RESUMEN
Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage5-9, revealing that stem-cell identity is an induced rather than a hardwired property.
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Linaje de la Célula , Intestinos/citología , Células Madre/citología , Animales , Diferenciación Celular , Reprogramación Celular , Femenino , Feto/citología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Intestinos/crecimiento & desarrollo , Masculino , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Regeneración , Nicho de Células MadreRESUMEN
BACKGROUND: Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival. METHODS AND RESULTS: Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression. CONCLUSION: Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Humanos , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/efectos adversos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , OncogenesRESUMEN
Selective modification of peptides and proteins is of foremost importance for the development of biopharmaceuticals and exploring biochemical pathways, as well as other applications. Here, we present a study on the development of a general and easily applicable selective method for N-terminal acylation of biomolecules, applying a new type of phenol esters. Key to the success was the development of highly tunable phenol activators bearing in the ortho-position, sulfonic acid or sulfonamide, acting as a steric shield for hydrolysis, and electron-withdrawing groups in the other ortho- and para-position for controlling the reactivity of the activated phenol esters. A library of heptapeptides, testing all 20 natural amino acids positioned at the N-terminal, were acylated in a selective manner at the N-terminus. The majority showed high conversion and excellent Nα-selectivity. Several biologically relevant biomolecules, including DesB30 insulin and human growth hormone, could also be modified at the N-terminal in a highly selective way, exemplified by either a fluorophore or a fatty acid sidechain. Finally, taking advantage of the possibility to accurately adjust the reactivity of the phenol esters, we present a potential strategy for the construction of dual active biopharmaceuticals through the employment of a bifunctional acylation linker and demonstrate its use in the creation of a GLP-1 insulin analogue, coupled through the lysine residue of GLP-1 and the N-terminal PheB1 amine of DesB30 insulin.
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Productos Biológicos , Ésteres , Acilación , Secuencia de Aminoácidos , Péptido 1 Similar al Glucagón , Humanos , Insulina , Péptidos/química , Fenol , Proteínas/metabolismoRESUMEN
This paper describes the discovery, synthesis, and use of novel water-soluble acylation reagents for efficient and selective modification, cross-linking, and labeling of proteins and peptides, as well as for their use in the effective modification of sepharose beads under pH control in aqueous media. The reagents are based on a 2,4-dichloro-6-sulfonic acid phenol ester core combined with a variety of linker structures. The combination of these motifs leads to an ideal balance between hydrolytic stability and reactivity. At high pH, good to excellent conversions (up to 95%) and regioselectivity (up to 99:1 Nε/Nα amine ratio) in the acylation were realized, exemplified by the chemical modification of incretin peptides and insulin. At neutral pH, an unusually high preference toward the N-terminal phenylalanine in an insulin derivative was observed (>99:1 Nα/Nε), which is up until now unprecedented in the literature for more elaborate reagents. In addition, the unusually high hydrolytic stability of these reagents and their ability to efficiently react at low concentrations (28 µM or 0.1 mg/mL) are exemplified with a hydroxy linker-based reagent and are a unique feature of this work.
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FenolRESUMEN
Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.
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Diferenciación Celular , Proliferación Celular , Células Epiteliales/fisiología , Mucosa Intestinal/fisiología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Animales , Drosophila , Regulación de la Expresión Génica , Humanos , Ratones , Vía de Señalización WntRESUMEN
A series of pharmaceutically relevant small molecules and biopharmaceuticals bearing aliphatic carboxamides have been successfully labeled with carbon-13. Key to the success of this novel carbon isotope labeling technique is the observation that 13 C-labeled NiII -acyl complexes, formed from a 13 CO insertion step with NiII -alkyl intermediates, rapidly react in less than one minute with 2,2'-dipyridyl disulfide to quantitatively form the corresponding 2-pyridyl thioesters. Either the use of 13 C-SilaCOgen or 13 C-COgen allows for the stoichiometric addition of isotopically labeled carbon monoxide. Subsequent one-pot acylation of a series of structurally diverse amines provides the desired 13 C-labeled carboxamides in good yields. A single electron transfer pathway is proposed between the NiII -acyl complexes and the disulfide providing a reactive NiIII -acyl sulfide intermediate, which rapidly undergoes reductive elimination to the desired thioester. By further optimization of the reaction parameters, reaction times down to only 11â min were identified, opening up the possibility of exploring this chemistry for carbon-11 isotope labeling. Finally, this isotope labeling strategy could be adapted to the synthesis of 13 C-labeled liraglutide and insulin degludec, representing two antidiabetic drugs.
