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Updated: Apr 3, 2026
Cell Type-specific Gene Expression Profiling in the Mouse Liver
Published on: September 17, 2019
Single-cell fixed RNA profiling uncovers SEMA4D and LMCD1 as therapeutic targets in a liver fibrosis model
Pham Minh Duc1, Le Thi Thanh Thuy2, Hoang Hai2
1Department of Hepatology, Graduate School of Medicine, Osaka Metropolitan University, Osaka, Japan; Division of Gastroenterology, Department of Internal Medicine, Hanoi Medical University, Hanoi, Vietnam; Department of Gastroenterology, Hanoi Medical University Hospital, Hanoi, Vietnam.
Background & Aims:
Single-cell fixed RNA profiling (FLEX) is a novel technique that captures RNA expression in frozen tissues at a single-cell resolution. We applied FLEX to mouse model of liver fibrosis progression and regression to identify novel antifibrotic targets.
Methods:
Mice were administered intraperitoneal thioacetamide for 10 weeks to induce fibrosis, regression was assessed 2 weeks after cessation. The livers were fixed, dissociated, and analysed using FLEX. Molecular validation included immunoblotting, immunohistochemistry, gene silencing/overexpression, cytokine/phosphokinase arrays, and human liver samples.
Results:
Approximately 40,000 liver cells were profiled, integrated, and annotated into 10 major cell types using lineage-specific markers. Pericentral signature restoration in hepatocytes, scar-resolving genes (Mmp14 and Ctsl), fenestrae restoration in liver sinusoidal endothelial cells, anti-inflammatory Kupffer cells, reduced fibrogenic cholangiocytes, and recovery-associated immune cell subsets were observed. During fibrosis, monocyte-derived macrophages secrete semaphorin-4D (SEMA4D), which binds to Plexin B2 on hepatic stellate cells (HSCs). SEMA4D+ cells were upregulated in mouse fibrotic livers (n = 6, p <0.05). Recombinant SEMA4D induces type I collagen in HSCs, whereas humanised monoclonal IgG4 SEMA4D blockade (VX15/2503) attenuates fibrosis in vivo (n = 5, p <0.05). LIM and cysteine-rich domains 1 (LMCD1) was enriched in fibrotic HSCs and suppressed during regression. LMCD1 knockdown reduced the expression of fibrotic protein, while LMCD1 overexpression promoted the expression of fibrotic protein via AKT/mTOR signalling. LMCD1 and SEMA4D are localised in the fibrotic septa and are correlated with the fibrotic stage of MASLD (n = 34, p <0.05) and HCV (n = 76, p <0.05) in humans.
Conclusions:
This FLEX-based single-cell atlas revealed critical transcriptional programs and cell-cell interactions, identifying SEMA4D and LMCD1 as promising therapeutic targets for liver fibrosis.
Impact And Implications:
In this study, we applied a novel technique, single-cell fixed RNA profiling, to profile 38,136 cells obtained from control, thioacetamide-induced liver fibrosis, and regression-phase mouse livers, generating a high-resolution atlas of liver fibrosis progression and regression. We identified the transcriptomic patterns of regressed cell subpopulations and uncovered two key therapeutic targets: the macrophage-derived factor semaphorin-4D (SEMA4D) and the hepatic stellate cell-specific transcription factor LIM and cysteine-rich domains 1 (LMCD1). Treatment with a humanised monoclonal IgG4 antibody against SEMA4D significantly alleviated liver fibrosis in the thioacetamide-induced mouse model. SEMA4D and LMCD1 expression correlated with the metabolic dysfunction-associated steatotic liver disease-related fibrosis stage, and the attenuation of SEMA4D and LMCD1 after HCV-sustained virologic response reduced the risk of progression to hepatocellular carcinoma.
Insights
Single-cell fixed RNA profiling identified semaphorin-4D (SEMA4D) and LIM and cysteine-rich domains 1 (LMCD1) as key targets for liver fibrosis. Blocking SEMA4D reduced fibrosis in mice, showing therapeutic potential.
Area of Science:
- Single-cell transcriptomics
- Liver disease research
- Drug target identification
Background:
- Liver fibrosis is a progressive condition characterized by scar tissue accumulation.
- Novel therapeutic targets are needed to treat liver fibrosis effectively.
- Single-cell fixed RNA profiling (FLEX) offers high-resolution RNA expression analysis in frozen tissues.
Purpose of the Study:
- To apply FLEX to a mouse model of liver fibrosis to identify antifibrotic targets.
- To generate a single-cell atlas of liver fibrosis progression and regression.
- To validate SEMA4D and LMCD1 as potential therapeutic targets.
Main Methods:
- Induction of liver fibrosis in mice using thioacetamide, followed by regression assessment.
- Single-cell RNA profiling using FLEX on liver tissues.
- Molecular validation including immunoblotting, immunohistochemistry, gene manipulation, and analysis of human liver samples.
Main Results:
- Profiling of approximately 40,000 liver cells revealed 10 major cell types and identified scar-resolving genes and restored cellular functions during regression.
- Monocyte-derived macrophages secrete semaphorin-4D (SEMA4D), which promotes fibrosis by acting on hepatic stellate cells (HSCs).
- LIM and cysteine-rich domains 1 (LMCD1) was enriched in fibrotic HSCs and promoted fibrosis via AKT/mTOR signaling. SEMA4D blockade attenuated fibrosis in vivo.
Conclusions:
- FLEX analysis provided a high-resolution atlas of liver fibrosis, revealing critical transcriptional programs and cell-cell interactions.
- SEMA4D and LMCD1 were identified as promising therapeutic targets for liver fibrosis.
- Expression of SEMA4D and LMCD1 correlated with fibrosis severity in human liver diseases (MASLD and HCV).
