RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily affects the lung but can also cause gastrointestinal (GI) symptoms. In vitro experiments confirmed that SARS-CoV-2 robustly infects intestinal epithelium. However, data on infection of adult gastric epithelium are sparse and a side-by-side comparison of the infection in the major segments of the GI tract is lacking. We provide this direct comparison in organoid-derived monolayers and demonstrate that SARS-CoV-2 robustly infects intestinal epithelium, while gastric epithelium is resistant to infection. RNA sequencing and proteome analysis pointed to angiotensin-converting enzyme 2 (ACE2) as a critical factor, and, indeed, ectopic expression of ACE2 increased susceptibility of gastric organoid-derived monolayers to SARS-CoV-2. ACE2 expression pattern in GI biopsies of patients mirrors SARS-CoV-2 infection levels in monolayers. Thus, local ACE2 expression limits SARS-CoV-2 expression in the GI tract to the intestine, suggesting that the intestine, but not the stomach, is likely to be important in viral replication and possibly transmission.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Mucosa Gástrica , Mucosa Intestinal , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , SARS-CoV-2/fisiología , Humanos , COVID-19/virología , COVID-19/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/virología , Tropismo Viral , Organoides/virología , Organoides/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Replicación Viral , AnimalesRESUMEN
The epithelium of the gastrointestinal tract has been extensively characterized using advanced histological and RNA sequencing techniques, which has revealed great cellular diversity. Pathogens, such as viruses and bacteria, are highly adapted to their host and often exhibit not only species-specificity, but also a preference or tropism for specific gastrointestinal segments or even cell types - some of these preferences are so specific, that these pathogens still cannot be cultured in the lab. Organoid technology now provides a tool to generate human cell types, which enables the study of host cell tropism. Focusing on the gastrointestinal tract, we provide an overview about cellular differentiation in vivo and in organoids and how differentiation in organoids and their derived models is used to advance our understanding of viral, bacterial, and parasitic infection. We emphasize that it is central to understand the composition of the model, as the alteration of culture conditions yields different cell types which affects infection. We examine future directions for wider application of cellular heterogeneity and potential advanced model systems for gastrointestinal tract infection studies.
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The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.
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Curriculum , Alfabetización , Células MadreRESUMEN
The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Hormonas Peptídicas , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Quimiotaxis , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Humanos , Hormonas Peptídicas/metabolismo , Tropismo , Urea/metabolismo , Factores de Virulencia/metabolismoRESUMEN
In November 2021, the Institute for Regenerative Medicine (IRM) and the Institute for Immunology (IFI) at the University of Pennsylvania, USA, joined forces and organized a symposium featuring external speakers as well as locally based scientists to discuss how the immune system influences tissue stem cell biology. As we review here, the presentations highlighted emerging concepts in the field, revealing how tissue-specific immune cell activation can guide stem cells in regeneration and repair.
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Comunicación Celular , Medicina Regenerativa , Células MadreRESUMEN
Infectious diseases are a major threat worldwide. With the alarming rise of antimicrobial resistance and emergence of new potential pathogens, a better understanding of the infection process is urgently needed. Over the last century, the development of in vitro and in vivo models has led to remarkable contributions to the current knowledge in the field of infection biology. However, applying recent advances in organoid culture technology to research infectious diseases is now taking the field to a higher level of complexity. Here, we describe the current methods available for the study of infectious diseases using organoid cultures.
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Biología , OrganoidesRESUMEN
Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.
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Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Organoides/virología , Receptor EphA2/metabolismo , Estómago/virología , Internalización del Virus , Células Epiteliales/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Humanos , Organoides/metabolismo , Estómago/fisiología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologíaRESUMEN
The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell-derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development.
