGFP mice. Statistical analyses were performed using one‐way ANOVA with Tukey HSD test. ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C) Liver sections of CCL₄‐treated Gucy1a1‐EGFP mice were stained with collagen1 and CD31. Stage‐1, stage‐2, and stage‐3 HSCs were defined based on their relative distance to the collagen‐1‐positive stage‐3 aHSCs. Zoomed‐in image showing the morphology of qHSCs and aHSCs at stage 1, stage 2, and stage 3. Arrow indicate the skeletonized morphology of individual aHSCs. (D) Morphological comparison of qHSCs and HSCs at stage 1, stage 2, and stage 3. Statistical analyses were performed using one‐way ANOVA with Tukey HSD test. ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

FIG. 5

PFs are the major source of collagen‐producing myofibroblasts in BDL liver. (A) Heatmap depicting the top 50 differentially expressed genes in the qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, stage‐3 aHSCs, and PFs of CCL₄‐treated and BDL livers (sort by average logFC in descending order). The right panel shows the representative genes. (B) UMAP visualization of qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, stage‐3 aHSCs, and portal fibroblasts pooled from sham, CCL₄, and BDL mice. The right panel shows the 3‐D UMAP representation of the five cell clusters. (C) Violin plot showing the expression levels of Thy1, Fbln1, Mfap4, Eln, Dpt, Gas6, Col1a1, and Col1a2 in aHSCs and PFs in the livers of CCL₄ or BDL mice. (D) UMAP visualization of qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, stage‐3 aHSCs, and portal fibroblasts in the individual livers of control, CCL₄, and BDL mice. (E) Proportion of qHSCs, stage‐1 aHSCs, stage‐2 aHSCs, stage‐3 aHSCs, and portal fibroblasts in the individual livers of control, CCL₄, and BDL mice. (F) Normalized percentage of PFs in sham, CCL₄, and BDL livers.

FIG. 6

Lineage tracing reveals HSCs contribute to the collagen‐producing myofibroblasts in CCL₄ liver instead of BDL liver. (A) Liver sections of control, CCL₄, or BDL‐treated Gucy1a1‐EGFP mice were stained with Collagen‐1 and Thy1. (B) Liver sections from bile duct‐ligated Gucy1a1‐EGFP::Gucy1a1‐CreERT2::ROSA26‐LSL‐tdTomato mice were stained with Thy1. (C) Liver sections from bile duct‐ligated Gucy1a1‐EGFP::Gucy1a1‐CreERT2::ROSA26‐LSL‐tdTomato mice were stained with collagen‐1.

FIG. 7

Differential responses of Riociguat treatment in CCL₄‐ and BDL‐induced liver fibrosis. (A) The correlation of the expression of Gucy1a1 and Gucy1b1 in HSCs and PFs. (B) The expression of Gucy1a1 and Gucy1b1 in individual HSCs and portal fibroblasts were overlaid on the UMAP. Dot lines outline the different subclusters according to Figure 4B. (C) Schematic overview of the experimental design. CCL₄ was injected twice a week for 3 weeks and Riociguat (10 mg/kg body weight) was administered twice a day by oral gavage. Representative images of liver sections from control, CCL₄‐, and CCL₄+ Riociguat‐treated mice were stained with sirius red to show the collagen deposit. (D) Sirius red positive area, hydroxyproline concentration, and serum ALT and AST enzymatic activity in the livers of control, CCL₄‐, and CCL₄+ Riociguat‐treated mice were quantitated (Corn oil + vehicle, n = 4; CCL₄ + vehicle, n = 6; CCL₄ + Riociguat, n = 6). Statistical analyses were performed using one‐way ANOVA with Tukey HSD test. ns: not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. (E) Schematic overview of the experimental design of the BDL experiment. Vehicle or Riociguat (10 mg/kg body weight) was administered twice a day for 10 days. Representative images of liver sections of vehicle‐ or Riociguat‐treated BDL mice were stained with sirius red. (F) Quantification of sirius red positive area, hydroxyproline concentration, ALT and AST enzymatic activity, and total bilirubin (T‐BIL) concentration in the serum of vehicle‐ or Riociguat‐treated BDL mice (sham + vehicle, n = 4; BDL + vehicle, n = 6; BDL + Riociguat, n = 5). Statistical analyses were performed using one‐way ANOVA with Tukey HSD test. ns: not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.