A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities.

BACKGROUND: Primary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function. METHODS: By mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions. RESULTS: The novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors. CONCLUSIONS: This study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.

Sex hormones differently regulate lipid metabolism genes in primary human hepatocytes.

BACKGROUND: Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is higher in men than in women. Hormonal and genetic causes may account for the sex differences in MASLD. Current human in vitro liver models do not sufficiently take the influence of biological sex and sex hormones into consideration. METHODS: Primary human hepatocytes (PHHs) were isolated from liver specimen of female and male donors and cultured with sex hormones (17ß-estradiol, testosterone and progesterone) for up to 72 h. mRNA expression levels of 8 hepatic lipid metabolism genes were analyzed by RT-qPCR. Sex hormones and their metabolites were determined in cell culture supernatants by LC-MS analyses. RESULTS: A sex-specific expression was observed for LDLR (low density lipoprotein receptor) with higher mRNA levels in male than female PHHs. All three sex hormones were metabolized by PHHs and the effects of hormones on gene expression levels varied depending on hepatocyte sex. Only in female PHHs, 17ß-estradiol treatment affected expression levels of PPARA (peroxisome proliferator-activated receptor alpha), LIPC (hepatic lipase) and APOL2 (apolipoprotein L2). Further changes in mRNA levels of female PHHs were observed for ABCA1 (ATP-binding cassette, sub-family A, member 1) after testosterone and for ABCA1, APOA5 (apolipoprotein A-V) and PPARA after progesterone treatment. Only the male PHHs showed changing mRNA levels for LDLR after 17ß-estradiol and for APOA5 after testosterone treatment. CONCLUSIONS: Male and female PHHs showed differences in their expression levels of hepatic lipid metabolism genes and their responsiveness towards sex hormones. Thus, cellular sex should be considered, especially when investigating the pathophysiological mechanisms of MASLD.

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS: Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS: We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS: We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION: We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19.

The AP-1 adaptor complex is essential for intracellular trafficking of the ORF2 capsid protein and assembly of Hepatitis E virus.

Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.

Analysis of host factor networks during hepatitis B virus infection in primary human hepatocytes.

BACKGROUND: Chronic hepatitis B virus (HBV) infection affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects. METHODS: PHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7 days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qRT-PCR was performed to validate the RNA-sequencing results, ensuring consistent findings. RESULTS: We observed significant alterations in the expression patterns of 149 genes from days 1 to 7 following HBV infection (R2 > 0.7, q < 0.05). Functional analysis of these genes identified RNA-binding proteins involved in mRNA metabolism and the regulation of alternative splicing during HBV infection. Results from qRT-PCR experiments and the analysis of two validation datasets suggest that RBM14 and RPL28 may serve as potential biomarkers for HBV-associated HCC. CONCLUSIONS: Transcriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.

Induction of steatosis in primary human hepatocytes recapitulates key pathophysiological aspects of metabolic dysfunction-associated steatotic liver disease.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease. Owing to limited available treatment options, novel pre-clinical models for target selection and drug validation are warranted. We have established and extensively characterized a primary human steatotic hepatocyte in vitro model system that could guide the development of treatment strategies for MASLD. METHODS: Cryopreserved primary human hepatocytes from five donors varying in sex and ethnicity were cultured with free fatty acids in a 3D collagen sandwich for 7 days and the development of MASLD was followed by assessing classical hepatocellular functions. As proof of concept, the effects of the drug firsocostat (GS-0976) on in vitro MASLD phenotypes were evaluated. RESULTS: Incubation with free fatty acids induced steatosis, insulin resistance, mitochondrial dysfunction, inflammation, and alterations in prominent human gene signatures similar to patients with MASLD, indicating the recapitulation of human MASLD in this system. The application of firsocostat rescued clinically observed fatty liver disease pathologies, highlighting the ability of the in vitro system to test the efficacy and potentially characterize the mode of action of drug candidates. CONCLUSIONS: Altogether, our human MASLD in vitro model system could guide the development and validation of novel targets and drugs for the treatment of MASLD. IMPACT AND IMPLICATIONS: Due to low drug efficacy and high toxicity, clinical treatment options for metabolic dysfunction-associated steatotic liver disease (MASLD) are currently limited. To facilitate earlier stop-go decisions in drug development, we have established a primary human steatotic hepatocyte in vitro model. As the model recapitulates clinically relevant MASLD characteristics at high phenotypic resolution, it can serve as a pre-screening platform and guide target identification and validation in MASLD therapy.

Deciphering the phospho-signature induced by hepatitis B virus in primary human hepatocytes.

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted 2- and 7-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both time points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

Examining the hepatotoxic potential of cannabidiol, cannabidiol-containing hemp extract, and cannabinol at consumer-relevant exposure concentrations in primary human hepatocytes.

