Intestinal epithelial cells (IECs) are capable of mounting an adequate antimicrobial inflammatory response to pathogens while tolerating commensals. The underlying regulatory mechanisms of immune sensitivity remain incompletely understood, particularly in the context of human IECs. To enhance our understanding of the immune response of IECs to bacterial epithelial barrier breach, we investigated whether epithelial responsiveness is contingent on cell identity and cell polarization. We exposed human intestinal organoids to bacterial antigens to study their immune responses. Notable discrepancies were observed in the specific reactions exhibited by intestinal stem cells (ISCs) and enterocytes. It was determined that basolateral exposure of IECs to bacterial antigens resulted in a robust response, whereas apical exposure elicited a significantly more modest response. We identified ISCs as the responders, while the reaction of enterocytes was found to be attenuated. The regulation of bacterial responsiveness in enterocytes occurs at multiple levels, including the modulation of NFκB activation and post-transcriptional control of mRNA stability. Our findings demonstrate that differentiated non-responsive enterocytes can be sensitized to bacterial antigens through the activation of the WNT pathway. These findings extend the crucial role of WNT signaling for intestinal epithelial homeostasis and regulation of stem cell maintenance, proliferation, differentiation, and tissue architecture in the gut. Additionally, they reveal a new function of WNT signaling in regulating microbial responses within the intestinal environment.

Plasticity is needed during development and homeostasis to generate diverse cell types from stem and progenitor cells. Following differentiation, plasticity must be restricted in specialized cells to maintain tissue integrity and function. For this reason, specialized cell identity is stable under homeostatic conditions; however, cells in some tissues regain plasticity during injury-induced regeneration. While precise gene expression controls these processes, the regulatory mechanisms that restrict or promote cell plasticity are poorly understood. Here we use the mouse small intestine as a model system to study cell plasticity. We find that H3K36 methylation reinforces expression of cell-type-associated genes to maintain specialized cell identity in intestinal epithelial cells. Depleting H3K36 methylation disrupts lineage commitment and activates regenerative gene expression. Correspondingly, we observe rapid and reversible remodelling of H3K36 methylation following injury-induced regeneration. These data suggest a fundamental role for H3K36 methylation in reinforcing specialized lineages and regulating cell plasticity and regeneration.

Inflammatory bowel disease is triggered by abnormalities in epithelial barrier function and immunological responses, although its pathogenesis is poorly understood. The dextran sodium sulphate (DSS)-induced colitis model has been used to examine inflammation in the colon. Damage to mucosa primality occurs in the large intestine and scarcely in the small intestine. To evaluate the effect on the ileum, we histologically analyzed the inflammatory and recovery phases in DSS model mice, and 40 kDa FITC-dextran was used to investigate barrier function. In the inflammatory phase, histological damage was insignificant. However, expanded crypts, hypertrophic goblet and Paneth cells, increased mucus production and secretion were observed. The cellular morphology was restored to that of the control in the recovery phase. According to in situ hybridization and lectin histochemistry, the expression of intestinal stem cell markers, secretory cell differentiation factors, and glycosylation of secretory granules in Paneth cells differed in the DSS model. DSS-treatment did not influence the barrier function in the ileum, and FITC-dextran did not diffuse via the paracellular pathway into the mucosa. However, cells incorporating FITC appeared even under normal conditions. The number of FITC-positive Paneth cells was lower in the DSS group than the control group. Our results showed morphological and functional alterations in ileal epithelial cells, especially secretory cells, in the DSS colitis model.

We focused on investigation of E. coli infection influence on the turnover and apoptosis of intestinal mucosa. We have verified changes in proliferation and apoptosis in epithelial lining as well as in lamina propria of jejunum and colon of germ-free (GF) piglets as healthy control group and GF piglets in which at 5th day their gut was colonized with E. coli bacteria (ECK group). According to our results we detected significant increase in proliferation of the epithelial cells only in the jejunum of the ECK group, indicating a higher sensitivity to colonization with E. coli. Significant changes in the TUNEL assay and immunohistochemistry of other studied markers (TNF-α, Caspase-3 and HSP-70) were noted only in the lamina propria mucosae of both intestinal segments in the ECK group. In conclusion, we found that the commensal gut microbiota plays a role in regulation of the turnover rate in the epithelial lining, but also in the cells in the lamina propria mucosae in both intestinal segments, and that the host response is dependent on the colonising bacteria.

A tri-protein complex containing NICD, RBPj and MAML1 binds DNA as monomer or as cooperative dimers to regulate transcription. Mice expressing Notch dimerization-deficient alleles (NDD) of Notch1 and Notch2 are sensitized to environmental insults but otherwise develop and age normally. Transcriptomic analysis of colonic spheroids uncovered no evidence of dimer-dependent target gene miss-regulation, confirmed impaired stem cell maintenance in-vitro, and discovered an elevated signature of epithelial innate immune response to symbionts, a likely underlying cause for heightened sensitivity in NDD mice. TurboID followed by quantitative nano-spray MS/MS mass-spectrometry analyses in a human colon carcinoma cell line expressing either NOTCH2DD or NOTCH2 revealed an unbalanced interactome, with reduced interaction of NOTCH2DD with the transcription machinery but relatively preserved interaction with the HDAC2 interactome suggesting modulation via cooperativity. To ask if HDAC2 activity contributes to Notch loss-of-function phenotypes, we used the HDAC2 inhibitor Valproic acid (VPA) and discovered it could prevent the intestinal consequences of NDD and gamma secretase inhibitors (DBZ or DAPT) treatment in mice and spheroids, suggesting synergy between HDAC activity and pro-differentiation program in intestinal stem cells.

