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Two experiments examining repetition priming in face recognition are reported. They employed eight rather than the more usual two presentation trials so that the prediction made by Logan's (1988) instance model of power function speedup of response time (RT) distributions could be examined. In Experiment 1, we presented the same photograph on each trial; in Experiment 2, we presented photographs of varying poses. Both experiments showed repetition priming effects for familiar and unfamiliar faces, power function speedup for both the mean and the standard deviation of RT and the power function speedup of the quanties of the RT distributions. We argue that our findings are consistent with the predictions made by the instance model and provide an explanatory challenge for alternative theoretical approaches. [ABSTRACT FROM AUTHOR]

Fat accumulation, de novo lipogenesis, and glycolysis are key drivers of hepatocyte reprogramming and the consequent metabolic dysfunction-associated steatotic liver disease (MASLD). Here we report that obesity leads to dysregulated expression of hepatic protein-tyrosine phosphatases (PTPs). PTPRK was found to be increased in steatotic hepatocytes in both humans and mice, and correlates positively with PPARγ-induced lipogenic signaling. High-fat-fed PTPRK knockout male and female mice have lower weight gain and reduced hepatic fat accumulation. Phosphoproteomic analysis in primary hepatocytes and hepatic metabolomics identified fructose-1,6-bisphosphatase 1 and glycolysis as PTPRK targets in metabolic reprogramming. Mechanistically, PTPRK-induced glycolysis enhances PPARγ and lipogenesis in hepatocytes. Silencing PTPRK in liver cancer cell lines reduces colony-forming capacity and high-fat-fed PTPRK knockout mice exposed to a hepatic carcinogen develop smaller tumours. Our study defines the role of PTPRK in the regulation of hepatic glycolysis, lipid metabolism, and tumour development in obesity., (© 2024. The Author(s).)

To accurately study human organ function and disease 'in the dish', it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver, which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however, severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore, the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study, we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore, we screened a compound library to identify additives that improved the maturity of in vitro -engineered human tissue, with the rationale that by taking such an approach, we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue.

The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.

Traditional drug development is a long and expensive process with high rates of failure. This has prompted the pharmaceutical industry to seek more efficient drug development frameworks, driving the emergence of organ-on-a-chip (OOC) based on microfluidic technologies. Unlike traditional animal experiments, OOC systems provide a more accurate simulation of human organ microenvironments and physiological responses, therefore offering a cost-effective and efficient platform for biomedical research, particularly in the development of new medicines. Additionally, OOC systems enable quick and real-time analysis, high-throughput experimentation, and automation. These advantages have shown significant promise in enhancing the drug development process. The success of an OOC system hinges on the integration of specific designs, manufacturing techniques, and biosensors to meet the need for integrated multiparameter datasets. This review focuses on the manufacturing, design, sensing systems, and applications of OOC systems, highlighting their design and sensing capabilities, as well as the technical challenges they currently face.

Hepatotropic viruses are amongst the most ubiquitous pathogens worldwide, causing significant morbidity and mortality. As hepatocytes are among the primary targets of these viruses, their ability to mount early effective innate defence responses is of major research interest. Interferon lambda (IFNL) is produced early in response to viral stimulation in other cell types, but hepatocyte production of this interferon is little investigated. Due to the difficulty and significant costs in obtaining and culturing human primary hepatocytes, surrogate systems are widely sought. Here we used induced pluripotent stem (iPS)-derived hepatocyte-like cells (HLCs) to investigate hepatic IFNL expression in response to viral-like ligands. We demonstrate that hepatocytes rely on cytoplasmic pattern recognition receptors (PRRs) such as Protein Kinase RNA-dependent (PKR) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR) for the detection of double stranded RNA . Stimulation of HLCs by viral-like RNA ligands activating cytosolic RNA sensors resulted in thousand fold increase of type III interferon gene expression. These results are in contrast with type I IFN expression, which was induced to a lower extent. Concomitant induction of interferon stimulated genes, such as interferon-stimulated gene 15 (ISG15) and CXCL10, indicated the ability of HLCs to activate interferon-dependent activity. These results demonstrate that HLCs mount an innate antiviral response upon stimulation with viral-like RNA characterized by the induction of type III IFN., (© The Author(s) 2024. Published by Oxford University Press.)