Hemp extracts and consumer products containing cannabidiol (CBD) and/or other phytocannabinoids derived from hemp have entered the marketplace in recent years. CBD is an approved drug in the United States for the treatment of certain seizure disorders. While effects of CBD in the liver have been well characterized, data on the effects of other cannabinoids and hemp extracts in the liver and methods for studying these effects in vitro are limited. This study examined the hepatotoxic potential of CBD, CBD concentration-matched hemp extract, and cannabinol (CBN), at consumer-relevant concentrations determined by in silico modeling, in vitro using primary human hepatocytes. Primary human hepatocytes exposed to between 10-nM and 25-µM CBD, CBN, or hemp extract for 24 and 48 h were evaluated by measuring lactate dehydrogenase release, apoptosis, albumin secretion, urea secretion, and mitochondrial membrane potential. Cell viability was not significantly affected by CBD, CBN, or the hemp extract at any of the concentrations tested. Exposure to hemp extract induced a modest but statistically significant decrease in albumin secretion, urea secretion, and mitochondrial membrane potential at the highest concentration tested whereas CBD only induced a modest but statistically significant decrease in albumin secretion compared with vehicle control. Although this study addresses data gaps in the understanding of cannabinoid hepatoxicity in vitro, additional studies will be needed to determine how these results correlate with relevant consumer exposure and the biological effects of cannabinoids in human liver.

Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models.

Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.

The differences in drug disposition gene induction by rifampicin and rifabutin are unlikely due to different effects on important pregnane X receptor (NR1I2) splice variants.

Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 µM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 µM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 µM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 µM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.

Single-cell metabolic profiling reveals subgroups of primary human hepatocytes with heterogeneous responses to drug challenge.

BACKGROUND: Xenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes are the gold standard model for the assessment of drug efficacy, safety, and toxicity in the early phases of drug development. Recent advances in single-cell genomics demonstrate liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte specialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis. RESULTS: Here, we investigate the metabolic capacity of individual human hepatocytes in vitro. We assess how chronic accumulation of lipids enhances cellular heterogeneity and impairs the metabolisms of drugs. Using a phenotyping five-probe cocktail, we identify four functional subgroups of hepatocytes responding differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism-related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminishes the drug-related metabolic capacity of hepatocytes. CONCLUSIONS: Our results demonstrate that, upon a metabolic challenge such as exposure to drugs or intracellular fat accumulation, hepatocyte subgroups display different and heterogeneous transcriptional responses.

In vitro testing of host-targeting small molecule antiviral matriptase/TMPRSS2 inhibitors in 2D and 3D cell-based assays.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic strongly stimulated the development of small molecule antivirals selectively targeting type II transmembrane serine proteases (TTSP), required for the host-cell entry of numerous viruses. A set of 3-amidinophenylalanine derivatives (MI-21, MI-472, MI-477, MI-485, MI-1903 and MI-1904), which inhibit the cleavage of certain viral glycoproteins was characterized in 2D and 3D primary human hepatocyte models on collagen- and Matrigel-coating using a CCK-8 assay to evaluate their cytotoxicity, a resorufin-based method to detect redox imbalances, fluorescence and ultrafiltration experiments to evaluate their interactions with human serum albumin (HSA) and α-acidic glycoprotein (AGP), and luminescence measurement to assess CYP3A4 modulation. For elucidation of selectivity of the applied compounds towards matriptase, transmembrane serine protease 2 (TMPRRS2), thrombin and factor Xa (FXa) Ki values were determined. It was proven that cell viability was only deteriorated by inhibitor MI-1903, and redox status was not influenced by administration of the selected inhibitors at 50 µM for 24 h. MI-472 and MI-477 formed relatively stable complexes with AGP. CYP3A4 inhibition was found to be strong in PHHs exposed to all inhibitors with the exception of MI-21, which seems to be a promising drug candidate also due to its better selectivity towards matriptase and TMPRSS2 over the blood clotting proteases thrombin and FXa. Our in vitro pharmacokinetic screening with these inhibitors helps to select the compounds with the best selectivity and safety profile suitable for a further preclinical characterization without animal sacrifice.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects.

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation.

Hepatic in vitro models that accurately replicate phenotypes and functionality of the human liver are needed for applications in toxicology, pharmacology and biomedicine. Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities. Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation. We demonstrate the manufacture of accurate liver microphysiological systems (MPS) via engineering of 3D stereolithography printed hydrogel chips with arrays of diffusion open synthetic vasculature channels at spacings approaching in vivo capillary distances. Chip designs are compatible with seeding of cell suspensions or preformed liver cell spheroids. Importantly, primary human hepatocytes (PHH) and hiPSC-derived hepatocyte-like cells remain viable, exhibit improved molecular phenotypes compared to isogenic monolayer and static spheroid cultures and form interconnected tissue structures over the course of multiple weeks in perfused culture. 3D optical oxygen mapping of embedded sensor beads shows that the liver MPS recapitulates oxygen gradients found in the acini, which translates into zone-specific acet-ami-no-phen toxicity patterns. Zonation, here naturally generated by high cell densities and associated oxygen and nutrient utilization along the flow path, is also documented by spatial proteomics showing increased concentration of periportal- versus perivenous-associated proteins at the inlet region and vice versa at the outlet region. The presented microperfused liver MPS provides a promising platform for the mesoscale culture of human liver cells at phenotypically relevant densities and oxygen exposures. STATEMENT OF SIGNIFICANCE: A full 3D tissue culture platform is presented, enabled by massively parallel arrays of high-resolution 3D printed microperfusion hydrogel channels that functionally mimics tissue vasculature. The platform supports long-term culture of liver models with dimensions of several millimeters at physiologically relevant cell densities, which is difficult to achieve with other methods. Human liver models are generated from seeded primary human hepatocytes (PHHs) cultured for two weeks, and from seeded spheroids of hiPSC-derived human liver-like cells cultured for two months. Both model types show improved functionality over state-of-the-art 3D spheroid suspensions cultured in parallel. The platform can generate physiologically relevant oxygen gradients driven by consumption rather than supply, which was validated by visualization of embedded oxygen-sensitive microbeads, which is exploited to demonstrate zonation-specific toxicity in PHH liver models.

HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes.

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

PURPOSE: Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. METHODS: Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. RESULTS: Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. CONCLUSION: The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Dicloxacillin-warfarin drug-drug interaction-A register-based study and in vitro investigations in 3D spheroid primary human hepatocytes.

AIMS: Dicloxacillin is used to treat staphylococcal infections and we have previously shown that dicloxacillin is an inducer of cytochrome P450 enzymes (CYPs). Here, we employed a translational approach to investigate the effect of a treatment with dicloxacillin on warfarin efficacy in Danish registries. Furthermore, we assessed dicloxacillin as an inducer of CYPs in vitro. METHODS: We conducted a register-based study and analysed international normalized ratio (INR) levels in chronic warfarin users before and after short- and long-term use of dicloxacillin (n = 1023) and flucloxacillin (n = 123). Induction of CYPs were investigated in a novel liver model of 3D spheroid primary human hepatocytes at the level of mRNA, and protein and enzyme activity. RESULTS: Short- and long-term dicloxacillin treatments decreased INR levels by -0.65 (95% confidence interval [CI]: -0.57 to -0.74) and -0.76 (95% CI: -0.50 to -1.02), respectively. More than 90% of individuals experienced subtherapeutic INR levels (below 2) after long-term dicloxacillin treatment. Flucloxacillin decreased INR levels by -0.37 (95% CI: -0.14 to -0.60). In 3D spheroid primary human hepatocytes, the maximal induction of CYP3A4 mRNA, protein and enzyme activity by dicloxacillin were 4.9-, 2.9- and 2.4-fold, respectively. Dicloxacillin also induced CYP2C9 mRNA by 1.7-fold. CONCLUSION: Dicloxacillin induces CYPs and reduces the clinical efficacy of warfarin in patients. This effect is substantially exacerbated during long-term treatment with dicloxacillin. The in vitro results corroborated this drug-drug interaction and correlated to the clinical findings. Caution is warranted for warfarin patients that initiate dicloxacillin or flucloxacillin, especially for a long-term treatment of endocarditis.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Potency ranking of pyrrolizidine alkaloids in metabolically competent human liver cancer cells and primary human hepatocytes using a genotoxicity test battery.

Pyrrolizidine alkaloids (PAs) occur as contaminants in plant-based foods and herbal medicines. Following metabolic activation by cytochrome P450 (CYP) enzymes, PAs induce DNA damage, hepatotoxicity and can cause liver cancer in rodents. There is ample evidence that the chemical structure of PAs determines their toxicity. However, more quantitative genotoxicity data are required, particularly in primary human hepatocytes (PHH). Here, the genotoxicity of eleven structurally different PAs was investigated in human HepG2 liver cells with CYP3A4 overexpression and PHH using an in vitro test battery. Furthermore, the data were subject to benchmark dose (BMD) modeling to derive the genotoxic potency of individual PAs. The cytotoxicity was initially determined in HepG2-CYP3A4 cells, revealing a clear structure-toxicity relationship for the PAs. Importantly, experiments in PHH confirmed the structure-dependent toxicity and cytotoxic potency ranking of the tested PAs. The genotoxicity markers γH2AX and p53 as well as the alkaline Comet assay consistently demonstrated a structure-dependent genotoxicity of PAs in HepG2-CYP3A4 cells, correlating well with their cytotoxic potency. BMD modeling yielded BMD values in the range of 0.1-10 µM for most cyclic and open diesters, followed by the monoesters. While retrorsine showed the highest genotoxic potency, monocrotaline and lycopsamine displayed the lowest genotoxicity. Finally, experiments in PHH corroborated the genotoxic potency ranking, and revealed genotoxic effects even in the absence of detectable cytotoxicity. In conclusion, our findings strongly support the concept of grouping PAs into potency classes and help to pave the way for a broader acceptance of relative potency factors in risk assessment.

Effect of Preoperative Chemotherapy on the Isolation Outcome of Primary Human Hepatocytes.

Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pretreated and untreated donor. Furthermore, the transcription levels of cytochrome P450 (CYP) 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens before isolation seem to perform as well as hepatocytes without preoperative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies. Impact statement Isolated primary human hepatocytes are an essential resource for pharmacological and toxicological studies. Our results present further evidence that isolated hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies-especially when fluoropyrimidine-based regimens are used.