Toxoplasma gondii is the causative agent of toxoplasmosis, a common zoonotic disease affecting vertebrates with high global incidence. For the parasite to disseminate throughout the body, it crosses the intestinal barrier, triggering inflammatory reactions. This study aimed to assess the tissue response in the ileum and colon of mice following chronic infection with T. gondii. Fourteen mice were divided into two groups: the infected group received 1000 T. gondii oocysts via gavage, and after 60 days, the mice were euthanized. The ileum and colon were collected and processed for histological analysis, inflammatory marker measurement and myenteric neuron analysis. Chronic infection resulted in a significant increase in intraepithelial lymphocytes and mast cells, as well as morphometric changes such as increased total intestinal wall thickness of the ileum, crypt depth, collagen fiber area, and a decrease in myeloperoxidase activity, without altering nitric oxide levels. While the number of myenteric neurons remained unchanged, there was an increase in vasoactive intestinal peptide expression. These results suggest persistence intestinal inflammatory stimuli in chronic T. gondii infection.

Intestinal ischemia and reperfusion injury (IRI) is a deadly and common condition. Death is associated with sepsis due to insufficient epithelial repair, requiring stem cell-driven regeneration, typically beginning 48 h after injury. Animal models are critical to advancing this field. To effectively study epithelial healing, models must survive clinically relevant intestinal ischemic injury extending to the crypt. Although mouse models are indispensable to intestinal research, their application for studying epithelial repair following severe IRI may be limited. Ischemic injury was induced in mouse and porcine jejunum for up to 3 h, with up to 72 h of reperfusion. Histologic damage was scored by Chiu-Park grade, and animal survival was assessed. Findings were compared between species. A mouse IRI literature review was performed to evaluate the purported degree of injury, duration of recovery, and reported survival rates. In mice and pigs, 3 h of ischemia induced severe, reliable injury extending into the crypt. However, at 48 h, mouse survival was only 23.5% compared with 100% survival in pigs. In literature, ischemia was induced for >1 h in only 4 of 102 mouse studies and none to 3 h. Recovery was attempted for 48 h in only six reports. Forty-seven studies reported intestinal crypt injury. Of those that featured histologic intestinal crypt damage, survival rates at 48 h ranged from 10 to 50% (median 30%). Mouse models are not ideal for studying intestinal stem cell-mediated recovery from severe IRI. Alternative large animal models, like pigs, are recommended.NEW & NOTEWORTHY Additional research is needed to improve recovery from severe intestinal ischemia. The selection of the ideal animal model is critical to facilitating this work. Based on our experimentation and literature review, porcine models, with increased translatability and an improved ability to survive both prolonged ischemia and the recovery period, appear to be the most appropriate choice for future studies.

Aging is a process that accompanies a decline in the function of various tissues and organs, especially affecting intestinal health. Agarose oligosaccharide (AOS) can prolong the lifespan of organisms and protect the intestine in the previous study. It was examined to evaluate the effects of AOS on intestinal health, and the potential associations between intestinal homeostasis and health status were further validated.

D-galactose-induced aging mice were used to investigate the role of AOS in promoting intestinal health by determining intestinal physiology, microbiota and stem cells.

AOS supplementation decreased the clinical frailty index of aging mice with increasing intestinal length and crypt depth; moreover, it decreased the average flatulence index and PCNA protein content in the intestine. Besides, AOS contributed to the diversity of the gut microbiota by increasing the relative abundance of Bacteroidetes and other bacteria that could produce short-chain fatty acids. Furthermore, AOS affected the expression of proinflammatory factors in aging mice, promoting the proliferative equilibrium of intestinal stem cells.

These findings confirmed that AOS could improve intestinal health in aging mice by maintaining intestinal homeostasis, which provides new insights into the potential application of AOS as a prebiotic.

Since their discovery, 3D cell cultures have emerged as powerful tools across various basic, translational research, and industrial discovery projects. One such application is in the physiological and pathophysiological modeling of pancreatic exocrine functions, which addresses critical clinical challenges, including acute and chronic pancreatitis. While several methods now exist for generating epithelial organoids (derived from induced pluripotent, embryonic, or adult stem cells), the advent of patient-derived organoids (PDOs) with controlled polarity has introduced a new frontier in pancreatic research. This advancement has significantly expanded the methodological arsenal available for studying human pancreatic epithelial secretion. In this article, we present basic protocols and a troubleshooting guide for an advanced culture method that results in an apical-to-basal polarity switch. Alongside the protocols, we emphasize a comprehensive cost breakdown, an aspect often challenging to estimate when implementing new techniques. By sharing the technical nuances and financial implications of these protocols, we aim to encourage researchers to transition from rodent models to primary human epithelial cells wherever feasible. This aligns with the U.S. Environmental Protection Agency's efforts to accelerate the translation of significant scientific findings to address major clinical needs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment and maintenance of pancreatic PDOs Basic Protocol 2: Cryopreservation and thawing of pancreatic PDOs Basic Protocol 3: Inducing polarity switching in pancreatic PDOs.

