Bioengineering vascularized liver tissue for biomedical research and application.

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.

In utero exposures to perfluoroalkyl substances and the human fetal liver metabolome in Scotland: a cross-sectional study.

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.

XBP1 as a novel molecular target to attenuate drug resistance in hepatocellular carcinoma.

INTRODUCTION: Despite improvements in clinical management of hepatocellular carcinoma (HCC), prognosis remains poor with a 5-year survival rate less than 40%. Drug resistance in HCC makes it challenging to treat; therefore, it is imperative to develop new therapeutic strategies. Higher expression of X-box binding protein 1 (XBP1) in tumor cells is highly correlated with poor prognosis. In tumor cells, XBP1 modulates the unfolded protein response (UPR) to restore homeostasis in endoplasmic reticulum. Targeting XBP1 could be a promising therapeutic strategy to overcome HCC resistance and improve the survival rate of patients. AREAS COVERED: This review provides the recent evidence that indicates XBP1 is involved in HCC drug resistance via DNA damage response, drug inactivation, and inhibition of apoptosis. In addition, the potential roles of XBP1 in inducing resistance in HCC cells were highlighted, and we showed how its inhibition could sensitize tumor cells to controlled cell death. EXPERT OPINION: Due to the diversity in molecular mechanism of multidrug-resistance, targeting one specific pathway is inadequate. XBP1 inhibition could be a potential therapeutic target to overcome verity of resistance mechanisms. The main function of this transcription factor in HCC treatment response is an attractive area for further studies and should be discussed more.

The Influence of Sex Hormones in Liver Function and Disease.

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.

In Vivo Tumorigenicity of the 20q11.21 Amplicon in an Engraftment Model of hPSCs and Differentiated Liver Cells.

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.

Serum-Free Production of Human Stem Cell-Derived Liver Spheres for Cancer Metastasis Research.

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.

Chemically cross-linked hydrogels from repetitive protein arrays.

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.

Identification of an FXR-modulated liver-intestine hybrid state in iPSC-derived hepatocyte-like cells.

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.

Nuclear factor programming improves stem-cell-derived hepatocyte phenotype.

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.

Maternal over-the-counter analgesics use during pregnancy and adverse perinatal outcomes: cohort study of 151 141 singleton pregnancies.

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.

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival.

Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.

Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease.

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.

Serum-Free Production of Three-Dimensional Hepatospheres from Pluripotent Stem Cells.

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.

Dimethyl fumarate reduces hepatocyte senescence following paracetamol exposure.

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-ß 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.

Modeling human hepatic steatosis in pluripotent stem cell-derived hepatocytes.

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).

Protocol for automated production of human stem cell derived liver spheres.

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).

Mathematical modelling of oxygen gradients in stem cell-derived liver tissue.

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.

A human pluripotent stem cell model for the analysis of metabolic dysfunction in hepatic steatosis.

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. 13C 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.

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

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.