Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

Isthmus progenitor cells contribute to homeostatic cellular turnover and support regeneration following intestinal injury.

The currently accepted intestinal epithelial cell organization model proposes that Lgr5+ crypt-base columnar (CBC) cells represent the sole intestinal stem cell (ISC) compartment. However, previous studies have indicated that Lgr5+ cells are dispensable for intestinal regeneration, leading to two major hypotheses: one favoring the presence of a quiescent reserve ISC and the other calling for differentiated cell plasticity. To investigate these possibilities, we studied crypt epithelial cells in an unbiased fashion via high-resolution single-cell profiling. These studies, combined with in vivo lineage tracing, show that Lgr5 is not a specific ISC marker and that stemness potential exists beyond the crypt base and resides in the isthmus region, where undifferentiated cells participate in intestinal homeostasis and regeneration following irradiation (IR) injury. Our results provide an alternative model of intestinal epithelial cell organization, suggesting that stemness potential is not restricted to CBC cells, and neither de-differentiation nor reserve ISC are drivers of intestinal regeneration.

Redefining intestinal stemness: The emergence of a new ISC population.

In tissue homeostasis, intestinal stem cells (ISCs) undergo continuous self-renewal to sustain rapid cellular turnover. In this issue of Cell, Capdevila et al.1 and Malagola, Vasciaveo, et al.2 identify a new ISC population in the upper crypt that can generate Lgr5+ stem cells during homeostasis.

Inhibition of a cyclic nucleotide-gated channel on neuronal cilia activates unfolded protein response in intestinal cells to promote longevity.

Ciliary defects are linked to ciliopathies, but impairments in the sensory cilia of Caenorhabditis elegans neurons extend lifespan, a phenomenon with previously unclear mechanisms. Our study reveals that neuronal cilia defects trigger the unfolded protein response of the endoplasmic reticulum (UPRER) within intestinal cells, a process dependent on the insulin/insulin-like growth factor 1 (IGF-1) signaling transcription factor and the release of neuronal signaling molecules. While inhibiting UPRER doesn't alter the lifespan of wild-type worms, it normalizes the extended lifespan of ciliary mutants. Notably, deactivating the cyclic nucleotide-gated (CNG) channel TAX-4 on the ciliary membrane promotes lifespan extension through a UPRER-dependent mechanism. Conversely, constitutive activation of TAX-4 attenuates intestinal UPRER in ciliary mutants. Administering a CNG channel blocker to worm larvae activates intestinal UPRER and increases adult longevity. These findings suggest that ciliary dysfunction in sensory neurons triggers intestinal UPRER, contributing to lifespan extension and implying that transiently inhibiting ciliary channel activity may effectively prolong lifespan.

Current applications of intestinal organoids: a review.

In the past decade, intestinal organoid technology has paved the way for reproducing tissue or organ morphogenesis during intestinal physiological processes in vitro and studying the pathogenesis of various intestinal diseases. Intestinal organoids are favored in drug screening due to their ability for high-throughput in vitro cultivation and their closer resemblance to patient genetic characteristics. Furthermore, as disease models, intestinal organoids find wide applications in screening diagnostic markers, identifying therapeutic targets, and exploring epigenetic mechanisms of diseases. Additionally, as a transplantable cellular system, organoids have played a significant role in the reconstruction of damaged epithelium in conditions such as ulcerative colitis and short bowel syndrome, as well as in intestinal material exchange and metabolic function restoration. The rise of interdisciplinary approaches, including organoid-on-chip technology, genome editing techniques, and microfluidics, has greatly accelerated the development of organoids. In this review, VOSviewer software is used to visualize hot co-cited journal and keywords trends of intestinal organoid firstly. Subsequently, we have summarized the current applications of intestinal organoid technology in disease modeling, drug screening, and regenerative medicine. This will deepen our understanding of intestinal organoids and further explore the physiological mechanisms of the intestine and drug development for intestinal diseases.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius).

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Improved Preservation of Mouse Intestinal Tissue Using a Formalin/Acetic Acid Fixative and Quantitative Histological Analysis Using QuPath.