Neural stem cells (NSCs) are progenitor cell populations generating glial cells and neurons and endowed with long-lasting self-renewal and differentiation potential. While some neural progenitors (NPs) in the embryonic nervous system are also long-lived and match this definition, the term NSC classically refers to such progenitor types in the adult. With the discovery of extensive NSC populations in the adult brain of Danio rerio (zebrafish) and of their high neurogenic activity, including for neuronal regeneration, this model organism has become a powerful tool to characterize and mechanistically dissect NSC properties. On these bases, this article will consider NSCs in the adult zebrafish brain, with a focus on its most extensively characterized domain, the telencephalon (notably its dorsal part, the pallium). Whenever necessary, we will also refer to other brain subdivisions, embryonic processes, and the mouse adult brain, whether for comparative purposes or because more information is available in these other systems.

The cause of duodenal atresia (DA) is not known. Tandler's "solid cord" hypothesis conflicts with current biological evidence. In humans, a genetic aetiology is supported by the association with Trisomy 21. Interruption of Fgf10 is the strongest genetic link to DA in mice, demonstrating an increased incidence and severity as embryos mature. This project aimed to develop an organoid model to facilitate ex vivo DA research on the FGF10/FGFR2b signalling pathway. We hypothesised that DA morphology represents an evolving spectrum of disease and that Fgf10 knockout organoids would vary in growth pattern compared to wild-type.

Organoids were cultured from the duodenum of E12.5 Fgf10 knockout, heterozygous and wild-type embryos, using an air-liquid interface with Growth Factor reduced Matrigel. Organoids were photographed every 48 h to observe growth. Organoids were isolated and fixed after 14 days, then stained with DAPI, KI-67, and cytokeratin to demonstrate proliferation and differentiation.

Wild-type duodenum developed into crypt-forming organoids. Fgf10 heterozygous duodenum failed to progress beyond the development stage of spheroids. Fgf10 knockout duodenum failed to demonstrate any growth. Wholemount staining showed the greatest cell proliferation and differentiation in wild-type tissue.

This research presents a novel concept for the growth of embryonic gastrointestinal tissue to inform normal biology. The small sample numbers and restricted culture duration limit longer-term growth analysis. While this model serves as a potential ex vivo setting for future research, that research should consider organoid models with greater standardisation and other gastrointestinal regions.

Animal/laboratory study.

The intestine is populated by a complex and dynamic assortment of microbes, collectively called gut microbiota, that interact with the host and contribute to its metabolism and physiology. Diet is considered a key regulator of intestinal microbiota, as ingested nutrients interact with and shape the resident microbiota composition. Furthermore, recent studies underscore the interplay of dietary and microbiota-derived nutrients, which directly impinge on intestinal stem cells regulating their turnover to ensure a healthy gut barrier. Although advanced sequencing methodologies have allowed the characterization of the human gut microbiome, mechanistic studies assessing diet-microbiota-host interactions depend on the use of genetically tractable models, such as Drosophila melanogaster. In this review, we first discuss the similarities between the human and fly intestines and then we focus on the effects of diet and microbiota on nutrient-sensing signaling cascades controlling intestinal stem cell self-renewal and differentiation, as well as disease. Finally, we underline the use of the Drosophila model in assessing the role of microbiota in gut-related pathologies and in understanding the mechanisms that mediate different whole-body manifestations of gut dysfunction.

1. This study was conducted to determine the effects of graded levels of phytase on the performance, egg quality and gut health of white laying hens.2. Treatments consisted of a negative control (NC) diet containing 0.14% available phosphorus (avP), positive control (PC) diet containing 0.35% avP provided via dicalcium phosphate (DCP) and DCP replaced in the PC by with three graded levels of phytase derived from Komagataella phaffii at 500 (PC-500), 750 (PC-750) and 1000 (PC-1000) FTU/kg which provided 0.176%, 0.188% and 0.200% of avP, respectively.3. Egg production, feed intake, feed conversion ratio and jejunal morphometry were negatively affected in NC-fed birds (p < 0.05). Considering the whole period, birds fed a diet supplemented with graded levels of phytase shared the same egg production and feed intake levels with PC birds (p < 0.05). Feed conversion ratio was significantly lowered by 4.9%, 1.6% and 7.6% in hens fed on diets PC-500, PC-750 and PC-1000, respectively compared to those fed the PC (p < 0.05).4. Neither of the dietary treatments affected cracked eggs, dirty eggs, eggshell breaking strength and eggshell thickness. Dietary supplementation of phytase significantly increased villus surface area by 15%, 36% and 40% in PC-500, PC-750 and PC-1000 birds, respectively compared to PC (p < 0.05).5. A significant increase in lactobacillus count was observed in line with increasing the level of phytase (p < 0.05). Dietary treatments had no effect on the caecal coliform or aerobic populations. Furthermore, phytase supplementation significantly increased the concentrations of total caecal short-chain fatty acid (SCFA; p < 0.01).6. In conclusion, along with improving performance parameters, the inclusion of phytase in laying hen diets can ameliorate intestinal morphology and stimulate caecal microflora and increase SCFA concentrations.