The liver performs a wide range of biological functions that are essential to body homeostasis. Damage to liver tissue can result in reduced organ function, and if chronic in nature can lead to organ scarring and progressive disease. Currently, donor liver transplantation is the only longterm treatment for end-stage liver disease. However, orthotopic organ transplantation suffers from several drawbacks that include organ scarcity and lifelong immunosuppression. Therefore, new therapeutic strategies are required. One promising strategy is the engineering of implantable and vascularized liver tissue. This resource could also be used to build the next generation of liver tissue models to better understand human health, disease and aging in vitro . This article reviews recent progress in the field of liver tissue bioengineering, including microfluidic-based systems, bio-printed vascularized tissue, liver spheroids and organoid models, and the induction of angiogenesis in vivo .

Background: Perfluoroalkyl and polyfluoroalkyl substances are classed as endocrine disrupting compounds but continue to be used in many products such as firefighting foams, flame retardants, utensil coatings, and waterproofing of food packaging. Perfluoroalkyl exposure aberrantly modulates lipid, metabolite, and bile acid levels, increasing susceptibility to onset and severity of metabolic diseases, such as diabetes and metabolic dysfunction-associated steatotic liver disease. To date, most studies in humans have focused on perfluoroalkyl-exposure effects in adults. In this study we aimed to show if perfluoroalkyls are present in the human fetal liver and if they have metabolic consequences for the human fetus., Methods: In this cross-sectional study, human fetal livers from elective termination of pregnancies at the Aberdeen Pregnancy Counselling Service, Aberdeen, UK, were analysed by both targeted (bile acids and perfluoroalkyl substances) and combined targeted and untargeted (lipids and polar metabolites) mass spectrometry based metabolomic analyses, as well as with RNA-Seq. Only fetuses from normally progressing pregnancies (determined at ultrasound scan before termination), terminated for non-medical reasons, from women older than 16 years, fluent in English, and between 11 and 21 weeks of gestation were collected. Women exhibiting considerable emotional distress or whose fetuses had anomalies identified at ultrasound scan were excluded. Stringent bioinformatic and statistical methods such as partial correlation network analysis, linear regression, and pathway analysis were applied to this data to investigate the association of perfluoroalkyl exposure with hepatic metabolic pathways., Findings: Fetuses included in this study were collected between Dec 2, 2004, and Oct 27, 2014. 78 fetuses were included in the study: all 78 fetuses were included in the metabolomics analysis (40 female and 38 male) and 57 fetuses were included in the RNA-Seq analysis (28 female and 29 male). Metabolites associated with perfluoroalkyl were identified in the fetal liver and these varied with gestational age. Conjugated bile acids were markedly positively associated with fetal age. 23 amino acids, fatty acids, and sugar derivatives in fetal livers were inversely associated with perfluoroalkyl exposure, and the bile acid glycolithocholic acid was markedly positively associated with all quantified perfluoroalkyl. Furthermore, 7α-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis rate, was strongly positively associated with perfluoroalkyl levels and was detectable as early as gestational week 12., Interpretation: Our study shows direct evidence for the in utero effects of perfluoroalkyl exposure on specific key hepatic products. Our results provide evidence that perfluoroalkyl exposure, with potential future consequences, manifests in the human fetus as early as the first trimester of gestation. Furthermore, the profiles of metabolic changes resemble those observed in perinatal perfluoroalkyl exposures. Such exposures are already linked with susceptibility, initiation, progression, and exacerbation of a wide range of metabolic diseases., Funding: UK Medical Research Council, Horizon Europe Program of the European Union, Seventh Framework Programme of the European Union, NHS Grampian Endowments grants, European Partnership for the Assessment of Risks from Chemicals, Swedish Research Council, Formas, Novo Nordisk Foundation, and the Academy of Finland., Competing Interests: Declaration of interests DCH is a founder, director, and shareholder in Stemnovate and Stimuliver. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Prof David C Hay is a founder, shareholder and director of Stimuliver ApS and Stemnovate Limited., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The liver performs a multitude of bodily functions, whilst retaining the ability to regenerate damaged tissue. In this review, we discuss sex steroid biology, regulation of mammalian liver physiology and the development of new model systems to improve our understanding of liver biology in health and disease. A major risk factor for the development of liver disease is hepatic fibrosis. Key drivers of this process are metabolic dysfunction and pathologic activation of the immune system. Although non-alcoholic fatty liver disease (NAFLD) is largely regarded as benign, it does progress to non-alcoholic steatohepatitis in a subset of patients, increasing their risk of developing cirrhosis and hepatocellular carcinoma. NAFLD susceptibility varies across the population, with obesity and insulin resistance playing a strong role in the disease development. Additionally, sex and age have been identified as important risk factors. In addition to the regulation of liver biochemistry, sex hormones also regulate the immune system, with sexual dimorphism described for both innate and adaptive immune responses. Therefore, sex differences in liver metabolism, immunity and their interplay are important factors to consider when designing, studying and developing therapeutic strategies to treat human liver disease. The purpose of this review is to provide the reader with a general overview of sex steroid biology and their regulation of mammalian liver physiology.

