Inflammatory bowel disease (IBD) is an inflammatory disease of the intestine whose primary pathological presentation is the destruction of the intestinal epithelium. The intestinal epithelium, located between the lumen and lamina propria, transmits luminal microbial signals to the immune cells in the lamina propria, which also modulate the intestinal epithelium. In IBD patients, intestinal epithelial cells (IECs) die dysfunction and the mucosal barrier is disrupted, leading to the recruitment of immune cells and the release of cytokines. In this review, we describe the structure and functions of the intestinal epithelium and mucosal barrier in the physiological state and under IBD conditions, as well as the patterns of epithelial cell death and how immune cells modulate the intestinal epithelium providing a reference for clinical research and drug development of IBD. In addition, according to the targeting of epithelial apoptosis and necroptotic pathways and the regulation of immune cells, we summarized some new methods for the treatment of IBD, such as necroptosis inhibitors, microbiome regulation, which provide potential ideas for the treatment of IBD. This review also describes the potential for integrating AI-driven approaches into innovation in IBD treatments.

Gene set analysis methods have played a major role in generating biological interpretations of omics data such as gene expression datasets. However, most methods focus on detecting homogenous pattern changes in mean expression while methods detecting pattern changes in variance remain poorly explored. While a few studies attempted to use gene-level variance analysis, such approach remains under-utilized. When comparing two phenotypes, gene sets with distinct changes in subgroups under one phenotype are overlooked by available methods although they reflect meaningful biological differences between two phenotypes. Multivariate sample-level variance analysis methods are needed to detect such pattern changes.

We used ranking schemes based on minimum spanning tree to generalize the Cramer-Von Mises and Anderson-Darling univariate statistics into multivariate gene set analysis methods to detect differential sample variance or mean. We characterized the detection power and Type I error rate of these methods in addition to two methods developed earlier using simulation results with different parameters. We applied the developed methods to microarray gene expression dataset of prednisolone-resistant and prednisolone-sensitive children diagnosed with B-lineage acute lymphoblastic leukemia and bulk RNA-sequencing gene expression dataset of benign hyperplastic polyps and potentially malignant sessile serrated adenoma/polyps. One or both of the two compared phenotypes in each of these datasets have distinct molecular subtypes that contribute to within phenotype variability and to heterogeneous differences between two compared phenotypes. Our results show that methods designed to detect differential sample variance provide meaningful biological interpretations by detecting specific hallmark gene sets associated with the two compared phenotypes as documented in available literature.

The results of this study demonstrate the usefulness of methods designed to detect differential sample variance in providing biological interpretations when biologically relevant but heterogeneous changes between two phenotypes are prevalent in specific signaling pathways. Software implementation of the methods is available with detailed documentation from Bioconductor package GSAR. The available methods are applicable to gene expression datasets in a normalized matrix form and could be used with other omics datasets in a normalized matrix form with available collection of feature sets.

Glial cells of the enteric nervous system (ENS) interact closely with the intestinal epithelium and secrete signals that influence epithelial cell proliferation and barrier formation in vitro. Whether these interactions are important in vivo, however, is unclear because previous studies reached conflicting conclusions (Prochera and Rao, 2023). To better define the roles of enteric glia in steady state regulation of the intestinal epithelium, we characterized the glia in closest proximity to epithelial cells and found that the majority express the gene Proteolipid protein 1 (PLP1) in both mice and humans. To test their functions using an unbiased approach, we genetically depleted PLP1+ cells in mice and transcriptionally profiled the small and large intestines. Surprisingly, glial loss had minimal effects on transcriptional programs and the few identified changes varied along the gastrointestinal tract. In the ileum, where enteric glia had been considered most essential for epithelial integrity, glial depletion did not drastically alter epithelial gene expression but caused a modest enrichment in signatures of Paneth cells, a secretory cell type important for innate immunity. In the absence of PLP1+ glia, Paneth cell number was intact, but a subset appeared abnormal with irregular and heterogenous cytoplasmic granules, suggesting a secretory deficit. Consistent with this possibility, ileal explants from glial-depleted mice secreted less functional lysozyme than controls with corresponding effects on fecal microbial composition. Collectively, these data suggest that enteric glia do not exert broad effects on the intestinal epithelium but have an essential role in regulating Paneth cell function and gut microbial ecology.