Colorectal cancer (CRC) represents the second leading cause of cancer incidence and the third leading cause of cancer deaths worldwide. There is currently a lack of understanding of the onset of CRC, hindering the development of effective prevention strategies, early detection methods and the selection of appropriate therapies. This article outlines the key aspects of host genetics currently known about the origin and development of CRC. The organisation of the colonic crypts is described. It discusses how the transformation of a normal cell to a cancer cell occurs and how that malignant cell can populate an entire colonic crypt, promoting colorectal carcinogenesis. Current knowledge about the cell of origin of CRC is discussed, and the two morphological pathways that can give rise to CRC, the classical and alternative pathways, are presented. Due to the molecular heterogeneity of CRC, each of these pathways has been associated with different molecular mechanisms, including chromosomal and microsatellite genetic instability, as well as the CpG island methylator phenotype. Finally, different CRC classification systems are described based on genetic, epigenetic and transcriptomic alterations, allowing diagnosis and treatment personalisation.

Viruses are obligate intracellular parasites, and they exploit the cellular pathways and resources of their respective host cells to survive and successfully multiply. The strategies of viruses concerning how to take advantage of the metabolic capabilities of host cells for their own replication can vary considerably. The most common metabolic alterations triggered by viruses affect the central carbon metabolism of infected host cells, in particular glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. The upregulation of these processes is aimed to increase the supply of nucleotides, amino acids, and lipids since these metabolic products are crucial for efficient viral proliferation. In detail, however, this manipulation may affect multiple sites and regulatory mechanisms of host-cell metabolism, depending not only on the specific viruses but also on the type of infected host cells. In this review, we report metabolic situations and reprogramming in different human host cells, tissues, and organs that are favorable for acute and persistent SARS-CoV-2 infection. This knowledge may be fundamental for the development of host-directed therapies.

Colorectal cancer (CRC) represents a significant global health burden, with high incidence and mortality rates worldwide. Recent progress in research highlights the distinct clinical and molecular characteristics of colon versus rectal cancers, underscoring tumor location's importance in treatment approaches. This article provides a comprehensive review of our current understanding of CRC epidemiology, risk factors, molecular pathogenesis, and management strategies. We also present the intricate cellular architecture of colonic crypts and their roles in intestinal homeostasis. Colorectal carcinogenesis multistep processes are also described, covering the conventional adenoma-carcinoma sequence, alternative serrated pathways, and the influential Vogelstein model, which proposes sequential APC, KRAS, and TP53 alterations as drivers. The consensus molecular CRC subtypes (CMS1-CMS4) are examined, shedding light on disease heterogeneity and personalized therapy implications.

The gastrointestinal tract (GI) is the largest immune organ whose function is controlled by a complex network of neurons from the enteric nervous system (ENS) as well as the sympathetic and parasympathetic system. Evolving evidence indicates that cross-communication between gut-innervating neurons and immune cells regulates many essential physiological functions including protection against mucosal infections. We previously demonstrated that following paraoxon treatment, 70 % of the mice were able to survive an oral infection with S. typhimurium, a virulent strain of Salmonella enterica serovar Typhimurium. The present study aims to investigate the effect that rivastigmine, a reversible AChE inhibitor used for the treatment of neurodegenerative diseases, has on the murine immune defenses of the intestinal mucosa. Our findings show that, similar to what is observed with paraoxon, administration of rivastigmine promoted the release of secretory granules from goblet and Paneth cells, resulting in increased mucin layer. Surprisingly, however, and unlike paraoxon, rivastigmine treatment did not affect overall mortality of infected mice. In order to investigate the mechanistic basis for the differential effects observed between paraoxon and rivastigmine, we used multi-color flowcytometric analysis to characterize the immune cell landscape in the intraepithelial (IE) and lamina propria (LP) compartments of intestinal mucosa. Our data indicate that treatment with paraoxon, but not rivastigmine, led to an increase of resident CD3+CD8+ T lymphocytes in the ileal mucosa (epithelium and lamina propria) and CD11b- CD11c+ dendritic cells in the LP. Our findings indicate the requirement for persistent cholinergic pathway engagement to effect a change in the cellular landscape of the mucosal tissue that is necessary for protection against lethal bacterial infections. Moreover, optimal protection requires a collaboration between innate and adaptive mucosal immune responses in the intestine.

Terminal differentiation requires massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4,000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identify dynamic occupancy of RNA polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II occupancy and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, chromatin immunoprecipitation sequencing (ChIP-seq), and immunoprecipitation (IP) mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II occupancy at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms that drive differentiation gene expression and find that pause-release of Pol II and post-transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in renewing adult tissue.

The intestinal barrier is mainly formed by a monolayer of epithelial cells, which forms a physical barrier to protect the gut tissues from external insults and provides a microenvironment for commensal bacteria to colonize while ensuring immune tolerance. Moreover, various immune cells are known to significantly contribute to intestinal barrier function by either directly interacting with epithelial cells or by producing immune mediators. Fulfilling this function of the gut barrier for mucosal homeostasis requires not only the intrinsic regulation of intestinal epithelial cells (IECs) but also constant communication with immune cells and gut microbes. The reciprocal interactions between IECs and immune cells modulate mucosal barrier integrity. Dysregulation of barrier function could lead to dysbiosis, inflammation, and tumorigenesis. In this overview, we provide an update on the characteristics and functions of IECs, and how they integrate their functions with tissue immune cells and gut microbiota to establish gut homeostasis.

Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.

The intestine is a visceral organ that integrates absorption, metabolism, and immunity, which is vulnerable to external stimulus. Researchers in the fields such as food science, immunology, and pharmacology have committed to developing appropriate in vitro intestinal cell models to study the intestinal absorption and metabolism mechanisms of various nutrients and drugs, or pathogenesis of intestinal diseases. In the past three decades, the intestinal cell models have undergone a significant transformation from conventional two-dimensional cultures to three-dimensional (3D) systems, and the achievements of 3D cell culture have been greatly contributed by the fabrication of different scaffolds. In this review, we first introduce the developing trend of existing intestinal models. Then, four types of scaffolds, including Transwell, hydrogel, tubular scaffolds, and intestine-on-a-chip, are discussed for their 3D structure, composition, advantages, and limitations in the establishment of intestinal cell models. Excitingly, some of the in vitro intestinal cell models based on these scaffolds could successfully mimic the 3D structure, microenvironment, mechanical peristalsis, fluid system, signaling gradients, or other important aspects of the original human intestine. Furthermore, we discuss the potential applications of the intestinal cell models in drug screening, disease modeling, and even regenerative repair of intestinal tissues. This review presents an overview of state-of-the-art scaffold-based cell models within the context of intestines, and highlights their major advances and applications contributing to a better knowledge of intestinal diseases. Impact statement The intestine tract is crucial in the absorption and metabolism of nutrients and drugs, as well as immune responses against external pathogens or antigens in a complex microenvironment. The appropriate experimental cell model in vitro is needed for in-depth studies of intestines, due to the limitation of animal models in dynamic control and real-time assessment of key intestinal physiological and pathological processes, as well as the "R" principles in laboratory animal experiments. Three-dimensional (3D) scaffold-based cell cultivation has become a developing tendency because of the superior cell proliferation and differentiation and more physiologically relevant environment supported by the customized 3D scaffolds. In this review, we summarize four types of up-to-date 3D cell culture scaffolds fabricated by various materials and techniques for a better recapitulation of some essential physiological and functional characteristics of original intestines compared to conventional cell models. These emerging 3D intestinal models have shown promising results in not only evaluating the pharmacokinetic characteristics, security, and effectiveness of drugs, but also studying the pathological mechanisms of intestinal diseases at cellular and molecular levels. Importantly, the weakness of the representative 3D models for intestines is also discussed.

Pulmonary diseases like asthma, chronic obstructive pulmonary disorder, lung fibrosis, and lung cancer pose a significant burden to global human health. Many of these complications arise as a result of exposure to particulate matter (PM), which has been examined in several preclinical and clinical trials for its effect on several respiratory diseases. Particulate matter of size less than 2.5 μm (PM2.5) has been known to inflict unforeseen repercussions, although data from epidemiological studies to back this are pending. Conventionally utilized two-dimensional (2D) cell culture and preclinical animal models have provided insufficient benefits in emulating the in vivo physiological and pathological pulmonary conditions. Three-dimensional (3D) structural models, including organ-on-a-chip models, have experienced a developmental upsurge in recent times. Lung-on-a-chip models have the potential to simulate the specific features of the lungs. With the advancement of technology, an emerging and advanced technique termed microfluidic organ-on-a-chip has been developed with the aim of identifying the complexity of the respiratory cellular microenvironment of the body. In the present Review, the role of lung-on-a-chip modeling in reproducing pulmonary complications has been explored, with a specific emphasis on PM2.5-induced pulmonary complications.

Colorectal cancer (CRC) is the second-highest cause of cancer-associated mortality among both men and women worldwide. One of the risk factors for CRC is obesity, which is correlated with a high-fat diet prevalent in Western dietary habits. The association between an obesogenic high-fat diet and CRC has been established for several decades; however, the mechanisms by which a high-fat diet increases the risk of CRC remain unclear. Recent studies indicate that gut microbiota strongly influence the pathogenesis of both high-fat diet-induced obesity and CRC. The gut microbiota is composed of hundreds of bacterial species, some of which are implicated in CRC. In particular, the expansion of facultative anaerobic Enterobacteriaceae, which is considered a microbial signature of intestinal microbiota functional imbalance (dysbiosis), is associated with both high-fat diet-induced obesity and CRC. Here, we review the interaction between the gut microbiome and its metabolic byproducts in the context of colorectal cancer (CRC) during high-fat diet-induced obesity. In addition, we will cover how a high-fat diet can drive the expansion of genotoxin-producing Escherichia coli by altering intestinal epithelial cell metabolism during gut inflammation conditions.