Human pluripotent stem cells (hPSCs) are a promising source of somatic cells for clinical applications and disease modelling. However, during culture they accumulate genetic aberrations such as amplification of 20q11.21 which occurs in approximately 20% of extensively cultured hPSC lines and confers a BCL2L1-mediated survival advantage. During the production of the large number of cells required for transplantation and therapy these aberrations may become unavoidable which has important safety implications for therapies and may also impact upon disease modelling. Presently, these risks are poorly understood; whilst it is apparent that large-scale genetic aberrations can pose an oncogenic risk, the risks associated with smaller, more insidious changes have not been fully explored. In this report, the effects of engraftment of human embryonic stem cells (hESCs) and hESC-derived hepatocyte-like cells (HLCs) with and without amplification of the 20q11.21 minimal amplicon and isochromosome 20q (i20q) in SCID-beige mice are presented. The cells were tracked in vivo using a luminescent reporter over a period of approximately four months. Intrasplenic injection of hESCs showed greater engraftment potential and the formation of more severely disruptive lesions in the liver and spleen of animals injected with cells containing 20q11.21 compared with i20q and wild type. HLCs with 20q11.21 engrafted more successfully and formed more severely disruptive lesions than wild type cells or cells with i20q. These results reinforce the notion that karyotyping of therapeutic hPSC is required for transplant, and suggest that screening for known common aberrations is necessary. Further work to identify commonly arising genetic aberrations should be performed and routine screening for hPSCs intended for therapeutic use should be used., Competing Interests: This work was funded by UK Regenerative Medicine PlatformThe authors declare no conflicts of interest., (Copyright © Journal of Stem Cells and Regenerative Medicine.)