Studies of the avian gastrointestinal tract, where nutrient absorption and key host-pathogen interactions occur, have been strongly enabled by the development of intestinal organoid models. Here we report a single cell transcriptomic atlas of intestinal organoid cells derived from embryos of broiler and layer chickens, capturing mesenchymal, epithelial, endothelial, immune and neuronal cell lineages. Eight inferred mesenchymal subpopulations reflect anatomically distinct intestinal layers, including fibroblasts, telocytes, myofibroblasts, smooth myocytes, pericytes, and interstitial cells of Cajal. Identified heterogeneity within the epithelial lineage included enterocytes, goblet cells, Paneth cells, tuft cells, and diverse enteroendocrine cell subtypes. Additionally, we identified candidate macrophages, monocytes, γδ T cells, NK cells and granulocytes. Layer and broiler organoids showed significant differences in cell-specific transcriptome, most pronounced in epithelial cells, pointing to divergent selection on intestinal physiology. Our analysis finally provides a catalogue of novel cell marker genes to enable future research of chicken intestinal organoids.

Enterocytes and four classic secretory cell types derive from intestinal epithelial stem cells. Based on morphology, location, and canonical markers, goblet and Paneth cells are considered distinct secretory types. Here, we report high overlap in their transcripts and sites of accessible chromatin, in marked contrast to those of their enteroendocrine or tuft cell siblings. Mouse and human goblet and Paneth cells express extraordinary fractions of few antimicrobial genes, which reflect specific responses to local niches. Wnt signaling retains some ATOH1+ secretory cells in crypt bottoms, where the absence of BMP signaling potently induces Paneth features. Cells that migrate away from crypt bottoms encounter BMPs and thereby acquire goblet properties. These phenotypes and underlying accessible cis-elements interconvert in post-mitotic cells. Thus, goblet and Paneth properties represent alternative phenotypic manifestations of a common signal-responsive terminal cell type. These findings reveal exquisite niche-dependent cell plasticity and cis-regulatory dynamics in likely response to antimicrobial needs.

Lacto-N-neotetraose (LNnT) is a prevalent neutral core human milk oligosaccharides (HMOs) recognized for its numerous benefits to infant health. In infant formula, galactooligosaccharide (GOS) are frequently used as substitutes for HMOs. However, the regulatory roles of LNnT and GOS in early intestinal development are not yet fully understood. This study aims to elucidate the effects of LNnT and GOS on intestinal development during early life. Our findings show that administering LNnT or GOS significantly increased the spleen and liver indices of infant mice at postnatal day 21. Immunofluorescence and qPCR analysis showed that feeding LNnT significantly promoted the proliferation and differentiation of intestinal stem cells (ISCs) in the colon of infant mice at postnatal day 21, and increased the expression of differentiation markers of goblet cells, intestinal epithelial cells, Paneth cells, and intestinal endocrine cells. Conversely, feeding GOS had no significant effect on the proliferation and differentiation of ISCs. Furthermore, intestinal microbiota analysis showed that LNnT increased the microbiota associated with intestinal regeneration and ISCs proliferation and differentiation in infant mice at postnatal day 21. In conclusion, LNnT promoted ISCs proliferation and differentiation in the colon and alters the composition and function of the intestinal microbiota to support intestinal development in infant mice.

The homeostasis of the intestinal epithelium relies on intricate yet insufficiently understood mechanisms of intestinal epithelial plasticity. Here, we elucidate the pivotal role of Frizzled5 (Fzd5), a Wnt pathway receptor, as a determinant of murine intestinal epithelial cell fate. Deletion of Fzd5 in Lgr5+ intestinal stem cells (ISCs) impairs their self-renewal, whereas its deletion in Krt19+ cells disrupts lineage generation, without affecting crypt integrity in either case. However, a broader deletion of Fzd5 across the epithelium leads to substantial crypt deterioration. Integrated analysis of single-cell RNA sequencing (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) identifies that Fzd5 governs chromatin accessibility, orchestrating the regulation of stem- and lineage-related gene expression mainly in ISCs and progenitor cells. In summary, our findings provide insights into the regulatory role of Fzd5 in governing intestinal epithelial plasticity.