The architecture and morphology of the intestinal tissue from mice or other small animals are difficult to preserve for histological and molecular analysis due to the fragile nature of this tissue. The intestinal mucosa consists of villi and crypts lined with epithelial cells. In between the epithelial folds extends the lamina propria, a loose connective tissue that contains blood and lymph vessels, fibroblasts, and immune cells. Underneath the mucosa are two layers of contractile smooth muscle and nerves. The tissue experiences significant changes during fixation, which can impair the reliability of histologic analysis. Poor-quality histologic sections are not suitable for quantitative image-based tissue analysis. This article offers a new fixative composed of neutral buffered formalin (NBF) and acetic acid, called FA. This fixative significantly improved the histology of mouse intestinal tissue compared to traditional NBF and enabled precise, reproducible histologic molecular analyses using QuPath software. Algorithmic training of QuPath allows for automated segmentation of intestinal compartments, which can be further interrogated for cellular composition and disease-related changes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Improved preservation of mouse intestinal tissue using a formalin/acetic acid fixative Support Protocol: Quantitative tissue analysis using QuPath.

Microbial metabolite steers intestinal stem cell fate under stress.

Recently in Cell Metabolism, Wei et al.1 unveiled a brain-to-gut pathway that conveys psychological stress to intestinal epithelial cells, leading to their dysfunction. This gut-brain axis involves a microbial metabolite, indole-3-acetate (IAA), as a niche signal that hampers mitochondrial respiration to skew intestinal stem cell (ISC) fate.

The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster.

Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, RasV12, esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a "specific" ISC/EB driver to examine ISC/EB-mediated animal physiology.

High-fat diet enhances cell proliferation and compromises intestinal permeability in a translational canine intestinal organoid model.

BACKGROUND: Emerging evidence underscores the responsiveness of the mammalian intestine to dietary cues, notably through the involvement of LGR5 + intestinal stem cells in orchestrating responses to diet-driven signals. However, the effects of high-fat diet (HFD) on these cellular dynamics and their impact on gut integrity remain insufficiently understood. Our study aims to assess the multifaceted interactions between palmitic acid (PA), cell proliferation, and the intestinal epithelial barrier using a canine colonoid model. Canine models, due to their relevance in simulating human intestinal diseases, offer a unique platform to explore the molecular mechanisms underlying HFD derived intestinal dysfunction. RESULTS: Canine colonoids were subjected to PA exposure, a surrogate for the effects of HFD. This intervention revealed a remarkable augmentation of cell proliferative activity. Furthermore, we observed a parallel reduction in transepithelial electrical resistance (TEER), indicating altered epithelium barrier integrity. While E-cadherin exhibited consistency, ZO-1 displayed a noteworthy reduction in fluorescence intensity within the PA-exposed group. CONCLUSIONS: By employing canine intestinal organoid systems, we provide compelling insights into the impact of PA on intestinal physiology. These findings underscore the importance of considering both cell proliferative activity and epithelial integrity in comprehending the repercussions of HFDs on intestinal health. Our study contributes to a deeper understanding of the consequences of HFD on intestinal homeostasis, utilizing valuable translational in vitro models derived from dogs.

TMT-Based Quantitative Proteomic Analysis Reveals the Key Role of Cell Proliferation and Apoptosis in Intestine Regeneration of Apostichopus japonicus.

Sea cucumbers are widely known for their powerful regenerative abilities, which allow them to regenerate a complete digestive tract within a relatively short time following injury or autotomy. Recently, even though the histological changes and cellular events in the processes of intestinal regeneration have been extensively studied, the molecular machinery behind this faculty remains unclear. In this study, tandem mass tag (TMT)-based quantitation was utilized to investigate protein abundance changes during the process of intestine regeneration. Approximately 538, 445, 397, 1012, and 966 differential proteins (DEPs) were detected (p < 0.05) between the normal and 2, 7, 12, 20, and 28 dpe stages, respectively. These DEPs also mainly focus on pathways of cell proliferation and apoptosis, which were further validated by 5-Ethynyl-2'-deoxyuridine (EdU) or Tunel-based flow cytometry assay. These findings provide a reference for a comprehensive understanding of the regulatory mechanisms of various stages of intestinal regeneration and provide a foundation for subsequent research on changes in cell fate in echinoderms.