Renewable and scalable human liver tissue platforms are a powerful tool to study organ physiology and model diseases, such as cancer. Stem cell-derived models provide an alternative to cell lines, which can display limited relevance to primary cells and tissue. Historically, two-dimensional (2D) cultures have been used to model liver biology as they are easy to scale and deploy. However, 2D liver models lack functional diversity and phenotypic stability in long-term culture. To address those issues, protocols for generating the three-dimensional (3D) tissue aggregates have been developed. Here, we describe a methodology to generate 3D liver spheres from pluripotent stem cells. Liver spheres are composed of three key liver cell types (hepatic progenitor cells, endothelial cells, and hepatic stellate cells) and have been used to study human cancer cell metastasis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Aims: Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLC) have enormous potential as a replacement for primary hepatocytes in drug screening, toxicology and cell replacement therapy, but their genome-wide expression patterns differ strongly from primary human hepatocytes (PHH)., Methods: We differentiated human induced pluripotent stem cells (hiPSC) via definitive endoderm to HLC and characterized the cells by single-cell and bulk RNA-seq, with complementary epigenetic analyses. We then compared HLC to PHH and publicly available data on human fetal hepatocytes (FH) ex vivo; we performed bioinformatics-guided interventions to improve HLC differentiation via lentiviral transduction of the nuclear receptor FXR and agonist exposure., Results: Single-cell RNA-seq revealed that transcriptomes of individual HLC display a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH - mostly intestinal genes - within the same cell. Bulk-level overrepresentation analysis, as well as regulon analysis at the single-cell level, identified sets of regulatory factors discriminating HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and increased the ability of bile canalicular secretion as well as lipid droplet formation, thereby increasing HLCs' similarity to PHH. The undesired non-liver gene expression was reproducibly decreased, although only by a moderate degree., Conclusion: In contrast to physiological hepatocyte precursor cells and mature hepatocytes, HLC co-express liver and hybrid genes in the same cell. Targeted modification of the FXR gene regulatory network improves their differentiation by suppressing intestinal traits whilst inducing hepatocyte features., Lay Summary: Generation of human hepatocytes from stem cells represents an active research field but its success is hampered by the fact that the stem cell-derived 'hepatocytes' still show major differences to hepatocytes obtained from a liver. Here, we identified an important reason for the difference, specifically that the stem cell-derived 'hepatocyte' represents a hybrid cell with features of hepatocytes and intestinal cells. We show that a specific protein (FXR) suppresses intestinal and induces liver features, thus bringing the stem cell-derived cells closer to hepatocytes derived from human livers., Competing Interests: Conflict of interest Patricio Godoy is also affiliated with F. Hoffmann-La Roche Ltd (Roche Innovation Center Basel, Basel, Switzerland), Barbara Küppers-Munther is affiliated with Takara Bio Europe AB (former Cellartis AB) (Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden). Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In this issue of Cell Stem Cell, Ma et al. demonstrate that the activation of the nuclear receptor thyroid hormone receptor beta (NR1A2) improves the differentiation status of hepatocyte-like cells derived from human pluripotent stem cells., Competing Interests: Declaration of interests D.H. is a founder, director, and shareholder in Stimuliver ApS and Stemnovate Limited., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Objectives: To identify any associations between in utero exposure to five over-the-counter (non-prescription) analgesics (paracetamol, ibuprofen, aspirin, diclofenac, naproxen) and adverse neonatal outcomes., Design: Retrospective cohort study using the Aberdeen Maternity and Neonatal Databank., Participants: 151 141 singleton pregnancies between 1985 and 2015., Main Outcome Measures: Premature delivery (<37 weeks), stillbirth, neonatal death, birth weight, standardised birthweight score, neonatal unit admission, APGAR score at 1 and 5 min, neural tube and amniotic band defects, gastroschisis and, in males, cryptorchidism and hypospadias., Results: 83.7% of women taking over-the-counter analgesics reported first trimester use when specifically asked about use at their first antenatal clinic visit. Pregnancies exposed to at least one of the five analgesics were significantly independently associated with increased risks for premature delivery <37 weeks (adjusted OR (aOR)=1.50, 95% CI 1.43 to 1.58), stillbirth (aOR=1.33, 95% CI 1.15 to 1.54), neonatal death (aOR=1.56, 95% CI 1.27 to 1.93), birth weight <2500 g (aOR=1.28, 95% CI 1.20 to 1.37), birth weight >4000 g (aOR=1.09, 95% CI 1.05 to 1.13), admission to neonatal unit (aOR=1.57, 95% CI 1.51 to 1.64), APGAR score <7 at 1 min (aOR=1.18, 95% CI 1.13 to 1.23) and 5 min (aOR=1.48, 95% CI 1.35 to 1.62), neural tube defects (aOR=1.64, 95% CI 1.08 to 2.47) and hypospadias (aOR=1.27, 95% CI 1.05 to 1.54 males only). The overall prevalence of over-the-counter analgesics use during pregnancy was 29.1%, however it rapidly increased over the 30-year study period, to include over 60% of women in the last 7 years of the study. This makes our findings highly relevant to the wider pregnant population., Conclusions: Over-the-counter (non-prescription) analgesics consumption during pregnancy was associated with a substantially higher risk for adverse perinatal health outcomes in the offspring. The use of paracetamol in combination with other non-steroidal anti-inflammatory drugs conferred the highest risk. The increased risks of adverse neonatal outcomes associated with non-prescribed, over-the-counter, analgesics use during pregnancy indicate that healthcare guidance for pregnant women regarding analgesic use need urgent updating., Competing Interests: Competing interests: DCH is a founder, director and shareholder in Stemnovate Limited. The remaining authors have no interests to disclose., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Biliary diseases can cause inflammation, fibrosis, bile duct destruction, and eventually liver failure. There are no curative treatments for biliary disease except for liver transplantation. New therapies are urgently required. We have therefore purified human biliary epithelial cells (hBECs) from human livers that were not used for liver transplantation. hBECs were tested as a cell therapy in a mouse model of biliary disease in which the conditional deletion of Mdm2 in cholangiocytes causes senescence, biliary strictures, and fibrosis. hBECs are expandable and phenotypically stable and help restore biliary structure and function, highlighting their regenerative capacity and a potential alternative to liver transplantation for biliary disease., Competing Interests: Declaration of interests S.J.F. and J.D.M.C. are founders and scientific advisors of Resolution Therapeutics Ltd (not related to this study). D.C.H. is a founder, director, and shareholder at Stemnovate Limited and Stimuliver ApS (not related to this study)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing renewable human liver tissue from stem cells has been pursued as a potential source of biological material for pharmaceutical and clinical endeavors. At present, two-dimensional differentiation procedures deliver tissue lacking long-term phenotypic and functional stability. Efforts to overcome these limiting factors have led to the development of protocols to generate three-dimensional cellular aggregates. Here we describe a methodology to generate 3D hepatospheres from human pluripotent stem cells using defined and commercially available reagents., (© 2021. Springer Science+Business Media, LLC.)

