Publications by authors named "Hans Clevers"

569 Publications

A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses.

Nat Commun 2021 09 17;12(1):5498. Epub 2021 Sep 17.

Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center, Utrecht, Netherlands.

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.
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http://dx.doi.org/10.1038/s41467-021-25729-7DOI Listing
September 2021

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

PLoS Pathog 2021 Sep 13;17(9):e1009927. Epub 2021 Sep 13.

Institute of Pharmacology and Structural Biology (IPBS), University of Toulouse, CNRS, Toulouse, France.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.
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http://dx.doi.org/10.1371/journal.ppat.1009927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8460005PMC
September 2021

Single-cell Ribo-seq reveals cell cycle-dependent translational pausing.

Nature 2021 Sep 8;597(7877):561-565. Epub 2021 Sep 8.

Oncode Institute, Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Utrecht, The Netherlands.

Single-cell sequencing methods have enabled in-depth analysis of the diversity of cell types and cell states in a wide range of organisms. These tools focus predominantly on sequencing the genomes, epigenomes and transcriptomes of single cells. However, despite recent progress in detecting proteins by mass spectrometry with single-cell resolution, it remains a major challenge to measure translation in individual cells. Here, building on existing protocols, we have substantially increased the sensitivity of these assays to enable ribosome profiling in single cells. Integrated with a machine learning approach, this technology achieves single-codon resolution. We validate this method by demonstrating that limitation for a particular amino acid causes ribosome pausing at a subset of the codons encoding the amino acid. Of note, this pausing is only observed in a sub-population of cells correlating to its cell cycle state. We further expand on this phenomenon in non-limiting conditions and detect pronounced GAA pausing during mitosis. Finally, we demonstrate the applicability of this technique to rare primary enteroendocrine cells. This technology provides a first step towards determining the contribution of the translational process to the remarkable diversity between seemingly identical cells.
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http://dx.doi.org/10.1038/s41586-021-03887-4DOI Listing
September 2021

Regulation of Solute Carriers OCT2 and OAT1/3 in the Kidney: A Phylogenetic, Ontogenetic and Cell Dynamic Perspective.

Physiol Rev 2021 09 6. Epub 2021 Sep 6.

Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

Over the course of more than 500 million years, the kidneys have undergone a remarkable evolution from primitive nephric tubes to intricate filtration-reabsorption systems that maintain homeostasis and remove metabolic end products from the body. The evolutionarily conserved solute carriers Organic Cation Transporter 2 (OCT2), and Organic Anion Transporters 1 and 3 (OAT1/3) coordinate the active secretion of a broad range of endogenous and exogenous substances, many of which accumulate in the blood of patients with kidney failure despite dialysis. Harnessing OCT2 and OAT1/3 through functional preservation or regeneration could alleviate the progression of kidney disease. Additionally, it would improve current in vitro test models that lose their expression in culture. With this review, we explore OCT2 and OAT1/3 regulation using different perspectives: phylogenetic, ontogenetic and cell dynamic. Our aim is to identify possible molecular targets to both help prevent or compensate for the loss of transport activity in patients with kidney disease, and to enable endogenous OCT2 and OAT1/3 induction in vitro in order to develop better models for drug development.
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http://dx.doi.org/10.1152/physrev.00009.2021DOI Listing
September 2021

Intestinal organoid cocultures with microbes.

Nat Protoc 2021 Aug 11. Epub 2021 Aug 11.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, the Netherlands.

Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
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http://dx.doi.org/10.1038/s41596-021-00589-zDOI Listing
August 2021

Disease modelling following organoid-based expansion of airway epithelial cells.

Am J Physiol Lung Cell Mol Physiol 2021 08 11. Epub 2021 Aug 11.

Department of Pulmonology, Leiden University Medical Center, Leiden, Netherlands.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion (3D) can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns, and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to (i) cellular composition; (ii) response to cigarette smoke exposure; (iii) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. Additionally, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.
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http://dx.doi.org/10.1152/ajplung.00234.2020DOI Listing
August 2021

Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids.

