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Purification protocol of hypertrophied hepatic stellate cells from CDAHFD mice liver for their transcriptomic characterization

ABSTRACT: Hepatic stellate cells (HSC) are known for their major role in hepatic fibrosis. Recently, it has been shown that different subpopulations of HSC co-exist during fibrogenesis and play different roles in the establishment of fibrosis. We previously highlighted, in murine model and human biopsies, a specific subpopulation of hypertrophied HSC (hypHSC) which exhibit exacerbated retinoid droplets and were closely associated to collagen fibers. The present study describes the purification protocol of hypHSC developed from a murine model of metabolic liver fibrosis. Liver dissociation followed by density gradient and fluorescence assisted cell sorting allowed us to obtain higlhly pure hypHSC preparations. Then, a transcriptomic analysis (bulk RNAseq) of hypHSCs versus quiescent HSCs purified from healthy mouse liver, was performed and showed that the hypHSCs molecular signature differs from HSC subtypes already described in the literature, with a “hybrid” profile of both inflammation and matrix remodeling. Our study highlights that a phenotype-to-molecular approach can provide complementary elements to single-cell molecular approaches.

ORGANISM(S): Mus musculus

PROVIDER: GSE294603 | GEO | 2026/05/15

REPOSITORIES: GEO

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Project description:Background & Aims: Persistent activation of hepatic stellate cells (HSCs) drives liver fibrosis, marked by myofibroblast activation (MFA) and epithelial-mesenchymal transition (EMT). Strategies to prevent or reverse this will be critical to treat liver fibrosis successfully. We previously showed that full-term, cell-free human amniotic fluid (cfAF) inhibits MFA and EMT in fibroblasts in vitro. We hypothesize that cfAF treatment can attenuate HSC activation and limit liver fibrosis. Methods: HSC activation was assessed using murine models of acute (DMN) and chronic (CCl4) liver damage, three-dimensional hepatic spheroids, and HSC cultures. EMT and MFA were induced in vitro using ethanol (spheroids) or TGFβ (HSCs) and evaluated via scratch assays, multi-omics approaches, and RNA/protein analyses. Results: cfAF treatment prevented weight loss in mice with chronic liver damage without adverse events. Histological analysis revealed a modest reduction in fibrosis with preserved liver architecture in both chronic and acute murine liver damage models. Gene expression profiling and immunostaining indicated cfAF administration to CCl4-treated mice led to reduced EMT- and MFA-related biomarkers along with changes in transcripts associated with liver metabolism, immune regulatory pathways, and cell signaling. In hepatic spheroids exposed to ethanol cfAF treatment lowered COL1A1 protein levels and smooth muscle actin (SMA) RNA abundance in a dose-dependent manner. Treating primary or LX2 hepatic stellate cells with cfAF strongly represses EMT, and multi-omics analyses revealed that it also attenuates TGFβ-induced MFA and the inflammatory phenotype. Thus cfAF treatment prevents liver fibrosis by safeguarding hepatic stellate cell activation. Conclusions: cfAF treatment limits liver fibrosis by repressing HSC activation in liver fibrosis models. These findings suggest cfAF may be a safe and effective therapy for reducing liver fibrosis and preventing the development of cirrhosis and/or hepatocellular carcinoma.

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Purification protocol of hypertrophied hepatic stellate cells from CDAHFD mice liver for their transcriptomic characterization

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