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.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

The development of complex in vitro hepatic systems and artificial liver devices has been hampered by the lack of reliable sources for relevant cell types, such as hepatic stellate cells (HSCs). Here we report efficient differentiation of human pluripotent stem cells into HSC-like cells (iPSC-HSCs). iPSC-HSCs closely resemble primary human HSCs at the transcriptional, cellular, and functional levels and possess a gene expression profile intermediate between that of quiescent and activated HSCs. Functional analyses revealed that iPSC-HSCs accumulate retinyl esters in lipid droplets and are activated in response to mediators of wound healing, similar to their in vivo counterparts. When maintained as 3D spheroids with HepaRG hepatocytes, iPSC-HSCs exhibit a quiescent phenotype but mount a fibrogenic response and secrete pro-collagen in response to known stimuli and hepatocyte toxicity. Thus, this protocol provides a robust in vitro system for studying HSC development, modeling liver fibrosis, and drug toxicity screening.

Liver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.

Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs.

Hepatic stellate cells activate upon liver injury and help at restoring damaged tissue by producing extracellular matrix proteins. A drastic increase in matrix proteins results in liver fibrosis and we hypothesize that this sudden increase leads to accumulation of proteins in the endoplasmic reticulum and its compensatory mechanism, the unfolded protein response. We indeed observe a very early, but transient induction of unfolded protein response genes during activation of primary mouse hepatic stellate cells in vitro and in vivo, prior to induction of classical stellate cell activation genes. This unfolded protein response does not seem sufficient to drive stellate cell activation on its own, as chemical induction of endoplasmic reticulum stress with tunicamycin in 3D cultured, quiescent stellate cells is not able to induce stellate cell activation. Inhibition of Jnk is important for the transduction of the unfolded protein response. Stellate cells isolated from Jnk knockout mice do not activate as much as their wild-type counterparts and do not have an induced expression of unfolded protein response genes. A timely termination of the unfolded protein response is essential to prevent endoplasmic reticulum stress-related apoptosis. A pathway known to be involved in this termination is the non-sense-mediated decay pathway. Non-sense-mediated decay inhibitors influence the unfolded protein response at early time points during stellate cell activation. Our data suggest that UPR in HSCs is differentially regulated between acute and chronic stages of the activation process. In conclusion, our data demonstrates that the unfolded protein response is a JNK1-dependent early event during hepatic stellate cell activation, which is counteracted by non-sense-mediated decay and is not sufficient to drive the stellate cell activation process. Therapeutic strategies based on UPR or NMD modulation might interfere with fibrosis, but will remain challenging because of the feedback mechanisms between the stress pathways.

A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals. Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)-the main producer of scar tissue during fibrosis-, but requires cultures in which at least hepatocytes are integrated. We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays. Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFβ can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips. We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models.

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.

Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis.

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.

Liver sinusoidal endothelial cells have a gatekeeper function in liver homeostasis by permitting substrates from the bloodstream into the space of Disse and regulating hepatic stellate cell activation status. Maintenance of LSEC's highly specialized phenotype is crucial for liver homeostasis. During liver fibrosis and cirrhosis, LSEC phenotype and functions are lost by processes known as capillarization and LSEC dysfunction. LSEC capillarization can be demonstrated by the loss of fenestrae (cytoplasmic pores) and the manifestation of a basement membrane. Currently, no protein or genetic markers can clearly distinguish healthy from damaged LSECs in acute or chronic liver disease. Single cell (sc)RNA sequencing efforts have identified several LSEC populations in mouse models for liver disease and in human cirrhotic livers. Still, there are no clearly defined genesets that can identify LSECs or dysfunctional LSEC populations in transcriptome data. Here, we developed genesets that are enriched in healthy and damaged LSECs which correlated very strongly with healthy and early stage- vs. advanced human liver diseases. A damaged LSEC signature comprised of Fabp4/5 and Vwf/a1 was established which could efficiently identify damaged endothelial cells in single cell RNAseq data sets. In LSECs from an acute CCl4 liver injury mouse model, Fabp4/5 and Vwf/a1 expression is induced within 1-3 days while in cirrhotic human livers these 4 genes are highly enriched in damaged LSECs. In conclusion, our newly developed gene signature of damaged LSECs can be applicable to a wide range of liver disease etiologies, implicating a common transcriptional alteration mechanism in LSEC damage.

Platelet Derived Growth Factor Receptor beta (PDGFRβ) has been associated to hepatic stellate cell activation and has been the target of multiple therapeutic studies. However, little is known concerning its use as a diagnostic agent.

Introduction: Chronic hepatitis B (HBV) and C (HCV) virus infection is associated with the activation of hepatic stellate cells (HSCs) toward a myofibroblastic phenotype, resulting in excessive deposition of extracellular matrix, the development of liver fibrosis, and its progression toward cirrhosis. The gold standard for the detection and staging of liver fibrosis remains the liver biopsy, which is, however, associated with some mild and severe drawbacks. Other non-invasive techniques evade these drawbacks, but lack inter-stage specificity and are unable to detect early stages of fibrosis. We investigated whether circulating vesicle-associated miRNAs can be used in the diagnosis and staging of liver fibrosis in HBV and HCV patients. Methods: Plasma samples were obtained from 14 healthy individuals and 39 early stage fibrotic patients (F0-F2) with chronic HBV or HCV infection who underwent transient elastography (Fibroscan). Extracellular vesicles were extracted from the plasma and the level of miRNA-122, -150, -192, -21, -200b, and -92a was analyzed by qRT-PCR in total plasma and circulating vesicles. Finally, these same miRNAs were also quantified in vesicles extracted from in vitro activating primary HSCs. Results: In total plasma samples, only miRNA-200b (HBV: p = 0.0384; HCV: p = 0.0069) and miRNA-122 (HBV: p < 0.0001; HCV: p = 0.0007) were significantly up-regulated during early fibrosis. In circulating vesicles, miRNA-192 (HBV: p < 0.0001; HCV: p < 0.0001), -200b (HBV: p < 0.0001; HCV: p < 0.0001), -92a (HBV: p < 0.0001; HCV: p < 0.0001), and -150 (HBV: p = 0.0016; HCV: p = 0.004) displayed a significant down-regulation in both HBV and HCV patients. MiRNA expression profiles in vesicles isolated from in vitro activating primary mouse HSCs resembled the miRNA expression profile in circulating vesicles. Conclusion: Our analysis revealed a distinct miRNA expression pattern in total plasma and its circulating vesicles. The expression profile of miRNAs in circulating vesicles of fibrotic patients suggests the potential use of these vesicle-associated miRNAs as markers for early stages of liver fibrosis.