Life Sci Alliance 2021 10 9;4(10). Epub 2021 Aug 9.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, Utrecht, the Netherlands

Prime editing is a recently reported genome editing tool using a nickase-cas9 fused to a reverse transcriptase that directly synthesizes the desired edit at the target site. Here, we explore the use of prime editing in human organoids. Common TP53 mutations can be correctly modeled in human adult stem cell-derived colonic organoids with efficiencies up to 25% and up to 97% in hepatocyte organoids. Next, we functionally repaired the cystic fibrosis CFTR-F508del mutation and compared prime editing to CRISPR/Cas9-mediated homology-directed repair and adenine base editing on the CFTR-R785* mutation. Whole-genome sequencing of prime editing-repaired organoids revealed no detectable off-target effects. Despite encountering varying editing efficiencies and undesired mutations at the target site, these results underline the broad applicability of prime editing for modeling oncogenic mutations and showcase the potential clinical application of this technique, pending further optimization.
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http://dx.doi.org/10.26508/lsa.202000940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8356249PMC
October 2021

Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification.

Stem Cell Reports 2021 Sep 5;16(9):2379-2394. Epub 2021 Aug 5.

Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Centre (UMC) Utrecht, 3584 CT Utrecht, the Netherlands; Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, the Netherlands. Electronic address:

The skin epidermis is a highly compartmentalized tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific compartments in the skin but none of them is specific to the IFE. Here, we identify Troy as a marker of IFE and HF infundibulum basal layer cells in developing and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells, as well as their progeny, confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.
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http://dx.doi.org/10.1016/j.stemcr.2021.07.007DOI Listing
September 2021

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195;

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
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http://dx.doi.org/10.1073/pnas.2023373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346827PMC
August 2021

Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.

Elife 2021 07 30;10. Epub 2021 Jul 30.

Department of Biochemistry, Erasmus University Medical Center, Rotterdam, Netherlands.

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.
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http://dx.doi.org/10.7554/eLife.60747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384419PMC
July 2021

Arrest of WNT/β-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries.

Proc Natl Acad Sci U S A 2021 Jul;118(30)

CNRS, Inserm, Institut de Biologie Valrose, Université Côte d'Azur, Parc Valrose, 06108 Nice Cedex 2, France;

Germ cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell-intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of , a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis.
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http://dx.doi.org/10.1073/pnas.2023376118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325354PMC
July 2021

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model.

Cell Rep 2021 Jul;36(3):109351

School of Life Sciences, Swiss Federal Institute of Technology in Lausanne (EPFL), 1015 Lausanne, Switzerland. Electronic address:

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.
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http://dx.doi.org/10.1016/j.celrep.2021.109351DOI Listing
July 2021

Integration of metabolomics, genomics, and immune phenotypes reveals the causal roles of metabolites in disease.

Genome Biol 2021 07 6;22(1):198. Epub 2021 Jul 6.

Oncode Institute, Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, 3584, CT, Utrecht, the Netherlands.

Background: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors.

Result: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target.

Conclusion: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.
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http://dx.doi.org/10.1186/s13059-021-02413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8259168PMC
July 2021

Advancing lung organoids for COVID-19 research.

Dis Model Mech 2021 06 28;14(6). Epub 2021 Jun 28.

Oncode Institute, Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Centre, Uppsalalaan 8, Utrecht 3584 CT, The Netherlands.

The COVID-19 pandemic has emphasised the need to develop effective treatments to combat emerging viruses. Model systems that poorly represent a virus' cellular environment, however, may impede research and waste resources. Collaborations between cell biologists and virologists have led to the rapid development of representative organoid model systems to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We believe that lung organoids, in particular, have advanced our understanding of SARS-CoV-2 pathogenesis, and have laid a foundation to study future pandemic viruses and develop effective treatments.
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http://dx.doi.org/10.1242/dmm.049060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272930PMC
June 2021

Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis.

EMBO Mol Med 2021 Jul 24;13(7):e13067. Epub 2021 Jun 24.

Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

Nephropathic cystinosis is a severe monogenic kidney disorder caused by mutations in CTNS, encoding the lysosomal transporter cystinosin, resulting in lysosomal cystine accumulation. The sole treatment, cysteamine, slows down the disease progression, but does not correct the established renal proximal tubulopathy. Here, we developed a new therapeutic strategy by applying omics to expand our knowledge on the complexity of the disease and prioritize drug targets in cystinosis. We identified alpha-ketoglutarate as a potential metabolite to bridge cystinosin loss to autophagy, apoptosis and kidney proximal tubule impairment in cystinosis. This insight combined with a drug screen revealed a bicalutamide-cysteamine combination treatment as a novel dual-target pharmacological approach for the phenotypical correction of cystinotic kidney proximal tubule cells, patient-derived kidney tubuloids and cystinotic zebrafish.
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http://dx.doi.org/10.15252/emmm.202013067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8261496PMC
July 2021

Organoids and organs-on-chips: Insights into human gut-microbe interactions.

Cell Host Microbe 2021 Jun;29(6):867-878

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584 CT Utrecht, the Netherlands; Oncode Institute, Hubrecht Institute, 3584 CT Utrecht, the Netherlands; The Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands. Electronic address:

The important and diverse roles of the gut microbiota in human health and disease are increasingly recognized. The difficulty of inferring causation from metagenomic microbiome sequencing studies and from mouse-human interspecies differences has prompted the development of sophisticated in vitro models of human gut-microbe interactions. Here, we review recent advances in the co-culture of microbes with intestinal and colonic epithelia, comparing the rapidly developing fields of organoids and organs-on-chips with other standard models. We describe how specific individual processes by which microbes and epithelia interact can be recapitulated in vitro. Using examples of bacterial, viral, and parasitic infections, we highlight the advantages of each culture model and discuss current trends and future possibilities to build more complex co-cultures.
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http://dx.doi.org/10.1016/j.chom.2021.04.002DOI Listing
June 2021

NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

Nature 2021 06 2;594(7863):430-435. Epub 2021 Jun 2.

Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.
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http://dx.doi.org/10.1038/s41586-021-03525-zDOI Listing
June 2021

Transcription factor Ascl2 promotes germinal center B cell responses by directly regulating AID transcription.

Cell Rep 2021 Jun;35(9):109188

Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China; Beijing Key Lab for Immunological Research on Chronic Diseases, Beijing 100084, China. Electronic address:

During germinal center (GC) reactions, activated B cells undergo clonal expansion and functional maturation to produce high-affinity antibodies and differentiate into plasma and memory cells, accompanied with class-switching recombination (CSR) and somatic hypermutation (SHM). Activation-induced cytidine deaminase (AID) is responsible for both CSR and SHM in GC B cells. Transcriptional mechanisms underlying AID regulation and GC B cell reactions are still not well understood. Here, we show that expression of Ascl2 transcription factor is upregulated in GC B cells. Ectopic expression of Ascl2 promotes GC B cell development and enhances antibody production and affinity maturation. Conversely, deletion of Ascl2 in B cells impairs the GC response. Genome-wide analysis reveals that Ascl2 directly regulates GC B cell-related genes, including AID; ectopic expression of AID in Ascl2-deficient B cells rescues their antibody defects. Thus, Ascl2 regulates AID transcription and promotes GC B cell responses.
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http://dx.doi.org/10.1016/j.celrep.2021.109188DOI Listing
June 2021

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids.

J Vis Exp 2021 04 30(170). Epub 2021 Apr 30.

Crown Bioscience Inc.;

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.
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http://dx.doi.org/10.3791/62495DOI Listing
April 2021

KSR1- and ERK-dependent translational regulation of the epithelial-to-mesenchymal transition.

Elife 2021 May 10;10. Epub 2021 May 10.

Eppley Institute, University of Nebraska Medical Center, Omaha, United States.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here, we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.
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http://dx.doi.org/10.7554/eLife.66608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8195604PMC
May 2021

Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.