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Células Epiteliales/inmunología , Tracto Gastrointestinal/inmunología , Inmunidad Innata , Organoides , Adulto , Animales , Bancos de Muestras Biológicas , Polaridad Celular , Predicción , Microbioma Gastrointestinal/inmunología , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Perfilación de la Expresión Génica , Células Caliciformes/inmunología , Humanos , Tolerancia Inmunológica , Recién Nacido , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/inmunología , Ratones , Modelos Inmunológicos , FN-kappa B/fisiología , Especificidad de Órganos , Organoides/citología , Organoides/inmunología , Células de Paneth/inmunología , Ganglios Linfáticos Agregados/inmunología , Células Madre/inmunología , Receptores Toll-Like/inmunologíaAsunto(s)
Investigación Biomédica/métodos , Organoides , Alternativas al Uso de Animales , Animales , Investigación Biomédica/ética , Investigación Biomédica/tendencias , Encéfalo/citología , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Células Madre Embrionarias/citología , Femenino , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Masculino , Ratones , Estudios Observacionales como Asunto , Organoides/citología , Organoides/efectos de los fármacos , Organoides/trasplante , Embarazo , Medicina Regenerativa/tendencias , Técnicas Reproductivas Asistidas/ética , Técnicas Reproductivas Asistidas/tendencias , Toxicología/métodos , Toxicología/tendenciasRESUMEN
OBJECTIVE: The epithelial layer of the GI tract is equipped with innate immune receptors to sense invading pathogens. Dysregulation in innate immune signalling pathways is associated with severe inflammatory diseases, but the responsiveness of GI epithelial cells to bacterial stimulation remains unclear. DESIGN: We generated 42 lines of human and murine organoids from gastric and intestinal segments of both adult and fetal tissues. Genome-wide RNA-seq of the organoids provides an expression atlas of the GI epithelium. The innate immune response in epithelial cells was assessed using several functional assays in organoids and two-dimensional monolayers of cells from organoids. RESULTS: Results demonstrate extensive spatial organisation of innate immune signalling components along the cephalocaudal axis. A large part of this organisation is determined before birth and independent of exposure to commensal gut microbiota. Spatially restricted expression of Toll-like receptor 4 (Tlr4) in stomach and colon, but not in small intestine, is matched by nuclear factor kappa B (NF-κB) responses to lipopolysaccharide (LPS) exposure. Gastric epithelial organoids can sense LPS from the basal as well as from the apical side. CONCLUSION: We conclude that the epithelial innate immune barrier follows a specific pattern per GI segment. The majority of the expression patterns and the function of TLR4 is encoded in the tissue-resident stem cells and determined primarily during development.
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Células Epiteliales/inmunología , Microbioma Gastrointestinal/inmunología , Inmunidad Innata/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Organoides/inmunología , Animales , Células Cultivadas , Humanos , Lipopolisacáridos/inmunología , Transducción de SeñalRESUMEN
Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate ß-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of ß-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Helicobacter pylori/patogenicidad , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , ADN/química , ADN/genética , Daño del ADN , Replicación del ADN/efectos de los fármacos , Floxuridina , Glicosiltransferasas/metabolismo , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/microbiología , Helicobacter pylori/metabolismo , Interacciones Huésped-Patógeno/fisiología , Humanos , Lipopolisacáridos/metabolismo , Mutación , FN-kappa B/genética , Proteínas Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patologíaRESUMEN
Correction for 'Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori' by Dana Westmeier et al., Nanoscale, 2018, 10, 1453-1463.
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During the last decades, the flourishing scientific field of molecular pathogenesis brought groundbreaking knowledge of the mechanisms of pathogenicity and the underlying bacterial virulence factors to cause infectious diseases. However, a major paradigm shift is currently occurring after it became increasingly evident that bacterial-host and host-host cell interactions including immune responses orchestrated by defined virulence factors are not the sole drivers of infectious disease development. Strong evidence has been collected that information and nutrient flow within complex microbial communities, as well as to and from host cells and matrices are equally important for successful infection. This particularly holds true for gastrointestinal (GI) pathogens and the GI microbiota interacting and communicating with each other as well as with the host GI mucus and mucosa. Gut-adapted pathogens appear to have developed powerful and specific strategies to interact with human GI mucus including the microbiota for nutrient acquisition, mucosal adhesion, inter-species communication and traversing the mucus barrier. This review covers the existing evidence on these topics and explores the mutual dynamics of host GI mucus, the mucosal habitat and incoming acute and chronic pathogens during GI infections. A particular focus is placed on the role of carbohydrates in diverse mucosal interaction, communication and competition processes. Novel techniques to analyze and synthesize mucus-derived carbohydrates and to generate mucus mimetics are introduced. Finally, open questions and future objectives for pathogen - host GI mucus research will be discussed.
