Single Cell Atlas: a single-cell multi-omics human cell encyclopedia.

Single-cell sequencing datasets are key in biology and medicine for unraveling insights into heterogeneous cell populations with unprecedented resolution. Here, we construct a single-cell multi-omics map of human tissues through in-depth characterizations of datasets from five single-cell omics, spatial transcriptomics, and two bulk omics across 125 healthy adult and fetal tissues. We construct its complement web-based platform, the Single Cell Atlas (SCA, www.singlecellatlas.org ), to enable vast interactive data exploration of deep multi-omics signatures across human fetal and adult tissues. The atlas resources and database queries aspire to serve as a one-stop, comprehensive, and time-effective resource for various omics studies.

Meta-analysis of the global distribution of clinically relevant CYP2C8 alleles and their inferred functional consequences.

BACKGROUND: CYP2C8 is responsible for the metabolism of 5% of clinically prescribed drugs, including antimalarials, anti-cancer and anti-inflammatory drugs. Genetic variability is an important factor that influences CYP2C8 activity and modulates the pharmacokinetics, efficacy and safety of its substrates. RESULTS: We profiled the genetic landscape of CYP2C8 variability using data from 96 original studies and data repositories that included a total of 33,185 unrelated participants across 44 countries and 43 ethnic groups. The reduced function allele CYP2C8*2 was most common in West and Central Africa with frequencies of 16-36.9%, whereas it was rare in Europe and Asia (< 2%). In contrast, CYP2C8*3 and CYP2C8*4 were common throughout Europe and the Americas (6.9-19.8% for *3 and 2.3-7.5% for *4), but rare in African and East Asian populations. Importantly, we observe pronounced differences (> 2.3-fold) between neighboring countries and even between geographically overlapping populations. Overall, we found that 20-60% of individuals in Africa and Europe carry at least one CYP2C8 allele associated with reduced metabolism and increased adverse event risk of the anti-malarial amodiaquine. Furthermore, up to 60% of individuals of West African ancestry harbored variants that reduced the clearance of pioglitazone, repaglinide, paclitaxel and ibuprofen. In contrast, reduced function alleles are only found in < 2% of East Asian and 8.3-12.8% of South and West Asian individuals. CONCLUSIONS: Combined, the presented analyses mapped the genetic and inferred functional variability of CYP2C8 with high ethnogeographic resolution. These results can serve as a valuable resource for CYP2C8 allele frequencies and distribution estimates of CYP2C8 phenotypes that could help identify populations at risk upon treatment with CYP2C8 substrates. The high variability between ethnic groups incentivizes high-resolution pharmacogenetic profiling to guide precision medicine and maximize its socioeconomic benefits, particularly for understudied populations with distinct genetic profiles.

Individualized pharmacotherapy utilizing genetic biomarkers and novel in vitro systems as predictive tools for optimal drug development and treatment.

In the area of drug development and clinical pharmacotherapy, a profound understanding of the pharmacokinetics and potential adverse reactions associated with the drug under investigation is paramount. Essential to this endeavour is a comprehensive understanding about interindividual variations in ADME genetics and the predictive capabilities of in vitro systems, shedding light on metabolite formation and the risk of adverse drug reactions (ADRs). Both the domains of pharmacogenomics and the advancement of in vitro systems are experiencing rapid expansion. Here we present an update on these burgeoning fields, providing an overview of their current status and illuminating potential future directions. Significance Statement There is a very rapid development in the area of pharmacogenomics and in vitro systems for predicting drug pharmacokinetics and risk for adverse drug reactions. We provide an update of the current status of pharmacogenomics and developed in vitro systems on these aspects aimed to achieve a better personalized pharmacotherapy.

Comparison of Human Long-Term Liver Models for Clearance Prediction of Slowly Metabolized Compounds.