The rapidly regenerating intestinal epithelium requires crypt intestinal stem cells (ISCs). Wnt/β-catenin signaling maintains crypt homeostasis and Lgr5+ ISCs, and WNT ligands bind Frizzled receptors (FZD1-10). Identifying specific FZD(s) essential for intestinal homeostasis has been elusive; however, bioengineered antagonists blocking Wnt binding to FZD5 and FZD8 deplete the gut epithelium in vivo, highlighting potential roles. Here, an epithelial-specific Fzd5 knockout (KO) elicited lethal pan-intestinal crypt and villus loss, whereas an Lgr5+ ISC-specific Fzd5 KO depleted Lgr5+ ISCs via premature differentiation and repressed Wnt target genes. Fzd5-null phenotypes were rescued by constitutive β-catenin activation in vivo and in both mouse and human enteroids. KO of Fzd5, not Fzd8, in enteroids ablated responsiveness to dual-specificity FZD5/FZD8-selective Wnt surrogate agonists, which ameliorated DSS-induced colitis in wild-type and Fzd8 KO mice. Overall, FZD5 is a dominant and essential regulator of crypt homeostasis, Lgr5+ ISCs, and intestinal response to Wnt surrogate agonists, with implications for therapeutic mucosal repair.

The intestinal stem cell (ISC) niche is vital for maintaining the integrity and function of the intestinal epithelium. ISC populations, characterized by their high proliferation and multipotency, reside within a specialized microenvironment at the base of crypts. Crypt base columnar (CBC) cells at the deepest part of crypts serve as replicating ISCs, while position 4 label-retaining cells (LRCs) located higher up in the crypts are also important for ISC maintenance during experiments. The interplay between CBCs, position 4 LRCs, transient amplifying (TA) cells and other niche components, including the pericrypt stromal cells, ensures a continuous supply of differentiated epithelial cells. Recent advancements in ISC biomarker studies have provided valuable insights into their molecular signatures, regulatory pathways and roles in the pathogenesis of intestinal disorders. Understanding the ISC niche has significant therapeutic implications, as manipulating ISC behaviors and regenerating damaged or diseased intestinal tissue show promise for novel therapeutic approaches. ISC organoids have also provided a platform for studying intestinal diseases and testing personalized therapies. This comprehensive review covers the anatomical composition, physiological regulation, ISC biomarker studies, contribution to intestinal disorder pathogenesis and potential therapeutic implications of the ISC niche.

Intestinal stem cells differentiate into absorptive enterocytes, characterised by increased brush border enzymes such as intestinal alkaline phosphatase (IAP), making up the majority (95%) of the terminally differentiated cells in the villus. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here, we show that the intestinal microRNA (miR)-27a-3p is an important regulator of intestinal epithelial cell proliferation and enterocyte differentiation. Repression of endogenous miR-27a-3p leads to increased enterocyte differentiation and decreased intestinal epithelial cell proliferation in mouse and human small intestinal organoids. Mechanistically, miR-27a-3p regulates intestinal cell differentiation and proliferation at least in part through the regulation of retinoic acid receptor α (RXRα), a modulator of Wnt/β-catenin signalling. Repression of miR-27a-3p increases the expression of RXRα and concomitantly, decreases the expression of active β-catenin and cyclin D1. In contrast, overexpression of miR-27a-3p mimic decreases the expression of RXRα and increases the expression of active β-catenin and cyclin D1. Moreover, overexpression of the miR-27a-3p mimic results in impaired enterocyte differentiation and increases intestinal epithelial cell proliferation. These alterations were attenuated or blocked by Wnt inhibition. Our study demonstrates an miR-27a-3p/RXRα/Wnt/β-catenin pathway that is important for the maintenance of enterocyte homeostasis in the small intestine.