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BACKGROUND & AIMS: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. METHODS: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. RESULTS: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline. CONCLUSIONS: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.
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Amidas/farmacología , Glucemia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Enteroendocrinas/efectos de los fármacos , Intolerancia a la Glucosa/tratamiento farmacológico , Hipoglucemiantes/farmacología , Íleon/efectos de los fármacos , Resistencia a la Insulina , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Células Madre/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismo , Animales , Biomarcadores/sangre , Glucemia/metabolismo , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Células Enteroendocrinas/metabolismo , Péptido 1 Similar al Glucagón/sangre , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/fisiopatología , Íleon/metabolismo , Insulina/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Organoides/efectos de los fármacos , Organoides/metabolismo , Fenotipo , Transducción de Señal/efectos de los fármacos , Células Madre/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
The epidermis is an integral part of our largest organ, the skin, and protects us against the hostile environment. It is a highly dynamic tissue that, during normal steady-state conditions, undergoes constant turnover. Multiple stem cell populations residing in autonomously maintained compartments facilitate this task. In this Review, we discuss stem cell behaviour during normal tissue homeostasis, regeneration and disease within the pilosebaceous unit, an integral structure of the epidermis that is responsible for hair growth and lubrication of the epithelium. We provide an up-to-date view of the pilosebaceous unit, encompassing the heterogeneity and plasticity of multiple discrete stem cell populations that are strongly influenced by external cues to maintain their identity and function.
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Transformación Celular Neoplásica/metabolismo , Células Epidérmicas , Epidermis/fisiología , Folículo Piloso/citología , Homeostasis/fisiología , Regeneración/fisiología , Células Madre/fisiología , Transformación Celular Neoplásica/genética , Folículo Piloso/embriología , HumanosRESUMEN
Human epidermal stem cells express high levels of ß1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1(+) cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1(+) cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1(+) cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of ß1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and ß1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment.
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Células Epidérmicas , Epidermis/fisiología , Perfilación de la Expresión Génica , Análisis de la Célula Individual/métodos , Biomarcadores/análisis , Biomarcadores/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Epidermis/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Perfilación de la Expresión Génica/métodos , Heterogeneidad Genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Análisis por Micromatrices , Modelos Biológicos , Reacción en Cadena de la Polimerasa/métodos , Células Madre/metabolismo , Células Madre/fisiología , Estudios de Validación como AsuntoRESUMEN
Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine-rich immunoglobulin repeats-1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re-expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E-cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition.
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Cadherinas/metabolismo , Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana/genética , Proteínas del Tejido Nervioso/genética , Lesiones Precancerosas/genética , Animales , Cadherinas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Inhibición de Contacto , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Homeostasis , Humanos , Pérdida de Heterocigocidad , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Complejos Multiproteicos , Proteínas del Tejido Nervioso/metabolismo , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Eliminación de Secuencia , Transducción de SeñalRESUMEN
The intestinal epithelium fulfills important physiological functions and forms a physical barrier to the intestinal lumen. Barrier function is regulated by several pathways, and its impairment contributes to the pathogenesis of inflammatory bowel disease (IBD), a chronic inflammatory condition affecting more than seven million people worldwide. Current treatment options specifically target inflammatory mediators and have led to improvement of clinical outcomes; however, a significant proportion of patients experience treatment failure. Pro-repair effects of inflammatory mediators on the epithelium are emerging. In this review we summarize current knowledge on involved epithelial pathways, identify open questions, and put recent findings into clinical perspective, and pro-repair effects. A detailed understanding of epithelial pathways integrating mucosal stimuli in homeostasis and inflammation is crucial for the development of novel, more targeted therapies.