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro .

Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-β signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo . We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties., Competing Interests: D.C.H is a co-founder and shareholder of Stemnovate Ltd. J.M.-R. and P.J.S.-L. are presently employed at Resolution Therapeutics. E.M.T., E.M.G., H.J., N.B. and A.E.K. are/were employees of Novo Nordisk Ltd. The rest of the authors certify that they have no conflicts of interest in the subject matter or materials discussed in this article. D.C.H. is serving as a guest editor on a special issue for iScience in 2021, and this issue is not related to the current article., (© 2021 The Author(s).)

This protocol describes how to produce human liver spheres from pluripotent stem cell-derived hepatic progenitors, endothelial cells, and hepatic stellate cells. Liver spheres form by self-assembly in microwells, generating up to 73 spheres per well of a 96-well plate. This process was automated using liquid handling and pipetting systems, permitting cost-effective scale-up and reducing sphere variability. In its current form, this system provides a powerful tool to generate human liver tissue for disease modeling and drug screening. For complete details on the use and execution of this protocol, please refer to Lucendo-Villarin et al. (2020) (https://doi.org/10.1088/1758-5090/abbdb2)., Competing Interests: Professor David Hay is a founder, director, and shareholder in Stemnovate Limited., (© 2021 The Author(s).)

This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021)., Competing Interests: Professor David Hay is a founder, director, and shareholder in Stemnovate Limited., (© 2021 The Author(s).)