The Wnt signaling pathway is critical in the onset and progression of gastrointestinal (GI) cancers. Anomalies in this pathway, often stemming from mutations in critical components such as adenomatous polyposis coli (APC) or β-catenin, lead to uncontrolled cell proliferation and survival. In the case of colorectal cancer, dysregulation of the Wnt pathway drives tumor initiation and growth. Similarly, aberrant Wnt signaling contributes to tumor development, metastasis, and resistance to therapy in other GI cancers, such as gastric, pancreatic, and hepatocellular carcinomas. Targeting the Wnt pathway or its downstream effectors has emerged as a promising therapeutic strategy for combating these highly aggressive GI malignancies. Here, we review the dysregulation of the Wnt signaling pathway in the pathogenesis of GI cancers and further explore the therapeutic potential of targeting the various components of the Wnt pathway. Furthermore, we summarize and integrate the preclinical evidence supporting the therapeutic efficacy of potent Wnt pathway inhibitors with completed and ongoing clinical trials in GI cancers. Additionally, we discuss the challenges of Wnt pathway-targeted therapies in GI cancers to overcome these concerns for effective clinical translation.

The role of the immune system in regulating tissue stem cells remains poorly understood, as does the relationship between immune-mediated tissue damage and regeneration. Graft vs. host disease (GVHD) occurring after allogeneic bone marrow transplantation (allo-BMT) involves immune-mediated damage to the intestinal epithelium and its stem cell compartment. To assess impacts of T-cell-driven injury on distinct epithelial constituents, we have performed single cell RNA sequencing on intestinal crypts following experimental BMT. Intestinal stem cells (ISCs) from GVHD mice have exhibited global transcriptomic changes associated with a substantial Interferon-γ response and upregulation of STAT1. To determine its role in crypt function, STAT1 has been deleted within murine intestinal epithelium. Following allo-BMT, STAT1 deficiency has resulted in reduced epithelial proliferation and impaired ISC recovery. Similarly, epithelial Interferon-γ receptor deletion has also attenuated proliferation and ISC recovery post-transplant. Investigating the mechanistic basis underlying this epithelial response, ISC STAT1 expression in GVHD has been found to correlate with upregulation of ISC c-Myc. Furthermore, activated T cells have stimulated Interferon-γ-dependent epithelial regeneration in co-cultured organoids, and Interferon-γ has directly induced STAT1-dependent c-Myc expression and ISC proliferation. These findings illustrate immunologic regulation of a core tissue stem cell program after damage and support a role for Interferon-γ as a direct contributor to epithelial regeneration.

In this Editorial review, we would like to focus on a very recent discovery showing the global autosomal gene regulation by Y- and inactivated X-chromosomal transcription factors, zinc finger gene on the Y chromosome (ZFY) and zinc finger protein X-linked (ZFX). ZFX and ZFY are both zinc-finger proteins that encode general transcription factors abundant in hematopoietic and embryonic stem cells. Although both proteins are homologs, interestingly, the regulation of self-renewal by these transcriptional factors is almost exclusive to ZFX. This fact implies that there are some differential roles between ZFX and ZFY in regulating the maintenance of self-renewal activity in stem cells. Besides the maintenance of stemness, ZFX overexpression or mutations may be linked to certain cancers. Although cancers and stem cells are double-edged swords, there is no study showing the link between ZFX activity and the telomere. Thus, stemness or cancers with ZFX may be linked to other molecules, such as Oct4, Sox2, Klf4, and others. Based on very recent studies and a few lines of evidence in the past decade, it appears that the ZFX is linked to the canonical Wnt signaling, which is one possible mechanism to explain the role of ZFX in the self-renewal of stem cells.

The study of fetal gut development is critical due to its substantial influence on immediate neonatal and long-term adult health. Current research largely focuses on microbiome colonization, gut immunity, and barrier function, alongside the impact of external factors on these phenomena. Limited research has been dedicated to the categorization of developing fetal gut cells. Our study aimed to enhance our understanding of fetal gut development by employing advanced machine-learning techniques on single-cell sequencing data. This dataset consisted of 62,849 samples, each characterized by 33,694 distinct gene features. Four feature ranking algorithms were utilized to sort features according to their significance, resulting in four feature lists. Then, these lists were fed into an incremental feature selection method to extract essential genes, classification rules, and build efficient classifiers. Several important genes were recognized by multiple feature ranking algorithms, such as FGG, MDK, RBP1, RBP2, IGFBP7, and SPON2. These features were key in differentiating specific developing intestinal cells, including epithelial, immune, mesenchymal, and vasculature cells of the colon, duo jejunum, and ileum cells. The classification rules showed special gene expression patterns on some intestinal cell types and the efficient classifiers can be useful tools for identifying intestinal cells.