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Bacterias/patogenicidad , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Glicosilación , Interacciones Huésped-Patógeno , Moco/metabolismo , Animales , Humanos , Mucosa Intestinal/microbiología , Factores de Virulencia/metabolismoRESUMEN
Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.
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Polaridad Celular/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Receptor Toll-Like 3/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Complejo 1 de Proteína Adaptadora/metabolismo , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Interferones/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virología , Ratones , Receptor Toll-Like 3/agonistas , Virus/inmunologíaRESUMEN
Precision medicine requires in vitro models which will both faithfully recapitulate the features of an individual's disease and enable drug testing on a wide variety of samples covering the greatest range of phenotypes possible for a particular disease. Organoid technology has immense potential to fulfill this demand, but it will be necessary to develop robust protocols that enable the generation of organoids in a dependable manner from nearly every patient. Here we provide a user's guide, including detailed step-by-step protocols, to the establishment, isolation and verification of gastric cancer organoids. Selection strategies include omission of growth factors, addition of drugs, isolation of distinct phenotypes and generation of monoclonal lines. For confirmation of cancer identity, we use sequencing, drug selection, karyotyping and histology. While we specify these protocols for human gastric cancer organoids here, the methods described are applicable to organoids derived from other tissues as well.
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Organoides/patología , Neoplasias Gástricas/patología , Genotipo , Humanos , Cariotipificación/métodos , Metafase , Mutación , Organoides/metabolismo , Medicina de Precisión , Proteína Smad4/genética , Neoplasias Gástricas/genética , Técnicas de Cultivo de Tejidos/métodosRESUMEN
Gastric cancer displays marked molecular heterogeneity with aggressive behavior and treatment resistance. Therefore, good in vitro models that encompass unique subtypes are urgently needed for precision medicine development. Here, we have established a primary gastric cancer organoid (GCO) biobank that comprises normal, dysplastic, cancer, and lymph node metastases (n = 63) from 34 patients, including detailed whole-exome and transcriptome analysis. The cohort encompasses most known molecular subtypes (including EBV, MSI, intestinal/CIN, and diffuse/GS, with CLDN18-ARHGAP6 or CTNND1-ARHGAP26 fusions or RHOA mutations), capturing regional heterogeneity and subclonal architecture, while their morphology, transcriptome, and genomic profiles remain closely similar to in vivo tumors, even after long-term culture. Large-scale drug screening revealed sensitivity to unexpected drugs that were recently approved or in clinical trials, including Napabucasin, Abemaciclib, and the ATR inhibitor VE-822. Overall, this new GCO biobank, with linked genomic data, provides a useful resource for studying both cancer cell biology and precision cancer therapy.
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Antineoplásicos/farmacología , Bancos de Muestras Biológicas , Ensayos de Selección de Medicamentos Antitumorales , Organoides/efectos de los fármacos , Organoides/patología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/patología , Aminopiridinas/farmacología , Bencimidazoles/farmacología , Benzofuranos/farmacología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Isoxazoles/farmacología , Masculino , Naftoquinonas/farmacología , Medicina de Precisión , Pirazinas/farmacología , Neoplasias Gástricas/clasificación , Neoplasias Gástricas/genéticaRESUMEN
Enteric bacteria may cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens without the need for specific functionalization. The NP-assembly that occurred rapidly was not influenced by variations in physiological temperature, though affected by the NPs' physico-chemical characteristics. Improved binding was observed for small NPs with a negative surface charge, whereas binding could be reduced by surface 'stealth' modifications. Employing human gastric epithelial cells and 3D-organoid models of the stomach, we show that NP-coating did not inhibit H. pylori's cellular attachment. However, even the assembly of non-bactericidal silica NPs attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. Here we demonstrate that NP binding to enteric bacteria may impact their pathobiology which could be further exploited to rationally modulate the (patho)biology of microbes by nanomaterials.