Mulberry Leaf-Derived Morin Activates ß-Catenin by Binding to Frizzled7 to Promote Intestinal Stem Cell Expansion upon Heat-Stable Enterotoxin b Injury.

Intestinal stem cells (ISCs) sustain epithelial renewal by dynamically altering behaviors of proliferation and differentiation in response to various nutrition and stress inputs. However, how ISCs integrate bioactive substance morin cues to protect against heat-stable enterotoxin b (STb) produced by Escherichia coli remains an uncertain question with implications for treating bacterial diarrhea. Our recent work showed that oral mulberry leaf-derived morin improved the growth performance in STb-challenged mice. Furthermore, morin supplementation reinstated the impaired small-intestinal epithelial structure and barrier function by stimulating ISC proliferation and differentiation as well as supporting intestinal organoid expansion ex vivo. Importantly, the Wnt/ß-catenin pathway, an ISC fate commitment signal, was reactivated by morin to restore the jejunal crypt-villus architecture in response to STb stimulation. Mechanically, the extracellular morin-initiated ß-catenin axis is dependent or partially dependent on the Wnt membrane receptor Frizzled7 (FZD7). Our data reveal an unexpected role of leaf-derived morin, which represents molecular signaling targeting the FZD7 platform instrumental for controlling ISC regeneration upon STb injury.

Gallic acid mitigates intestinal inflammation and loss of tight junction protein expression using a 2D-Caco-2 and RAW 264.7 co-culture model.

A 2D-intestinal epithelial Caco-2/RAW 264.7 macrophage co-culture model was developed to demonstrate the relative efficacy of different phenolic acids to mitigate changes in Caco-2 epithelial cell redox state initiated both directly by autoxidation products, H2O2, and indirectly through cell communication events originating from cytokine stimulated macrophage. An inducer cocktail (lipopolysaccharide + interferon gamma) was used to activate RAW 264.7 cells in the 2D- Caco-2/RAW co-culture and intracellular changes in Caco-2 cell redox signaling occurred in response to positive changes (p < 0.05) in inflammatory biomarkers derived in macrophage that included IL-6, TNF-α, nitric oxide and peroxynitrite, respectively. Phenolic acids varied in relative capacity to reduce NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cocktail inflamed induced macrophage. This response in addition to the relative predisposition of gallic acid (GA) to undergo autoxidation to generate H2O2 activity (p < 0.05), culminated in downstream cell signaling in Caco-2 nuclear factor erythroid 2-related factor (Nrf2) activity (increase 26.9 %), altered monolayer integrity (increase 33.7 %), and release of interleukin 8 (IL-8) (decrease 80.5 %) (p < 0.05). It can be concluded that the co-culture model described herein was useful to assess the importance of communication between cytokine stimulated macrophage and intestinal cells. Moreover, the relative unique efficacy of GA, compared to other phenolic acids tested to protect against activated macrophage induced changes related to intestinal dysfunction were particularly relevant to epithelial redox signaling, intestinal permeability and regulation of tight junction proteins. This study concludes that phenolic acids are not equal in the capacity to protect against intestinal cell dysfunction despite some indication of biological activity.

Effect of Porosity on the Colonization of Digital Light-Processed 3D Hydrogel Constructs toward the Development of a Functional Intestinal Model.

With the rapid increase of the number of patients with gastrointestinal diseases in modern society, the need for the development of physiologically relevant in vitro intestinal models is key to improve the understanding of intestinal dysfunctions. This involves the development of a scaffold material exhibiting physiological stiffness and anatomical mimicry of the intestinal architecture. The current work focuses on evaluating the scaffold micromorphology of gelatin-methacryloyl-aminoethyl-methacrylate-based nonporous and porous intestinal 3D, intestine-like constructs, fabricated via digital light processing, on the cellular response. To this end, Caco-2 intestinal cells were utilized in combination with the constructs. Both porous and nonporous constructs promoted cell growth and differentiation toward enterocyte-like cells (VIL1, ALPI, SI, and OCLD expression showed via qPCR, ZO-1 via immunostaining). The porous constructs outperformed the nonporous ones regarding cell seeding efficiency and growth rate, confirmed by MTS assay, live/dead staining, and TEER measurements, due to the presence of surface roughness.