Lgr5+ intestinal stem cells (ISCs) are crucial for the intestinal epithelium renewal and regeneration after injury. However, the mechanism underlying the interplay between Wnt and BMP signaling in this process is not fully understood. Here we report that Bcl11b, which is downregulated by BMP signaling, enhances Wnt signaling to maintain Lgr5+ ISCs and thus promotes the regeneration of the intestinal epithelium upon injury. Loss of Bcl11b function leads to a significant decrease of Lgr5+ ISCs in both intestinal crypts and cultured organoids. Mechanistically, BMP suppresses the expression of Bcl11b, which can positively regulate Wnt target genes by inhibiting the function of the Nucleosome Remodeling and Deacetylase (NuRD) complex and facilitating the β-catenin-TCF4 interaction. Bcl11b can also promote intestinal epithelium repair after injuries elicited by both irradiation and DSS-induced inflammation. Furthermore, Bcl11b deletion prevents proliferation and tumorigenesis of colorectal cancer cells. Together, our findings suggest that BMP suppresses Wnt signaling via Bcl11b regulation, thus balancing homeostasis and regeneration in the intestinal epithelium.

Lgr5-positive cells located in the basal layer of crypts have self-regenerative and proliferative differentiation potentials of intestinal stem cells (ISCs), maintaining a balance of regeneration-repair in mucosal epithelium. However, the mechanisms of mucosal repair that are regulated by ISCs in ulcerative colitis (UC) remain unclear.

Colon tissues from patients with UC were collected to test β-catenin and Notch1 expression by using Western blot and quantitative real-time polymerase chain reaction (PCR). β-cateninfl/fl mice, β-cateninTg mice, and Dll1tm1 Gos mice were used to cross with Lgr5-EGFP-IRES-creERT2 mice to generate mice of different genotypes, altering the activation of Wnt/β-catenin and Dll1-mediated Notch signaling in ISCs in vivo. Dextran sulfate sodium (DSS) was used to induce a colitis mice model. Intestinal organoids were isolated and cultured to observe the proliferation and differentiation levels of ISCs.

β-catenin and Notch1 expression were significantly increased in the inflamed colon tissues from patients with UC. Wnt/β-catenin activation and Dll1-mediated Notch pathway inhibition in Lgr5-positive stem cells promoted the expressions of E-cadherin, CK20, and CHGA in colonic organoids and epithelium, implying the promotion of colonic epithelial integrity. Activation of Wnt/β-catenin and suppression of Dll1-mediated Notch pathway in Lgr5-positive ISCs alleviated the DSS-induced intestinal mucosal inflammation in mice.

Lgr5-positive ISCs are characterized by self-renewal and high dividend potential, which play an important role in the injury and repair of intestinal mucosa. More importantly, the Wnt/β-catenin signaling pathway cooperates with the Notch signaling pathway to maintain the function of the Lgr5-positive ISCs.

Crohn's disease (CD) is a complex and heterogeneous condition with no perfect preclinical model or cure. To address this, we explore adult stem cell-derived organoids that retain their tissue identity and disease-driving traits. We prospectively create a biobank of CD patient-derived organoid cultures (PDOs) from colonic biopsies of 53 subjects across all clinical subtypes and healthy subjects. Gene expression analyses enabled benchmarking of PDOs as tools for modeling the colonic epithelium in active disease and identified two major molecular subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD). Each subtype shows internal consistency in the transcriptome, genome, and phenome. The spectrum of morphometric, phenotypic, and functional changes within the "living biobank" reveals distinct differences between the molecular subtypes. Drug screens reverse subtype-specific phenotypes, suggesting phenotyped-genotyped CD PDOs can bridge basic biology and patient trials by enabling preclinical phase "0" human trials for personalized therapeutics.