A major bottleneck in the study of human liver physiology is the provision of stable liver tissue in sufficient quantity. As a result, current approaches to modelling human drug efficacy and toxicity rely heavily on immortalized human and animal cell lines. These models are informative but do possess significant drawbacks. To address the issues presented by those models, researchers have turned to pluripotent stem cells (PSCs). PSCs can be generated from defined genetic backgrounds, are scalable, and capable of differentiation to all the cell types found in the human body, representing an attractive source of somatic cells for in vitro and in vivo endeavours. Although unlimited numbers of somatic cell types can be generated in vitro, their maturation still remains problematic. In order to develop high fidelity PSC-derived liver tissue, it is necessary to better understand the cell microenvironment in vitro including key elements of liver physiology. In vivo a major driver of zonated liver function is the oxygen gradient that exists from periportal to pericentral regions. In this paper, we demonstrate how cell culture conditions for PSC-derived liver sphere systems can be optimised to recapitulate physiologically relevant oxygen gradients by using mathematical modelling. The mathematical model incorporates some often-understated features and mechanisms of traditional spheroid systems such as cell-specific oxygen uptake, media volume, spheroid size, and well dimensions that can lead to a spatially heterogeneous distribution of oxygen. This mathematical modelling approach allows for the calibration and identification of culture conditions required to generate physiologically realistic function within the microtissue through recapitulation of the in vivo microenvironment., Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: SDW contributed to this work whilst employed as an academic at Liverpool John Moores University. He has since taken up employment at Syngenta and has interests in the company. DCH is a co-founder, director and shareholder of Stemnovate Limited. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Analgesia during pregnancy is often necessary. Due to their widespread availability, many mothers opt to use over-the-counter (OTC) analgesics. Those analgesic compounds and their metabolites can readily cross the placenta and reach the developing foetus. Evidence for safety or associations with adverse health outcomes is conflicting, limiting definitive decision-making for healthcare professionals., Objective and Rationale: This review provides a detailed and objective overview of research in this field. We consider the global prevalence of OTC analgesia during pregnancy, explain the current mechanistic understanding of how analgesic compounds cross the placenta and reach the foetus, and review current research on exposure associations with offspring health outcomes., Search Methods: A comprehensive English language literature search was conducted using PubMed and Scopus databases. Different combinations of key search terms were used including 'over-the-counter/non-prescription analgesics', 'pregnancy', 'self-medication', 'paracetamol', 'acetaminophen', 'diclofenac', 'aspirin', 'ibuprofen', 'in utero exposure', 'placenta drug transport', 'placental transporters', 'placenta drug metabolism' and 'offspring outcomes'., Outcomes: This article examines the evidence of foetal exposure to OTC analgesia, starting from different routes of exposure to evidence, or the lack thereof, linking maternal consumption to offspring ill health. There is a very high prevalence of maternal consumption of OTC analgesics globally, which is increasing sharply. The choice of analgesia selected by pregnant women differs across populations. Location was also observed to have an effect on prevalence of use, with more developed countries reporting the highest consumption rates. Some of the literature focuses on the association of in utero exposure at different pregnancy trimesters and the development of neurodevelopmental, cardiovascular, respiratory and reproductive defects. This is in contrast to other studies which report no associations., Wider Implications: The high prevalence and the challenges of reporting exact consumption rates make OTC analgesia during pregnancy a pressing reproductive health issue globally. Even though some healthcare policy-making authorities have declared the consumption of some OTC analgesics for most stages of pregnancy to be safe, such decisions are often based on partial review of literature. Our comprehensive review of current evidence highlights that important knowledge gaps still exist. Those areas require further research in order to provide pregnant mothers with clear guidance with regard to OTC analgesic use during pregnancy., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13 C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models., Competing Interests: Professor David Hay is a founder, shareholder, and director in Stemnovate Limited. All other authors declare that they have no competing interests., (© 2020 The Author(s).)

The debate about possible adverse effects of bisphenol A (BPA) has been ongoing for decades. Bisphenol F (BPF) and S (BPS) have been suggested as "safer" alternatives. In the present study we used hepatocyte-like cells (HLCs) derived from the human embryonic stem cell lines Man12 and H9 to compare the three bisphenol derivatives. Stem cell-derived progenitors were produced using an established system and were exposed to BPA, BPF and BPS for 8 days during their transition to HLCs. Subsequently, we examined cell viability, inhibition of cytochrome P450 (CYP) activity, and genome-wide RNA profiles. Sub-cytotoxic, inhibitory concentrations (IC 50 ) of CYP3A were 20, 9.5 and 25 µM for BPA, BPF and BPS in Man12 derived HLCs, respectively. The corresponding concentrations for H9-derived HLCs were 19, 29 and 31 µM. These IC 50 concentrations were used to study global expression changes in this in vitro study and are higher than unconjugated BPA in serum of the general population. A large overlap of up- as well as downregulated genes induced by the three bisphenol derivatives was seen. This is at least 28-fold higher compared to randomly expected gene expression changes. Moreover, highly significant correlations of expression changes induced by the three bisphenol derivatives were obtained in pairwise comparisons. Dysregulated genes were associated with reduced metabolic function, cellular differentiation, embryonic development, cell survival and apoptosis. In conclusion, no major differences in cytochrome inhibitory activities of BPA, BPF and BPS were observed and gene expression changes showed a high degree of similarity., (Copyright © 2020 Lucendo-Villarin et al.)