The accurate prediction of human clearance is an important task during drug development. The proportion of low clearance compounds has increased in drug development pipelines across the industry since such compounds may be dosed in lower amounts and at lower frequency. Such compounds present new challenges to in vitro systems used for clearance extrapolation. In this study we compared the accuracy of clearance predictions of suspension culture to four different long-term stable in vitro liver models, including HepaRG sandwich culture, the Hµrel stochastic co-culture, the Hepatopac micropatterned co-culture (MPCC) and a micro-array spheroid culture. Hepatocytes in long-term stable systems remained viable and active over several days of incubation. Although intrinsic clearance values were generally high in suspension culture, clearance of low turnover compounds could frequently not be determined using this method. Metabolic activity and intrinsic clearance values from HepaRG cultures were low and, consequently, many compounds with low turnover did not show significant decline despite long incubation times. Similarly, stochastic co-cultures occasionally failed to show significant turnover for multiple low and medium turnover compounds. Among the different methods, MPCCs and spheroids provided the most consistent measurements. Notably, all culture methods resulted in underprediction of clearance, this could however be compensated for by regression correction. Combined, the results indicate that spheroid culture as well as the MPCC system provide adequate in vitro tools for human extrapolation for compounds with low metabolic turnover. Significance Statement In this study, we compared suspension cultures, HepaRG sandwich cultures, the Hµrel liver stochastic co-cultures, the Hepatopac micropatterned co-cultures (MPCC) and micro-array spheroid cultures for low clearance determination and prediction. Overall, HepaRG and suspension cultures showed modest value for the low determination and prediction of clearance compounds. The micro-array spheroid culture resulted in the most robust clearance measurements, whereas using the MPCC resulted in the most accurate prediction for low clearance compounds.

Consequences of Amyloid-ß Deficiency for the Liver.

The hepatic content of amyloid beta (Aß) decreases drastically in human and rodent cirrhosis highlighting the importance of understanding the consequences of Aß deficiency in the liver. This is especially relevant in view of recent advances in anti-Aß therapies for Alzheimer's disease (AD). Here, it is shown that partial hepatic loss of Aß in transgenic AD mice immunized with Aß antibody 3D6 and its absence in amyloid precursor protein (APP) knockout mice (APP-KO), as well as in human liver spheroids with APP knockdown upregulates classical hallmarks of fibrosis, smooth muscle alpha-actin, and collagen type I. Aß absence in APP-KO and deficiency in immunized mice lead to strong activation of transforming growth factor-ß (TGFß), alpha secretases, NOTCH pathway, inflammation, decreased permeability of liver sinusoids, and epithelial-mesenchymal transition. Inversely, increased systemic and intrahepatic levels of Aß42 in transgenic AD mice and neprilysin inhibitor LBQ657-treated wild-type mice protect the liver against carbon tetrachloride (CCl4 )-induced injury. Transcriptomic analysis of CCl4 -treated transgenic AD mouse livers uncovers the regulatory effects of Aß42 on mitochondrial function, lipid metabolism, and its onco-suppressive effects accompanied by reduced synthesis of extracellular matrix proteins. Combined, these data reveal Aß as an indispensable regulator of cell-cell interactions in healthy liver and a powerful protector against liver fibrosis.

Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells.

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

Population-specific variations in KCNH2 predispose patients to delayed ventricular repolarization upon dihydroartemisinin-piperaquine therapy.

Dihydroartemisinin-piperaquine is efficacious for the treatment of uncomplicated malaria and its use is increasing globally. Despite the positive results in fighting malaria, inhibition of the Kv11.1 channel (hERG; encoded by the KCNH2 gene) by piperaquine has raised concerns about cardiac safety. Whether genetic factors could modulate the risk of piperaquine-mediated QT prolongations remained unclear. Here, we first profiled the genetic landscape of KCNH2 variability using data from 141,614 individuals. Overall, we found 1,007 exonic variants distributed over the entire gene body, 555 of which were missense. By optimizing the gene-specific parametrization of 16 partly orthogonal computational algorithms, we developed a KCNH2-specific ensemble classifier that identified a total of 116 putatively deleterious missense variations. To evaluate the clinical relevance of KCNH2 variability, we then sequenced 293 Malian patients with uncomplicated malaria and identified 13 variations within the voltage sensing and pore domains of Kv11.1 that directly interact with channel blockers. Cross-referencing of genetic and electrocardiographic data before and after piperaquine exposure revealed that carriers of two common variants, rs1805121 and rs41314375, experienced significantly higher QT prolongations (ΔQTc of 41.8 ms and 61 ms, respectively, vs 14.4 ms in controls) with more than 50% of carriers having increases in QTc >30 ms. Furthermore, we identified three carriers of rare population-specific variations who experienced clinically relevant delayed ventricular repolarization. Combined, our results map population-scale genetic variability of KCNH2 and identify genetic biomarkers for piperaquine-induced QT prolongation that could help to flag at-risk patients and optimize efficacy and adherence to antimalarial therapy.

Inflammatory, metabolic, and sex-dependent gene-regulatory dynamics of microglia and macrophages in neonatal hippocampus after hypoxia-ischemia.

