**1. 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. Interestingly, a variety of different functions have been assigned to HSCs and/or MFB, ranging from extracellular matrix production, mechanical properties (e.g., contraction and vascular resistance regulation in the liver), and lipid metabolism to

immune regulation [4], raising the question about yet unrecognized, functionally diverse subsets of HSCs/MFB. The aim of this study was therefore the evaluation of the heterogeneity of resting HSCs and activated MFB, both in vivo and in vitro, by single cell RNA sequencing (scRNASeq) analysis. 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. We could identify S100 calcium binding protein A6 (S100A6) as a key marker of activated MFB. Furthermore, we found that in vitro activated MFB lack key functions of in vivo activated MFB as the production of various chemokines, such as CC-chemokine ligand 2 (CCL2) and CXC-chemokine ligand 1 (*CCL1*).

Taken together, our data demonstrate the heterogeneity of activated MFB in vivo and highlight the differences of in vivo and in vitro activated MFB, leading to a better understanding of HSCs to MFB trans0differentation during liver fibrosis.