Liver disease represents an increasing cause of global morbidity and mortality. Currently, liver transplant is the only treatment curative for end-stage liver disease. Donor organs cannot meet the demand and therefore scalable treatments and new disease models are required to improve clinical intervention. Pluripotent stem cells represent a renewable source of human tissue. Recent advances in three-dimensional cell culture have provided the field with more complex systems that better mimic liver physiology and function. Despite these improvements, current cell-based models are variable in performance and expensive to manufacture at scale. This is due, in part, to the use of poorly defined or cross-species materials within the process, severely affecting technology translation. To address this issue, we have developed an automated and economical platform to produce liver tissue at scale for modelling disease and small molecule screening. Stem cell derived liver spheres were formed by combining hepatic progenitors with endothelial cells and stellate cells, in the ratios found within the liver. The resulting tissue permitted the study of human liver biology 'in the dish' and could be scaled for screening. In summary, we have developed an automated differentiation system that permits reliable self-assembly of human liver tissue for biomedical application. Going forward we believe that this technology will not only serve as an in vitro resource, and may have an important role to play in supporting failing liver function in humans., (Creative Commons Attribution license.)

Hepatocytes are key players in the innate immune response to liver pathogens but are challenging to study because of inaccessibility and a short half-life. Recent advances in in vitro differentiation of hepatocyte-like cells (HLCs) facilitated studies of hepatocyte-pathogen interactions. Here, we aimed to define the anti-viral innate immune potential of human HLCs with a focus on toll-like receptor (TLR)-expression and the presence of a metabolic switch. We analysed cytoplasmic pattern recognition receptor (PRR)- and endosomal TLR-expression and activity and adaptation of HLCs to an inflammatory environment. We found that transcript levels of retinoic acid inducible gene I (RIG-I), melanoma differentiation antigen 5 (MDA5), and TLR3 became downregulated during differentiation, indicating the acquisition of a more tolerogenic phenotype, as expected in healthy hepatocytes. HLCs responded to activation of RIG-I by producing interferons (IFNs) and IFN-stimulated genes. Despite low-level expression of TLR3, receptor expression was upregulated in an inflammatory environment. TLR3 signalling induced expression of proinflammatory cytokines at the gene level, indicating that several PRRs need to interact for successful innate immune activation. The inflammatory responsiveness of HLCs was accompanied by the downregulation of cytochrome P450 3A and 1A2 activity and decreased serum protein production, showing that the metabolic switch seen in primary hepatocytes during anti-viral responses is also present in HLCs., Competing Interests: Prof. D.C.H. is a founder, director, and shareholder in Stemnovate Limited. The other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4α) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4α locus.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl 4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl 4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl 4 -induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.

Background & Aims: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known., Methods: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles., Results: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues., Conclusions: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols., Competing Interests: G.R., S.B., W.C., J.T. and T.B. are employees of Novartis Pharma AG. D.C.H is a co-founder and shareholder in Stemnovate Limited. This does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have nothing to disclose.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.  Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

Non-alcoholic fatty liver disease (NAFLD) is estimated to affect 24% of the global adult population. NAFLD is a major risk factor for the development of cirrhosis and hepatocellular carcinoma, as well as being strongly associated with type 2 diabetes and cardiovascular disease. It has been proposed that up to 88% of obese adults have NAFLD, and with global obesity rates increasing, this disease is set to become even more prevalent. Despite intense research in this field, the molecular processes underlying the pathology of NAFLD remain poorly understood. Hepatic intracellular lipid accumulation may lead to dysregulated tricarboxylic acid (TCA) cycle activity and associated alterations in metabolite levels. The TCA cycle metabolites alpha-ketoglutarate, succinate and fumarate are allosteric regulators of the alpha-ketoglutarate-dependent dioxygenase family of enzymes. The enzymes within this family have multiple targets, including DNA and chromatin, and thus may be capable of modulating gene transcription in response to intracellular lipid accumulation through alteration of the epigenome. In this review, we discuss what is currently understood in the field and suggest areas for future research which may lead to the development of novel preventative or therapeutic interventions for NAFLD.