Infectious diseases are increasingly recognized as a major threat worldwide due to the rise of antimicrobial resistance and the emergence of novel pathogens. In vitro models that can adequately mimic in vivo gastrointestinal physiology are in high demand to elucidate mechanisms behind pathogen infectivity, and to aid the design of effective preventive and therapeutic interventions. There exists a trade-off between simple and high throughput models and those that are more complex and physiologically relevant. The complexity of the model used shall be guided by the biological question to be addressed. This review provides an overview of the structure and function of the intestine and the models that are developed to emulate this. Conventional models are discussed in addition to emerging models which employ engineering principles to equip them with necessary advanced monitoring capabilities for intestinal host-pathogen interrogation. Limitations of current models and future perspectives on the field are presented.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease, which is thought to be initiated by colon cancer stem cells, according to the cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcomes. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our MACC1 transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and the CSC-associated invasive phenotype based on patient-derived tumor organoids (PDOs), patient-derived xenografts (PDXs) and human CRC cell lines. We showed that CD44-enriched CSCs from PDO models express significantly higher levels of MACC1 and LGR5 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PDO and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with the CSC markers CD44, NANOG and LGR5 in PDO models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter, uncovering the long-known CSC marker LGR5 as a novel essential signaling mediator employed by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by a significant positive correlation of MACC1 with LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis inducer MACC1 acts as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs and prevent cancer recurrence and distant metastasis formation.

Propionate is a short-chain fatty acid that is generated upon microbiome-mediated fiber fermentation in the intestine. By modulating immune and metabolic pathways, propionate exerts many health benefits. Key bacterial species, such as Bacteroides thetaiotaomicron, generate propionate, but the biochemical pathways and specific functions remain undetermined. We identified a gene operon-encoding methylmalonyl-CoA mutase (MCM) that contributes to propionate biosynthesis in B. thetaiotaomicron. Colonization of germ-free mice with wild-type or MCM-deficient strains as well as in vitro examination demonstrated that MCM-mediated propionate production promotes goblet cell differentiation and mucus-related gene expression. Intestinal organoids lacking the propionate receptor, GPR41, showed reduced goblet cell differentiation upon MCM-mediated propionate production. Furthermore, although wild-type B. thetaiotaomicron alleviated DSS-induced intestinal inflammation, this effect was abolished in mice receiving the MCM-deficient strain but restored upon propionate supplementation. These data emphasize the critical role of MCM-mediated propionate biosynthesis in goblet cell differentiation, offering potential pathways to ameliorate colitis.

The maintenance of the functional integrity of the intestinal epithelium requires a tight coordination between cell production, migration, and shedding along the crypt-villus axis. Dysregulation of these processes may result in loss of the intestinal barrier and disease. With the aim of generating a more complete and integrated understanding of how the epithelium maintains homeostasis and recovers after injury, we have built a multi-scale agent-based model (ABM) of the mouse intestinal epithelium. We demonstrate that stable, self-organizing behaviour in the crypt emerges from the dynamic interaction of multiple signalling pathways, such as Wnt, Notch, BMP, ZNRF3/RNF43, and YAP-Hippo pathways, which regulate proliferation and differentiation, respond to environmental mechanical cues, form feedback mechanisms, and modulate the dynamics of the cell cycle protein network. The model recapitulates the crypt phenotype reported after persistent stem cell ablation and after the inhibition of the CDK1 cycle protein. Moreover, we simulated 5-fluorouracil (5-FU)-induced toxicity at multiple scales starting from DNA and RNA damage, which disrupts the cell cycle, cell signalling, proliferation, differentiation, and migration and leads to loss of barrier integrity. During recovery, our in silico crypt regenerates its structure in a self-organizing, dynamic fashion driven by dedifferentiation and enhanced by negative feedback loops. Thus, the model enables the simulation of xenobiotic-, in particular chemotherapy-, induced mechanisms of intestinal toxicity and epithelial recovery. Overall, we present a systems model able to simulate the disruption of molecular events and its impact across multiple levels of epithelial organization and demonstrate its application to epithelial research and drug development.

Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. We identified dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of- function, ChIP-seq and IP mass spectrometry, we demonstrate that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. We also explore alternate mechanisms which drive differentiation gene expression and find pause-release of Pol II and post- transcriptional mRNA stability regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue.

Intestinal epithelial stem cells (ISC) are responsible for epithelial regeneration and are critical to the intestine's ability to regain barrier function following injury. Evaluating ISC biomarker expression in cases of small intestinal strangulation (SIS) may provide insight into clinical progression.

Intestinal resection margins from cases of SIS were evaluated to determine if (1) evidence of injury could be identified using histomorphometry, (2) ISC biomarker expression was decreased in the proximal resection margin compared to control and distal resection margin, and (3) the ISC biomarker expression was associated with the number of preoperative risk factors negatively related to outcome, post-operative complications, or case outcome.

Retrospective cohort study.

