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Activated hepatic stellate cells (aHSC) are the main source of extra cellular matrix in liver fibrosis. Activation is classically divided in two phases: initiation and perpetuation. Currently, HSC-based therapeutic candidates largely focus on targeting the aHSCs in the perpetuation phase. However, the importance of HSC initiation during chronic liver disease (CLD) remains unclear. Here, we identified transcriptional programs of initiating and activated HSCs by RNA sequencing, using in vitro and in vivo mouse models of fibrosis. Importantly, we show that both programs are active in HSCs during murine and human CLD. In human cirrhotic livers, scar associated mesenchymal cells employ both transcriptional programs at the single cell level. Our results indicate that the transcriptional programs that drive the initiation of HSCs are still active in humans suffering from CLD. We conclude that molecules involved in the initiation of HSC activation, or in the maintenance of aHSCs can be considered equally important in the search for druggable targets of chronic liver disease.

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow ( CenTFinder ) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder , we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2 ) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Scarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells. To identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. Gene expression profiling confirmed early changes for known genes related to HSC activation such as alpha smooth muscle actin (Acta2), lysyl oxidase (Lox) and collagen, type I, alpha 1 (Col1a1). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. Insulin-like growth factor binding protein 3 (Igfbp3) was identified as a gene strongly affected by VPA treatment. During normal HSC activation Igfbp3 is up regulated and this can thus be prevented by VPA treatment in vitro and in vivo. siRNA-mediated silencing of Igfbp3 in primary mouse HSCs induced matrix metalloproteinase (Mmp) 9 mRNA expression and strongly reduced cell migration. The reduced cell migration after Igfbp3 knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents Igfbp3 transcription during activation of HSCs in vitro and in vivo.

Chronic cholangiopathies, such as primary and secondary sclerosing cholangitis, are progressive disease entities, associated with periportal accumulation of inflammatory cells, encompassing monocytes and macrophages, peribiliary extracellular matrix (ECM) deposition and ductular reaction (DR). This study aimed to elucidate the relevance of macrophages in the progression of chronic cholangiopathies through macrophage depletion in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model. One group of mice received a single i.p. injection of Clodronate encapsulated liposomes (CLOLipo) at day 7 of a 14 day DDC treatment, while control animals were co-treated with PBSLipo instead. Mice were sacrificed after 7 or respectively 14 days of treatment for immunohistochemical assessment of macrophage recruitment (F4/80), ECM deposition (Sirius Red, Laminin) and DR (CK19). Macrophage depletion during a 14 day DDC treatment resulted in a significant inhibition of ECM deposition. Porto-lobular migration patterns of laminin-rich ECM and ductular structures were significantly attenuated and a progression of DR was effectively inhibited by macrophage depletion. CLOLipo co-treatment resulted in a confined DR to portal regions without amorphous cell clusters. This study suggests that therapeutic options selectively directed towards macrophages might represent a feasible treatment for chronic cholestatic liver diseases.

Liver fibrosis majorly impacts global health, necessitating the development of in vitro models to study disease mechanisms and develop drug therapies. Relevant models should at least include hepatocytes and hepatic stellate cells (HSCs) and ideally use three-dimensional cultures to mimic in vivo conditions. Induced pluripotent stem cells (iPSCs) allow for patient-specific liver modelling, but current models based on iPSC-derived hepatocytes (iHepatocytes) and HSCs (iHSCs) still lack key functions. We developed organoids of iHepatocytes and iHSCs and compared them to HepaRG and primary HSC organoids. RNA sequencing analysis comparison of these cultures identified a potential role for the transcription factor RXRA in hepatocyte differentiation and HSC quiescence. Treating cells with the RXRA ligand 9-cis-retinoic acid (9CRA) promoted iHepatocyte metabolism and iHSC quiescence. In organoids, 9CRA enhanced fibrotic response to TGF-β and acetaminophen, highlighting its potential for refining iPSC-based liver fibrosis models to more faithfully replicate human drug-induced liver injury and fibrotic conditions.

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap- and Taz-deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells.

Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-κB inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-κB activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor β, connective tissue growth factor, platelet-derived growth factor β-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro , OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-κB inhibition via up-regulated IκBα. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.

Objective Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive. Design Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays. Results In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib. Conclusions Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

Local delivery of mRNA-based immunotherapy offers a promising avenue as it enables the production of specific immunomodulatory proteins that can stimulate the immune system to recognize and eliminate cancer cells while limiting systemic exposure and toxicities. Here, we develop and employ lipid-based nanoparticles (LNPs) to intratumorally deliver an mRNA mixture encoding the cytokines interleukin (IL)−21 and IL-7 and the immunostimulatory molecule 4-1BB ligand (Triplet LNP). IL-21 synergy with IL-7 and 4-1BBL leads to a profound increase in the frequency of tumor-infiltrating CD8 + T cells and their capacity to produce granzyme B and IFN-γ, leading to tumor eradication and the development of long-term immunological memory. Mechanistically, the efficacy of the Triplet LNP depends on tumor-draining lymph nodes to tumor CD8 + T-cell trafficking. Moreover, we highlight the therapeutic potential of the Triplet LNP in multiple tumor models in female mice and its superior therapeutic efficacy to immune checkpoint blockade. Ultimately, the expression of these immunomodulators is associated with better overall survival in patients with cancer. Local delivery of mRNA-based immunotherapy represents an option for cancer therapy. Here the authors report that intratumoral delivery of lipid nanoparticle-formulated mRNA encoding IL-21, IL-7, and 4-1BBL induces systemic anti-tumor immunity in preclinical cancer models.

Hepatic stellate cells (HSCs) are nonparenchymal liver cells responsible for extracellular matrix homeostasis and are the main cells involved in the development of liver fibrosis following injury. The lack of reliable sources of HSCs has hence limited the development of complex in vitro systems to model liver diseases and toxicity. Here we describe a protocol to differentiate human induced pluripotent stem cells (iPSCs) into hepatic stellate cells (iPSC-HSCs). The protocol is based on the addition of several growth factors important for liver development sequentially over 12 d. iPSC-HSCs present phenotypic and functional characteristics of primary HSCs and can be expanded or frozen and used to perform high-throughput in vitro studies. We also describe how to coculture iPSC-HSCs with hepatocytes, which self-assemble into three-dimensional (3D) hepatic spheroids. This protocol enables the generation of HSC-like cells for in vitro modeling and drug screening studies. Human iPSCs are differentiated into HSCs by culture with growth factors. They respond to fibrogenic stimuli, arising as a new source of HSC-like cells for in vitro modeling. Subsequent coculture with hepatocytes facilitates self-assembly into 3D hepatic spheroids.