Cell Stem Cell 2021 05;28(5):816-832

Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. Electronic address:

Hepatic, pancreatic, and biliary (HPB) organoids are powerful tools for studying development, disease, and regeneration. As organoid research expands, the need for clear definitions and nomenclature describing these systems also grows. To facilitate scientific communication and consistent interpretation, we revisit the concept of an organoid and introduce an intuitive classification system and nomenclature for describing these 3D structures through the consensus of experts in the field. To promote the standardization and validation of HPB organoids, we propose guidelines for establishing, characterizing, and benchmarking future systems. Finally, we address some of the major challenges to the clinical application of organoids.
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http://dx.doi.org/10.1016/j.stem.2021.04.005DOI Listing
May 2021

Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.

Cell Stem Cell 2021 08 13;28(8):1380-1396.e6. Epub 2021 Apr 13.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Oncode Institute, Jaarbeursplein 6, 3521 AL Utrecht, the Netherlands; Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands. Electronic address:

Cervical cancer is a common gynecological malignancy often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study the cervical epithelium and the cancers derived thereof. Here we describe a long-term culturing protocol for ecto- and endocervical epithelia that generates 3D organoids that stably recapitulate the two tissues of origin. As evidenced for HSV-1, organoid-based cervical models may serve to study sexually transmitted infections. Starting from Pap brush material, a small biobank of tumoroids derived from affected individuals was established that retained the causative human papillomavirus (HPV) genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutic agents and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches.
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http://dx.doi.org/10.1016/j.stem.2021.03.012DOI Listing
August 2021

The frontier of live tissue imaging across space and time.

Cell Stem Cell 2021 04;28(4):603-622

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA. Electronic address:

What you see is what you get-imaging techniques have long been essential for visualization and understanding of tissue development, homeostasis, and regeneration, which are driven by stem cell self-renewal and differentiation. Advances in molecular and tissue modeling techniques in the last decade are providing new imaging modalities to explore tissue heterogeneity and plasticity. Here we describe current state-of-the-art imaging modalities for tissue research at multiple scales, with a focus on explaining key tradeoffs such as spatial resolution, penetration depth, capture time/frequency, and moieties. We explore emerging tissue modeling and molecular tools that improve resolution, specificity, and throughput.
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http://dx.doi.org/10.1016/j.stem.2021.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034393PMC
April 2021

Exploring the human lacrimal gland using organoids and single-cell sequencing.

Cell Stem Cell 2021 07 16;28(7):1221-1232.e7. Epub 2021 Mar 16.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Center Utrecht, 3584 CT Utrecht, the Netherlands; Oncode Institute, Hubrecht Institute, 3584 CT Utrecht, the Netherlands. Electronic address:

The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.
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http://dx.doi.org/10.1016/j.stem.2021.02.024DOI Listing
July 2021

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Nat Protoc 2021 04 10;16(4):1936-1965. Epub 2021 Mar 10.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
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http://dx.doi.org/10.1038/s41596-020-00474-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221035PMC
April 2021

The Organoid Platform: Promises and Challenges as Tools in the Fight against COVID-19.

Stem Cell Reports 2021 03;16(3):412-418

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht, 3584 CT Utrecht, the Netherlands; Oncode Institute, Hubrecht Institute, 3584 CT Utrecht, the Netherlands. Electronic address:

Many pathogenic viruses that affect man display species specificity, limiting the use of animal models. Studying viral biology and identifying potential treatments therefore benefits from the development of in vitro cell systems that closely mimic human physiology. In the current COVID-19 pandemic, rapid scientific insights are of the utmost importance to limit its impact on public health and society. Organoids are emerging as versatile tools to progress the understanding of SARS-CoV-2 biology and to aid in the quest for novel treatments.
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http://dx.doi.org/10.1016/j.stemcr.2020.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940129PMC
March 2021

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

Cell Rep 2021 03;34(10):108819

Hubrecht Institute and Oncode Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, the Netherlands; Princess Maxima Center for Pediatric Oncology, Utrecht, the Netherlands. Electronic address:

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1 KRT15 stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.
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http://dx.doi.org/10.1016/j.celrep.2021.108819DOI Listing
March 2021

Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip.

Nat Protoc 2021 04 5;16(4):2023-2050. Epub 2021 Mar 5.

Hubrecht Institute-Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, the Netherlands.

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.
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http://dx.doi.org/10.1038/s41596-020-00479-wDOI Listing
April 2021
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