Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis.

Oliver Krenkel,Thomas Ritz,Jana Hundertmark,Frank Tacke,Ralf Weiskirchen

doi:10.3390/cells8050503

Abstract

Activation of hepatic stellate cells (HSCs) and their trans-differentiation towards collagen-secreting myofibroblasts (MFB) promote liver fibrosis progression. During chronic liver disease, resting HSCs become activated by inflammatory and injury signals. However, HSCs/MFB not only produce collagen, but also secrete cytokines, participate in metabolism, and have biomechanical properties. We herein aimed to characterize the heterogeneity of these liver mesenchymal cells by single cell RNA sequencing. In vivo resting HSCs or activated MFB were isolated from C57BL6/J mice challenged by carbon tetrachloride (CCl4) intraperitoneally for 3 weeks to induce liver fibrosis and compared to in vitro cultivated MFB. While resting HSCs formed a homogenous population characterized by high platelet derived growth factor receptor β (PDGFRβ) expression, in vivo and in vitro activated MFB split into heterogeneous populations, characterized by α-smooth muscle actin (α-SMA), collagens, or immunological markers. S100 calcium binding protein A6 (S100A6) was a universal marker of activated MFB on both the gene and protein expression level. Compared to the heterogeneity of in vivo MFB, MFB in vitro sequentially and only transiently expressed marker genes, such as chemokines, during culture activation. Taken together, our data demonstrate the heterogeneity of HSCs and MFB, indicating the existence of functionally relevant subsets in hepatic fibrosis.

Highlights

Hepatic stellate cell (HSC) activation and their trans-differentiation to myofibroblasts (MFB) due to chronic hepatic inflammation is a major hallmark feature of liver fibrosis [1]
We found that resting platelet derived growth factor receptor β- (PDGFR-β) positive HSCs show a high homogeneity, while activated α-smooth muscle actin- (α-SMA) positive MFB split into four different subpopulations, characterized by uniquely expressed gene patterns related to collagen synthesis or immunologic functions
The presence of liver fibrosis after three weeks of CCl4 treatment was confirmed by a hematoxylin and eosin (H&E) stain as well as α smooth muscle actin (α-SMA) immunohistochemistry on formalin-fixed and paraffin-embedded (FFPE)

Summary

Introduction

Hepatic stellate cell (HSC) activation and their trans-differentiation to myofibroblasts (MFB) due to chronic hepatic inflammation is a major hallmark feature of liver fibrosis [1]. Preventing or reversing excessive hepatic scarring is a major therapeutic target in treating chronic liver diseases, such as viral hepatitis and alcoholic and non-alcoholic steatohepatitis [2]. Resting HSC store lipids, such as retinol, can become activated following triggering signals released by damaged hepatocytes or activated local immune cells, such as e.g., Kupffer cells. Activating signals include transforming growth factor-β (TGF-β), platelet derived growth factors as well as various cytokines, such as interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) [3]. Activated MFB alter the composition and density of the extracellular matrix, by secreting collagens, and release inflammatory mediators, including chemokines and cytokines, thereby aggravating local inflammation. A variety of different functions have been assigned to HSCs and/or MFB, ranging from extracellular matrix production, mechanical

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