Neonatal hypoxia-ischemia (HI) is a major cause of perinatal death and long-term disabilities worldwide. Post-ischemic neuroinflammation plays a pivotal role in HI pathophysiology. In the present study, we investigated the temporal dynamics of microglia (CX3CR1GFP/+) and infiltrating macrophages (CCR2RFP/+) in the hippocampi of mice subjected to HI at postnatal day 9. Using inflammatory pathway and transcription factor (TF) analyses, we identified a distinct post-ischemic response in CCR2RFP/+ cells characterized by differential gene expression in sensome, homeostatic, matrisome, lipid metabolic, and inflammatory molecular signatures. Three days after injury, transcriptomic signatures of CX3CR1GFP/+ and CCR2RFP/+ cells isolated from hippocampi showed a partial convergence. Interestingly, microglia-specific genes in CX3CR1GFP/+ cells showed a sexual dimorphism, where expression returned to control levels in males but not in females during the experimental time frame. These results highlight the importance of further investigations on metabolic rewiring to pave the way for future interventions in asphyxiated neonates.

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 choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function.

Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments.

Intraocular liver spheroids for non-invasive high-resolution in vivo monitoring of liver cell function.

Longitudinal monitoring of liver function in vivo is hindered by the lack of high-resolution non-invasive imaging techniques. Using the anterior chamber of the mouse eye as a transplantation site, we have established a platform for longitudinal in vivo imaging of liver spheroids at cellular resolution. Transplanted liver spheroids engraft on the iris, become vascularized and innervated, retain hepatocyte-specific and liver-like features and can be studied by in vivo confocal microscopy. Employing fluorescent probes administered intravenously or spheroids formed from reporter mice, we showcase the potential use of this platform for monitoring hepatocyte cell cycle activity, bile secretion and lipoprotein uptake. Moreover, we show that hepatic lipid accumulation during diet-induced hepatosteatosis is mirrored in intraocular in vivo grafts. Here, we show a new technology which provides a crucial and unique tool to study liver physiology and disease progression in pre-clinical and basic research.

Next-generation sequencing in pharmacogenomics - fit for clinical decision support?

INTRODUCTION: The technological advances of sequencing methods during the past 20 years have fuelled the generation of large amounts of sequencing data that comprise common variations, as well as millions of rare and personal variants that would not be identified by conventional genotyping. While comprehensive sequencing is technically feasible, its clinical utility for guiding personalized treatment decisions remains controversial. AREAS COVERED: We discuss the opportunities and challenges of comprehensive sequencing compared to targeted genotyping for pharmacogenomic applications. Current pharmacogenomic sequencing panels are heterogeneous and clinical actionability of the included genes is not a major focus. We provide a current overview and critical discussion of how current studies utilize sequencing data either retrospectively from biobanks, databases or repurposed diagnostic sequencing, or prospectively using pharmacogenomic sequencing. EXPERT OPINION: While sequencing-based pharmacogenomics has provided important insights into genetic variations underlying the safety and efficacy of a multitude pharmacological treatments, important hurdles for the clinical implementation of pharmacogenomic sequencing remain. We identify gaps in the interpretation of pharmacogenetic variants, technical challenges pertaining to complex loci and variant phasing, as well as unclear cost-effectiveness and incomplete reimbursement. It is critical to address these challenges in order to realize the promising prospects of pharmacogenomic sequencing.

Pharmacogenomics Beyond Single Common Genetic Variants: The Way Forward.

Interindividual variability in genes encoding drug-metabolizing enzymes, transporters, receptors, and human leukocyte antigens has a major impact on a patient's response to drugs with regard to efficacy and safety. Enabled by both technological and conceptual advances, the field of pharmacogenomics is developing rapidly. Major progress in omics profiling methods has enabled novel genotypic and phenotypic characterization of patients and biobanks. These developments are paralleled by advances in machine learning, which have allowed us to parse the immense wealth of data and establish novel genetic markers and polygenic models for drug selection and dosing. Pharmacogenomics has recently become more widespread in clinical practice to personalize treatment and to develop new drugs tailored to specific patient populations. In this review, we provide an overview of the latest developments in the field and discuss the way forward, including how to address the missing heritability, develop novel polygenic models, and further improve the clinical implementation of pharmacogenomics.

Adjudin improves beta cell maturation, hepatic glucose uptake and glucose homeostasis.