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Inflamación , Enfermedades Inflamatorias del Intestino , Humanos , Inflamación/patología , Intestinos , Mucosa Intestinal , Homeostasis , Mediadores de Inflamación/metabolismo , FenotipoRESUMEN
The intestinal epithelium ensures uptake of vital nutrients and acts as a barrier between luminal contents and the underlying immune system. In inflammatory bowel diseases, such as ulcerative colitis (UC), this barrier is compromised, and patients experience debilitating symptoms. Here, we perform single-cell RNA profiling of epithelial cells and outline patterns of cell fate decisions in healthy individuals and UC patients. We demonstrate that patterns of hierarchical behavior are altered in UC patients and identify unique cellular states associated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation in ulcerated and non-ulcerated areas of the colonic epithelium. These transcriptional changes could be recapitulated in human colonic organoids, wherein cytokine-mediated activation of JAK/STAT led to the emergence of cell populations with augmented regenerative properties. Altogether, our findings indicate that intricate relationships between epithelial and cytokine signaling regulate cell fate during epithelial tissue regeneration in humans and have important implications for the understanding of UC biology.
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Colitis Ulcerosa , Mucosa Intestinal , Quinasas Janus , Factores de Transcripción STAT , Transducción de Señal , Humanos , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colitis Ulcerosa/genética , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Mucosa Intestinal/metabolismo , Células Epiteliales/metabolismo , Organoides/metabolismo , Análisis de la Célula Individual , Colon/metabolismo , Colon/patología , Citocinas/metabolismo , Diferenciación CelularRESUMEN
Hair follicle development and growth are regulated by Wnt signalling and depend on interactions between epidermal cells and a population of fibroblasts at the base of the follicle, known as the dermal papilla (DP). DP cells have a distinct gene expression signature from non-DP dermal fibroblasts. However, their origins are largely unknown. By generating chimeric mice and performing skin reconstitution assays we show that, irrespective of whether DP form during development, are induced by epidermal Wnt activation in adult skin or assemble from disaggregated cells, they are polyclonal in origin. While fibroblast proliferation is necessary for hair follicle formation in skin reconstitution assays, mitotically inhibited cells readily contribute to DP. Although new hair follicles do not usually develop in adult skin, adult dermal fibroblasts are competent to contribute to DP during hair follicle neogenesis, irrespective of whether they originate from skin in the resting or growth phase of the hair cycle or skin with ß-catenin-induced ectopic follicles. We propose that during skin reconstitution fibroblasts may be induced to become DP cells by interactions with hair follicle epidermal cells, rather than being derived from a distinct subpopulation of cells.
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Comunicación Celular , Diferenciación Celular , Dermis/citología , Folículo Piloso/citología , Cabello/crecimiento & desarrollo , Animales , Células Epidérmicas , Fibroblastos/citología , Cabello/citología , Ratones , Vía de Señalización WntRESUMEN
Tissues with a high turnover rate produce millions of cells daily and have abundant regenerative capacity. At the core of their maintenance are populations of stem cells that balance self-renewal and differentiation to produce the adequate numbers of specialized cells required for carrying out essential tissue functions. Here, we compare and contrast the intricate mechanisms and elements of homeostasis and injury-driven regeneration in the epidermis, hematopoietic system, and intestinal epithelium-the fastest renewing tissues in mammals. We highlight the functional relevance of the main mechanisms and identify open questions in the field of tissue maintenance.
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Mucosa Intestinal , Células Madre , Animales , Epidermis , Diferenciación Celular , Homeostasis , MamíferosRESUMEN
Generation of functionally mature organs requires exquisite control of transcriptional programs governing cell state transitions during development. Despite advances in understanding the behavior of adult intestinal stem cells and their progeny, the transcriptional regulators that control the emergence of the mature intestinal phenotype remain largely unknown. Using mouse fetal and adult small intestinal organoids, we uncover transcriptional differences between the fetal and adult state and identify rare adult-like cells present in fetal organoids. This suggests that fetal organoids have an inherent potential to mature, which is locked by a regulatory program. By implementing a CRISPR-Cas9 screen targeting transcriptional regulators expressed in fetal organoids, we establish Smarca4 and Smarcc1 as important factors safeguarding the immature progenitor state. Our approach demonstrates the utility of organoid models in the identification of factors regulating cell fate and state transitions during tissue maturation and reveals that SMARCA4 and SMARCC1 prevent precocious differentiation during intestinal development.
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Células Madre Adultas , Sistemas CRISPR-Cas , Animales , Ratones , Diferenciación Celular/genética , Feto , OrganoidesRESUMEN
The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12â, LGGâ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.