InR and Pi3K maintain intestinal homeostasis through STAT/EGFR and Notch signaling in enteroblasts.

To maintain the integrity of the adult gut, the proliferation and differentiation of stem cells must be strictly controlled. Several signaling pathways control the proliferation and differentiation of Drosophila intestinal epithelial cells. Although the modulatory effects of insulin pathway components on cell proliferation have been characterized, their specific role in which cell type and how these components interact with other regulatory signaling pathways remain largely unclear. In this study, we found that InR/Pi3K has major functions in enteroblasts (EBs) that were not previously described. The absence of InR/Pi3K in progenitors leads to a decrease in the number of EBs, while it has no significant effect on intestinal stem cells (ISCs). In addition, we found that InR/Pi3K regulates Notch activity in ISCs and EBs in an opposite way. This is also the reason for the decrease in EB. On the one hand, aberrantly low levels of Notch signaling in ISCs inhibit their proper differentiation into EBs; on the other hand, the higher Notch levels in EBs promote their excessive differentiation into enterocytes (ECs), leading to marked increases in abnormal ECs and decreased proliferation. Moreover, we found that Upd/JAK/STAT signaling acts as an effector or modifier of InR/Pi3K function in the midgut and cooperates with EGFR signaling to regulate cell proliferation. Altogether, our results demonstrate that InR and Pi3K are essential for coordinating stem cell differentiation and proliferation to maintain intestinal homeostasis.

Villus myofibroblasts are developmental and adult progenitors of mammalian gut lymphatic musculature.

Inside the finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic capillary, the lacteal, sending fats into the systemic blood circulation for energy production. Despite this vital function, mechanisms of formation, assembly alongside lacteals, and maintenance of villus smooth muscle are unknown. By combining single-cell RNA sequencing and quantitative lineage tracing of the mouse intestine, we identified a local hierarchy of subepithelial fibroblast progenitors that differentiate into mature smooth muscle fibers via intermediate contractile myofibroblasts. This continuum persists as the major mechanism for villus musculature renewal throughout adult life. The NOTCH3-DLL4 signaling axis governs the assembly of smooth muscle fibers alongside their adjacent lacteals and is required for fat absorption. Our studies identify the ontogeny and maintenance of a poorly defined class of intestinal smooth muscle, with implications for accelerated repair and recovery of digestive function following injury.

Nutrient metabolism in regulating intestinal stem cell homeostasis.

Intestinal stem cells (ISCs) are known for their remarkable proliferative capacity, making them one of the most active cell populations in the body. However, a high turnover rate of intestinal epithelium raises the likelihood of dysregulated homeostasis, which is known to cause various diseases, including cancer. Maintaining precise control over the homeostasis of ISCs is crucial to preserve the intestinal epithelium's integrity during homeostasis or stressed conditions. Recent research has indicated that nutrients and metabolic pathways can extensively modulate the fate of ISCs. This review will explore recent findings concerning the influence of various nutrients, including lipids, carbohydrates, and vitamin D, on the delicate balance between ISC proliferation and differentiation.

Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

Interaction of species A rotavirus VP4 with the cellular proteins vimentin and actin related protein 2 discovered by a proximity interactome assay.

IMPORTANCE: Rotavirus (RV) is an important zoonosis virus, which can cause severe diarrhea and extra-intestinal infection. To date, some proteins or carbohydrates have been shown to participate in the attachment or internalization of RV, including HGBAs, Hsc70, and integrins. This study attempted to indicate whether there were other proteins that would participate in the entry of RV; thus, the RV VP4-interacting proteins were identified by proximity labeling. After analysis and verification, it was found that VIM and ACTR2 could significantly promote the proliferation of RV in intestinal cells. Through further viral binding assays after knockdown, antibody blocking, and recombinant protein overexpression, it was revealed that both VIM and ACTR2 could promote RV replication.

Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut.

A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that downregulation of the conserved cholinergic enzyme acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific Egr5 (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+, which spreads in the epithelium through Innexin2-Innexin7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial Ca2+ currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury.