The liver is remarkable for its ability to regenerate after injury, yet when this process fails, acute liver failure (ALF) carries devastating outcomes. Traditional research methods, reliant on bulk tissue analysis, masked the diversity of cellular players driving both damage and repair. Now, advances in single-cell transcriptomics—especially single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics—are changing the landscape.
From Bulk Analysis to Single Cells
Bulk RNA sequencing averages signals across millions of cells, obscuring rare but crucial subpopulations. By contrast, single-cell transcriptomics captures gene expression profiles of individual cells, allowing researchers to pinpoint transient, rare, or injury-specific cell states.
Two main approaches dominate the field: (1) scRNA-seq, best for immune-focused studies using fresh tissue; (2) snRNA-seq, which isolates nuclei and enables the study of frozen samples, useful for rare cell detection. When combined with spatial transcriptomics (ST), which maps where cells are located within liver architecture, these tools provide unprecedented resolution of how different cells coordinate during injury and repair.
Hepatocytes: The Frontline of Repair
Hepatocytes, the primary functional cells of the liver, drive regeneration after acute damage. Single-cell studies revealed that surviving hepatocytes in zones 2 and 3 proliferate to replace necrotic tissue. Beyond this expected activity, researchers identified surprising cell states: (1) Fetal-like hepatocytes (Afp⁺Cdh17⁺Spp1⁺) emerge at injury-repair borders, resembling developmental cells with high biosynthetic activity; (2) Migratory Anxa2⁺ hepatocytes display motile morphologies with lamellipodia, directly moving into injured areas to close wounds. Knockdown experiments confirmed that Anxa2 is essential for hepatocyte migration and tissue repair. These discoveries overturn the long-held view that liver regeneration depends solely on resident proliferation, highlighting migration as a complementary repair strategy.
Hepatic Stellate Cells: More Than Fibrosis
Hepatic stellate cells (HSCs), often studied for their fibrogenic role in chronic disease, show strikingly dynamic behavior in ALI: (1) Early injury phase: Activated HSCs express Acta2 and cytokines without producing fibrosis-driving collagen (Col1a1), suggesting a contractile, immunomodulatory phenotype. (2) Repair phase: They transition toward extracellular matrix (ECM) remodeling and hepatocyte support, partly by secreting hepatocyte growth factor (HGF), a key promoter of regeneration. Spatial analyses revealed that periportal HSCs proliferate and migrate into damaged regions, suggesting they act as reservoirs for regenerative support. This positions HSCs as dual-function players—injury contributors and essential repair facilitators.
Endothelial Cells: Gatekeepers of Regeneration
Liver sinusoidal endothelial cells (LSECs) regulate zonation and regenerative signaling. Single-cell studies uncovered their crucial role in producing Wnt ligands (Wnt2, Wnt9b), which orchestrate hepatocyte proliferation after injury. Following acetaminophen overdose, pericentral LSECs sharply upregulate Wnt9b, while liver vascular endothelial cells (LVECs) increase Wnt2. After partial hepatectomy, both ligands surge within 24 hours in pericentral LSECs, likely stimulated by MIF- or CXCL12-CXCR7 signaling, alongside hemodynamic changes. Increased blood flow-induced shear stress stimulates HB-EGF production through YAP1 activation, reinforcing the pro-regenerative program. These findings suggest endothelial cells integrate mechanical and molecular cues to guide hepatocyte renewal.
Macrophages: Guardians and Repair Crews
Macrophages exhibit perhaps the most striking heterogeneity during ALI and regeneration. Single-cell profiling clarified the sequence of events: (1) Kupffer cells (KCs): Resident macrophages initiate inflammation by releasing cytokines and recruiting neutrophils. Some subsets, like Cxcl2⁺ KCs, amplify injury. (2) Monocyte-derived macrophages (MoMFs): Initially pro-inflammatory (Ly6Chigh), they later transition to restorative Ly6Clow states, aiding in tissue repair. (3) Trem2⁺ macrophages: Newly identified populations emerge during repair, showing enhanced phagocytic activity and roles in clearing dying cells. Interestingly, Trem2's role differs across contexts—protective in acetaminophen injury but detrimental in ischemia-reperfusion models. This heterogeneity reveals macrophages as highly adaptable, making them promising therapeutic targets to tip the balance from inflammation to repair.
The Technology Driving Discovery
Behind these insights lie major technical strides: (1) High-throughput sequencing platforms like 10x Chromium dominate scRNA-seq, while Stereo-seq and Visium HD achieve near single-cell resolution in spatial mapping; (2) Cell enrichment strategies (FACS, MACS) ensure rare or fragile cell populations are captured; (3) Bioinformatics advances, including deep learning annotation tools (e.g., scBERT, GPTCelltype), enable precise classification of closely related cell states. The next frontier lies in establishing comprehensive liver cell reference datasets to improve rare cell identification and harmonize findings across laboratories.
Therapeutic Implications
By mapping cellular heterogeneity, single-cell transcriptomics is not just descriptive but predictive for therapeutic innovation. Potential applications include: (1) Targeting Anxa2⁺ hepatocytes to enhance wound closure after acute liver damage; (2) Modulating HSC phenotypes to maximize regenerative functions while minimizing fibrotic activity; (3) Leveraging endothelial Wnt signaling as a regenerative cue; (4) Reprogramming macrophages toward Trem2⁺ reparative states to accelerate recovery. Such strategies could reduce reliance on liver transplantation and improve survival in patients with acute liver failure.
In conclusion, Dr. Wen and Professor Ju's review captures a transformative moment in hepatology research. By dissecting the liver one cell at a time, single-cell transcriptomics is exposing hidden players and pathways in injury and regeneration. These insights not only enrich our fundamental knowledge but also chart a path toward new therapies for acute liver diseases—a field where treatment options remain limited.
As the authors conclude, the promise of single-cell transcriptomics lies not only in understanding how the liver heals itself, but in harnessing that knowledge to develop precision interventions that improve patient outcomes.
See the article:
Wen Y, Ju C. New insights into liver injury and regeneration from single-cell transcriptomics. eGastroenterology 2025;3:e100202. doi:10.1136/egastro-2025-100202
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