Intestinal samples were obtained intraoperatively from resection margins of adult horses with SIS and horses euthanised for reasons unrelated to colic. Preoperative risk factors negatively related to outcome, post-operative complications, and case outcome were obtained from medical records. Horses were grouped as euthanised intraoperatively, postoperatively, or survived to discharge. Histomorphometry and immunofluorescence were performed to evaluate tissue architecture and ISC and progenitor cell number. Groups were compared using one-way ANOVA. Associations between biomarker expression and the number of preoperative risk factors and post-operative complications negatively related to outcome were determined using linear regression modelling.

Thirty-six cases of SIS were evaluated. Ki67+ cell counts were decreased in the proximal (mean = 15.45 cells; 95% CI = 10.27-20.63; SD = 4.17; p = 0.02) and distal resection margins (mean = 15.05; 95% CI = 8.46-21.64; SD = 4.141; p = 0.03) in horses euthanised postoperatively compared to control (mean = 23.62 cells; 95% CI = 19.42-27.83; SD = 5.883). In the distal resection margin, an increase in SOX9+ Ki67+ cells were associated with a decrease in the total number of preoperative risk factors negatively related to outcome (95% CI = 0.236-1.123; p = 0.008, SE = 0.1393).

Small population size.

Proliferating cell and ISC numbers may be associated with case outcome.

Pulmonary infections have been a leading etiology of morbidity and mortality worldwide. Upper and lower respiratory tract infections have multifactorial causes, which include bacterial, viral, and rarely, fungal infections. Moreover, the recent emergence of SARS-CoV-2 has created havoc and imposes a huge healthcare burden. Drug and vaccine development against these pulmonary pathogens like respiratory syncytial virus, SARS-CoV-2, Mycobacteria, etc., requires a systematic set of tools for research and investigation. Currently, in vitro 2D cell culture models are widely used to emulate the in vivo physiologic environment. Although this approach holds a reasonable promise over pre-clinical animal models, it lacks the much-needed correlation to the in vivo tissue architecture, cellular organization, cell-to-cell interactions, downstream processes, and the biomechanical milieu. In view of these inadequacies, 3D cell culture models have recently acquired interest. Mammalian embryonic and induced pluripotent stem cells may display their remarkable self-organizing abilities in 3D culture, and the resulting organoids replicate important structural and functional characteristics of organs such the kidney, lung, gut, brain, and retina. 3D models range from scaffold-free systems to scaffold-based and hybrid models as well. Upsurge in organs-on-chip models for pulmonary conditions has anticipated encouraging results. Complexity and dexterity of developing 3D culture models and the lack of standardized working procedures are a few of the setbacks, which are expected to be overcome in the coming times. Herein, we have elaborated the significance and types of 3D cell culture models for scrutinizing pulmonary infections, along with the in vitro techniques, their applications, and additional systems under investigation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Scribble is a master scaffold protein in apical-basal polarity. Current knowledge about the biological function of Scribble in colonic epithelial plasticity/regeneration during intestinal inflammation is limited. Here, we showed that the level of Scribble is decreased in inflammatory bowel disease (IBD) patients and mice with DSS-induced colitis. ScribΔIEC mice develops severe acute colitis with disrupted epithelial barrier integrity and impaired crypt stem cell's function. Mechanistically, Scribble suppressed the process of autophagy by modulating the stability of caspase-dependent degradation of Atg16L1 by directly interacting with Atg16L1 in a LRR domain-dependent manner in IECs and led to an accumulation of ROS both in intestinal stem cells and epithelial cells. In addition, further study indicates that dietary sphingomyelin alleviates DSS-induced colitis by increase the expression of Scribble, which suggests that Scribble may be the critical marker of IBD. Our study shows that Scribble deficiency is associated with the dysregulated autophagy and impaired maintenance of colonic stemness, and it may be a target for diagnosis and treatment of IBD.

The effect is studied of water-suspended soot microparticles on the actin cytoskeleton, apoptosis, and proliferation in the gill epithelium of pearl gourami. To this end, the fish are kept in aquariums with 0.005 g/L of soot for 5 and 14 days. Laser confocal microscopy is used to find that at the analyzed times of exposure to the pollutant zones appear in the gill epithelium, where the actin framework of adhesion belts dissociates and F-actin either forms clumps or concentrates perinuclearly. It is shown that the exposure to soot microparticles enhances apoptosis. On day 5, suppression of the proliferation of cells occurs, but the proliferation increases to the control values on day 14. Such a paradoxical increase in proliferation may be a compensatory process, maintaining the necessary level of gill function under the exposure to toxic soot. This process may occur until the gills' recovery reserve is exhausted. In general, soot microparticles cause profound changes in the actin cytoskeleton in gill cells, greatly enhance cell death, and influence cell proliferation as described. Together, these processes may cause gill dysfunction and affect the viability of fish.