AIMS/HYPOTHESIS: Recovering functional beta cell mass is a promising approach for future diabetes therapies. The aim of the present study is to investigate the effects of adjudin, a small molecule identified in a beta cell screen using zebrafish, on pancreatic beta cells and diabetes conditions in mice and human spheroids. METHODS: In zebrafish, insulin expression was examined by bioluminescence and quantitative real-time PCR (qPCR), glucose levels were examined by direct measurements and distribution using a fluorescent glucose analogue, and calcium activity in beta cells was analysed by in vivo live imaging. Pancreatic islets of wild-type postnatal day 0 (P0) and 3-month-old (adult) mice, as well as adult db/db mice (i.e. BKS(D)-Leprdb/JOrlRj), were cultured in vitro and analysed by qPCR, glucose stimulated insulin secretion and whole mount staining. RNA-seq was performed for islets of P0 and db/db mice. For in vivo assessment, db/db mice were treated with adjudin and subjected to analysis of metabolic variables and islet cells. Glucose consumption was examined in primary human hepatocyte spheroids. RESULTS: Adjudin treatment increased insulin expression and calcium response to glucose in beta cells and decreased glucose levels after beta cell ablation in zebrafish. Adjudin led to improved beta cell function, decreased beta cell proliferation and glucose responsive insulin secretion by decreasing basal insulin secretion in in vitro cultured newborn mouse islets. RNA-seq of P0 islets indicated that adjudin treatment resulted in increased glucose metabolism and mitochondrial function, as well as downstream signalling pathways involved in insulin secretion. In islets from db/db mice cultured in vitro, adjudin treatment strengthened beta cell identity and insulin secretion. RNA-seq of db/db islets indicated adjudin-upregulated genes associated with insulin secretion, membrane ion channel activity and exocytosis. Moreover, adjudin promoted glucose uptake in the liver of zebrafish in an insulin-independent manner, and similarly promoted glucose consumption in primary human hepatocyte spheroids with insulin resistance. In vivo studies using db/db mice revealed reduced nonfasting blood glucose, improved glucose tolerance and strengthened beta cell identity after adjudin treatment. CONCLUSIONS/INTERPRETATION: Adjudin promoted functional maturation of immature islets, improved function of dysfunctional islets, stimulated glucose uptake in liver and improved glucose homeostasis in db/db mice. Thus, the multifunctional drug adjudin, previously studied in various contexts and conditions, also shows promise in the management of diabetic states. DATA AVAILABILITY: Raw and processed RNA-seq data for this study have been deposited in the Gene Expression Omnibus under accession number GSE235398 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235398 ).

Compound Absorption in Polymer Devices Impairs the Translatability of Preclinical Safety Assessments.

Organotypic and microphysiological systems (MPS) that can emulate the molecular phenotype and function of human tissues, such as liver, are increasingly used in preclinical drug development. However, despite their improved predictivity, drug development success rates have remained low with most compounds failing in clinical phases despite promising preclinical data. Here, it is tested whether absorption of small molecules to polymers commonly used for MPS fabrication can impact preclinical pharmacological and toxicological assessments and contribute to the high clinical failure rates. To this end, identical devices are fabricated from eight different MPS polymers and absorption of prototypic compounds with different physicochemical properties are analyzed. It is found that overall absorption is primarily driven by compound hydrophobicity and the number of rotatable bonds. However, absorption can differ by >1000-fold between polymers with polydimethyl siloxane (PDMS) being most absorptive, whereas polytetrafluoroethylene (PTFE) and thiol-ene epoxy (TEE) absorbed the least. Strikingly, organotypic primary human liver cultures successfully flagged hydrophobic hepatotoxins in lowly absorbing TEE devices at therapeutically relevant concentrations, whereas isogenic cultures in PDMS devices are resistant, resulting in false negative safety signals. Combined, these results can guide the selection of MPS materials and facilitate the development of preclinical assays with improved translatability.

Wnt/ß-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

BACKGROUND AND AIMS: The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/ß-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear. METHODS: Organotypic 3D spheroid cultures of primary human or murine hepatocytes were used to identify the signaling network underlying cell cycle re-entry. Furthermore, we performed chemogenomic screening of a library enriched for epigenetic regulators and modulators of immune function to determine the importance of epigenomic control for human hepatocyte regeneration. RESULTS: Our results showed that, unlike in rodents, activation of Wnt/ß-catenin signaling is the major mitogenic cue for adult primary human hepatocytes. Furthermore, we identified TGFß inhibition and inflammatory signaling through NF-κB as essential steps for the quiescent-to-regenerative switch that allows Wnt/ß-catenin-induced proliferation of human cells. In contrast, growth factors, but not Wnt/ß-catenin signaling, triggered hyperplasia in murine hepatocytes. High-throughput screening in a human model confirmed the relevance of NFκB and revealed the critical roles of polycomb repressive complex 2, as well as of the bromodomain families I, II, and IV. CONCLUSIONS: This study revealed a network of NFκB, TGFß, and Wnt/ß-catenin that controls human hepatocyte regeneration in the absence of exogenous growth factors, identified novel regulators of hepatocyte proliferation, and highlighted the potential of organotypic culture systems for chemogenomic interrogation of complex physiological processes.