Single-Cell Gene Expression and Epigenetic Profiling Offer a New Way Forward for Liver Fibrosis Treatment

July 14, 2025 By Stuart P. Atkinson

Workflow, perturbations, and SERPINE1-TGFβ activation model. — Multi-omic profiling in normal, steatotic, and fibrotic livers pinpoints six HSC genes up in MASH, followed by cell, spheroid, and mouse experiments tying SERPINE1 to TGFβ activation. From Kim, et al.

Solving the Problem of Liver Fibrosis

Metabolic dysfunction-associated steatotic liver disease represents a spectrum of conditions that range from steatosis (metabolic dysfunction-associated steatotic liver or MASL) to steatohepatitis (metabolic dysfunction-associated steatohepatitis or MASH), with the latter involving significant levels of liver fibrosis (Kisseleva and Brenner). Importantly, highly plastic and heterogeneous populations of hepatic stellate cells play a critical role in the pathogenesis of MASH-associated fibrosis; liver injury can induce normally quiescent hepatic stellate cells to transdifferentiate into activated hepatic stellate cells/fibrogenic myofibroblasts, which produce the large amounts of extracellular matrix proteins that make up the fibrous scar (Liu et al. and Rosenthal et al.). Unfortunately, liver fibrosis can progress to cirrhosis and potentially even to liver failure if the underlying cause is not treated early in the course of the disease.

Researchers guided by Allen Wang, David A. Brenner, and Tatiana Kisseleva (University of California San Diego) sought to explore the possible basis of novel anti-fibrotic therapies that may aid liver disease treatment by identifying those genes (and exploring their regulation) in hepatic stellate cells critical to MASH-associated fibrosis. Reporting in a recent Journal of Hepatology article, the team now describes their comparison of single-cell transcriptomic and chromatin accessibility profiles from human hepatic stellate cells isolated from normal, MASL, and MASH livers, which aimed to identify upregulated genes in activated hepatic stellate cells and determine those that significantly impact the pathogenesis of MASH-associated fibrosis (Kim et al.). Overall, this exciting study identified hepatic stellate cell subclusters not detected by previous studies (Yashaswini et al. and Wang et al.) and characterized the mechanism by which these cells become activated in MASH livers, which included the transcriptional machinery that induces hepatic stellate cell transdifferentiation.

parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or " Paired-Tag " from Epigenome Technologies generates joint epigenetic and gene expression profiles at single-cell resolution. Paired-Tag can detect histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays and avoids the requirement for cell sorting. Applying Paired-Tag technology may enable researchers to take giant leaps forward in our understanding of gene regulatory mechanisms, identify novel therapeutic targets, and improve disease management; what additional insight could Paired-Tag have provided for this study of liver fibrosis?

UMAPs, composition chart, PCA, heatmap, and correlation matrix. — Single-cell RNA and ATAC maps compare healthy, MASL, and MASH livers—highlighting shifts in cell populations, PCA separation of samples, HSC gene expression patterns, and how the three conditions correlate. From Kim, et al.

Multiomic Analyses Reveal the Role of Activated Hepatic Stellate Cells in MASH

Kim et al. applied single-nucleus (sn)RNA-seq and snATAC-seq to a cohort of eighteen liver samples and then employed 2D human hepatic stellate cell cultures, 3D human liver spheroids, and hepatic stellate cell-specific gene knockout mice to evaluate their findings. The large, matching snRNA-seq and snATAC-seq analysis datasets revealed the presence of heterogeneous human hepatic stellate cells comprising specific subsets - quiescent, activated, and inflammatory phenotypes and identified a highly fibrogenic MASH-enriched activated hepatic stellate cell subcluster as a significant source for extracellular matrix-associated gene expression. While quiescent cells predominated in normal and MASL hepatic stellate cell populations, fibrogenic ECM-producing activated cells expanded only in MASH hepatic stellate cell populations. Chromatin accessibility and transcriptomic profiles of MASH activated hepatic stellate cells/myofibroblasts differed from those in normal and MASL hepatic stellate cells; however, MASL hepatic stellate cells exhibited unique characteristics despite displaying broad similarities to normal hepatic stellate cells, suggesting that steatosis does not represent a benign condition. Additionally, the chromatin accessibility data revealed significant changes when comparing normal and MASL hepatic stellate cells, suggesting that early epigenetic alterations may predict transcriptional responses in hepatic stellate cells. The upregulation of activation-specific and quiescence-specific genes in MASL hepatic stellate cells suggested they may be primed for or protected from activation.

The authors also discovered a set of genes -GAS7, SPON1, SERPINE1, LTBP2, KLF9, and EFEMP1 displaying increased chromatin accessibility and upregulated expression that drove the MASH-related activation of hepatic stellate cells; furthermore, the identified crosstalk between lineage-specific (JUNB/AP1), cluster-specific (RUNX1/2) and signal-specific (FOXA1/2) transcription factors that regulated the expression of these genes and, as such, the hepatic stellate cell phenotype.

Finally, the team explored the pathological relevance of the SERPINE1 gene via Dicer substrate small interfering RNA (dsiRNA)-based hepatic stellate cell-specific gene knockdown (Song et al.), the pharmacological inhibition of the SERPINE1 gene product (PA-1) in 3D human MASH liver spheroids, and hepatic stellate cell-specific Serpine1 knockout mice. SERPINE1 knockdown prevented fibrosis development in 3D human liver spheroids, while Serpine1 genetic deletion/PA-1 inhibition protected mice from MASH liver fibrosis. These findings represent the first description of the pathogenic role of activated human hepatic stellate cell-derived PAI-1 in MASH liver fibrosis development and suggest a way forward regarding developing novel treatment strategies for liver fibrosis treatment.

Boxplots of subcluster proportions, bar chart, donor heatmap, UMAP trajectory. — nRNA-seq of stellate cells shows how quiescent (Q) and activated (A1/A2) subclusters shift with fibrosis, with each donor’s cluster makeup and a pseudotime path from Q to A2. From Kim, et al.

A New Way Forward for MASH-associated Liver Fibrosis Treatment?

Overall, this exciting single-cell transcriptomic and epigenetic study identified novel genes/regulatory mechanisms underlying the activation of the hepatic stellate cells in MASH-affected livers that cause fibrosis; furthermore, they employed this knowledge to outlined potential strategies towards the development of much-needed anti-fibrotic therapies. Epigenetic and transcriptomic profiling at the single-cell level in human cells offers a means to push groundbreaking research forward ; can Epigenome Technologies help in this endeavor? Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Furthermore, Epigenome Technologies offers other single-cell products and services suitable for a wide range of research requirements.

For more on how single-cell gene expression and epigenetic profiling offer a way forward for liver fibrosis treatment, see the Journal of Hepatology, May 2025.