During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α's Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60-100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background : The liver plays a central role in human drug metabolism. To model drug metabolism, the major cell type of the liver, the hepatocyte, is commonly used. Hepatocytes can be derived from human and animal sources, including pluripotent stem cells. Cell-based models have shown promise in modeling human drug exposure. The assays used in those studies are normally 'snap-shot' in nature, and do not provide the complete picture of human drug exposure. Research design and methods : In this study, we employ stem cell-derived hepatocytes and impedance sensing to model human drug toxicity. This impedance-based stem cell assay reports hepatotoxicity in real time after treatment with compounds provided by industry. Results : Using electric cell-substrate impedance Sensing (ECIS), we were able to accurately measure drug toxicity post-drug exposure in real time and more quickly than gold standard biochemical assays. Conclusions : ECIS is robust and non-destructive methodology capable of monitoring human drug exposure with superior performance to current gold standard 'snapshot' assays. We believe that the methodology presented within this article could prove valuable in the quest to better predict off-target effects of drugs in humans.

Human pluripotent stem cells (hPSCs) have the potential to transform medicine. However, hurdles remain to ensure safety for such cellular products. Science-based understanding of the requirements for source materials is required as are appropriate materials. Leaders in hPSC biology, clinical translation, biomanufacturing and regulatory issues were brought together to define requirements for source materials for the production of hPSC-derived therapies and to identify other key issues for the safety of cell therapy products. While the focus of this meeting was on hPSC-derived cell therapies, many of the issues are generic to all cell-based medicines. The intent of this report is to summarize the key issues discussed and record the consensus reached on each of these by the expert delegates.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology 'in the dish', and in the future may permit the support of compromised liver function in humans.

Theoretical and practical advances in time-frequency analysis, in general, and the continuous wavelet transform (CWT), in particular, have increased over the last two decades. Although the Morlet wavelet has been the default choice for wavelet analysis, a new family of analytic wavelets, known as generalized Morse wavelets, which subsume several other analytic wavelet families, have been increasingly employed due to their time and frequency localization benefits and their utility in isolating and extracting quantifiable features in the time-frequency domain. The current paper describes two practical applications of analysing the features obtained from the generalized Morse CWT: (i) electromyography, for isolating important features in muscle bursts during skating, and (ii) electrocardiography, for assessing heart rate variability, which is represented as the ridge of the main transform frequency band. These features are subsequently quantified to facilitate exploration of the underlying physiological processes from which the signals were generated.This article is part of the theme issue 'Redundancy rules: the continuous wavelet transform comes of age'., (© 2017 The Author(s).)

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in 96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro , but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'., (© 2018 The Authors.)

Stem cell-derived hepatocyte-like cells (HLCs) offer great opportunities for studies of host-pathogen interactions and tissue regeneration, as well as hepatotoxicity. To reliably predict the outcome of infection or to enhance graft survival, a finely tuned innate immune system is essential. Hepatocytes have long been considered solely metabolic and their critical innate immune potential is only recently gaining attention. Viral infection studies show that pathogen detection by cytosolic receptors leads to interferon (IFN) induction in primary hepatocytes and HLCs. IFN expression in HLCs is characterized by strong expression of type III IFN and low expression of type I IFN which is also a characteristic of primary hepatocytes. The response to IFN differs in HLCs with lower interferon-stimulated gene (ISG)-expression levels than in primary hepatocytes. Tumour necrosis factor-alpha (TNF-α) signalling is less studied in HLCs, but appears to be functional. Expression of toll-like receptors (TLR) 2-5, 7 and 9 has been reported in primary hepatocytes but has been poorly studied in HLCs. In summary, although they retain some immature features, HLCs are in many ways superior to hepatoma cell lines for cell-based modelling. In this review, we will provide an overview of innate immune signalling in HLCs and how this compares with primary hepatocytes.This article is part of the themed issue 'Designer human tissue: coming to a lab near you'., (© 2018 The Author(s).)