Animal models have historically been poor preclinical predictors of gastrointestinal (GI) directed therapeutic efficacy and drug-induced GI toxicity. Human stem and primary cell-derived culture systems are a major focus of efforts to create biologically relevant models that enhance preclinical predictive value of intestinal efficacy and toxicity. The inherent variability in stem-cell-based complex cultures makes development of useful models a challenge; the stochastic nature of stem-cell differentiation interferes with the ability to build and validate robust, reproducible assays that query drug responses and pharmacokinetics. In this study, we aimed to characterize and reduce potential sources of variability in a complex stem cell-derived intestinal epithelium model, termed RepliGut® Planar, across cells from multiple human donors, cell lots, and passage numbers. Assessment criteria included barrier formation and integrity, gene expression, and cytokine responses. Gene expression and culture metric analyses revealed that controlling for stem/progenitor-cell passage number reduces variability and maximizes physiological relevance of the model. After optimizing passage number, donor-specific differences in cytokine responses were observed in a case study, suggesting biologic variability is observable in cell cultures derived from multiple human sources. Our findings highlight key considerations for designing assays that can be applied to additional primary-cell derived systems, as well as establish utility of the RepliGut® Planar platform for robust development of human-predictive drug-response assays.

The stem/progenitor cell pool is indispensable for the development, homeostasis and regeneration of the gastric epithelium, owing to its defining ability to self-renew whilst supplying the various functional epithelial lineages needed to digest food efficiently. A detailed understanding of the intricacies and complexities surrounding the behaviours and roles of these stem cells offers insights, not only into the physiology of gastric epithelial development and maintenance, but also into the pathological consequences following aberrations in stem cell regulation. Here, we provide an insightful synthesis of the existing knowledge on gastric epithelial stem cell biology, including the in vitro and in vivo experimental techniques that have advanced such studies. We highlight the contributions of stem/progenitor cells towards patterning the developing stomach, specification of the differentiated cell lineages and maintenance of the mature epithelium during homeostasis and following injury. Finally, we discuss gaps in our understanding and identify key research areas for future work.

Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.

Cellular quiescence is a dormant, non-dividing cell state characterized by significant shifts in physiology and metabolism. Quiescence plays essential roles in a wide variety of biological processes, ranging from microbial sporulation to human reproduction and wound repair. Moreover, when the regulation of quiescence is disrupted, it can drive cancer growth and compromise tissue regeneration after injury. In this Review, we examine the dynamic changes in metabolism that drive and support dormant and transiently quiescent cells, including spores, oocytes and adult stem cells. We begin by defining quiescent cells and discussing their roles in key biological processes. We then examine metabolic factors that influence cellular quiescence in both healthy and disease contexts, and how these could be leveraged in the treatment of cancer.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

The epithelial lining of the stomach undergoes rapid turnover to preserve its structural and functional integrity, a process driven by long-lived stem cells residing in the antral and corpus glands. Several subpopulations of gastric stem cells have been identified and their phenotypic and functional diversities linked to spatiotemporal specification of stem cells niches. Here, we review the biological features of gastric stem cells at various locations of the stomach under homeostatic conditions, as demonstrated by reporter mice, lineage tracing, and single cell sequencing. We also review the role of gastric stem cells in epithelial regeneration in response to injury. Moreover, we discuss emerging evidence demonstrating that accumulation of oncogenic drivers or alteration of stemness signaling pathways in gastric stem cells promotes gastric cancer. Given a fundamental role of the microenvironment, this review highlights the role reprogramming of niche components and signaling pathways under pathological conditions in dictating stem cell fate. Several outstanding issues are raised, such as the relevance of stem cell heterogeneity and plasticity, and epigenetic regulatory mechanisms, to Helicobacter pylori infection-initiated metaplasia-carcinogenesis cascades. With the development of spatiotemporal genomics, transcriptomics, and proteomics, as well as multiplexed screening and tracing approaches, we anticipate that more precise definition and characterization of gastric stem cells, and the crosstalk with their niche will be delineated in the near future. Rational exploitation and proper translation of these findings may bring forward novel modalities for epithelial rejuvenation and cancer therapeutics.

In colorectal cancer (CRC), the normal tissue adjacent to tumor (NAT) communicates actively with the tumor. Adult stem cells from the colon play a crucial role in the development of the colonic epithelium. In the tumor microenvironment, however, it is unclear what changes have occurred in colonic stem cells derived from NAT.

Using an intestinal stem cell culture system, we cultured colonic cells from NAT and paired CRC tissue, as well as cells from healthy tissue (HLT). Clonogenicity and differentiation ability were used to compare the function of clones from NAT, HLT and CRC tissues. RNA high-throughput sequencing of these clones was used to identify the molecular characteristics of NAT-derived clones. Coculture of clones from HLT and CRC was used to assess molecular changes.

We found that the morphological characteristics, clonogenic ability, and differentiation ability of NAT-derived clones were consistent with those of HLT-derived clones. However, NAT-derived clones changed at the molecular level. A number of genes were specifically activated in NAT. NAT-derived clones enriched pathways related to inflammation and fibrosis, including epithelial mesenchymal transition (EMT) pathway and TGF-beta signaling pathway. Our results also confirmed that NAT-derived clones could recruit fibroblasts in mice. In addition, HLT-derived clones showed high expression of FOSB when cocultured with tumor cells.

Our results demonstrate that colonic stem cells from NAT in the tumor microenvironment undergo changes at the molecular level, and these molecular characteristics can be maintained in vitro, which can induce fibrosis and an inflammatory response. Video Abstract.