Radiotherapy is a pivotal intervention for cancer patients, significantly impacting their treatment outcomes and survival prospects. Nevertheless, in the course of treating those with abdominal, pelvic, or retroperitoneal malignant tumors, the procedure inadvertently exposes adjacent intestinal tissues to radiation, posing risks of radiation-induced enteropathy upon reaching threshold doses. Stem cells within the intestinal crypts, through their controlled proliferation and differentiation, support the critical functions of the intestinal epithelium, ensuring efficient nutrient absorption while upholding its protective barrier properties. Intestinal stem cells (ISCs) regulation is intricately orchestrated by diverse signaling pathways, among which are the WNT, BMP, NOTCH, EGF, Hippo, Hedgehog and NF-κB, each contributing to the complex control of these cells' behavior. Complementing these pathways are additional regulators such as nutrient metabolic states, and the intestinal microbiota, all of which contribute to the fine-tuning of ISCs behavior in the intestinal crypts. It is the harmonious interplay among these signaling cascades and modulating elements that preserves the homeostasis of intestinal epithelial cells (IECs), thereby ensuring the gut's overall health and function. This review delves into the molecular underpinnings of how stem cells respond in the context of radiation enteropathy, aiming to illuminate potential biological targets for therapeutic intervention. Furthermore, we have compiled a summary of several current treatment methodologies. By unraveling these mechanisms and treatment methods, we aspire to furnish a roadmap for the development of novel therapeutics, advancing our capabilities in mitigating radiation-induced intestinal damage.

The β-catenin-dependent canonical Wnt signaling is pivotal in organ development, tissue homeostasis, and cancer. Here, we identified an upstream enhancer of Ctnnb1 - the coding gene for β-catenin, named ieCtnnb1 (intestinal enhancer of Ctnnb1), which is crucial for intestinal homeostasis. ieCtnnb1 is predominantly active in the base of small intestinal crypts and throughout the epithelia of large intestine. Knockout of ieCtnnb1 led to a reduction in Ctnnb1 transcription, compromising the canonical Wnt signaling in intestinal crypts. Single-cell sequencing revealed that ieCtnnb1 knockout altered epithelial compositions and potentially compromised functions of small intestinal crypts. While deletion of ieCtnnb1 hampered epithelial turnovers in physiologic conditions, it prevented occurrence and progression of Wnt/β-catenin-driven colorectal cancers. Human ieCTNNB1 drove reporter gene expression in a pattern highly similar to mouse ieCtnnb1. ieCTNNB1 contains a single-nucleotide polymorphism associated with CTNNB1 expression levels in human gastrointestinal epithelia. The enhancer activity of ieCTNNB1 in colorectal cancer tissues was stronger than that in adjacent normal tissues. HNF4α and phosphorylated CREB1 were identified as key trans-factors binding to ieCTNNB1 and regulating CTNNB1 transcription. Together, these findings unveil an enhancer-dependent mechanism controlling the dosage of Wnt signaling and homeostasis in intestinal epithelia.

Gene set analysis methods have played a major role in generating biological interpretations from omics data such as gene expression datasets. However, most methods focus on detecting homogenous pattern changes in mean expression and methods detecting pattern changes in variance remain poorly explored. While a few studies attempted to use gene-level variance analysis, such approach remains under-utilized. When comparing two phenotypes, gene sets with distinct changes in subgroups under one phenotype are overlooked by available methods although they reflect meaningful biological differences between two phenotypes. Multivariate sample-level variance analysis methods are needed to detect such pattern changes.

We use ranking schemes based on minimum spanning tree to generalize the Cramer-Von Mises and Anderson-Darling univariate statistics into multivariate gene set analysis methods to detect differential sample variance or mean. We characterize these methods in addition to two methods developed earlier using simulation results with different parameters. We apply the developed methods to microarray gene expression dataset of prednisolone-resistant and prednisolone-sensitive children diagnosed with B-lineage acute lymphoblastic leukemia and bulk RNA-sequencing gene expression dataset of benign hyperplastic polyps and potentially malignant sessile serrated adenoma/polyps. One or both of the two compared phenotypes in each of these datasets have distinct molecular subtypes that contribute to heterogeneous differences. Our results show that methods designed to detect differential sample variance are able to detect specific hallmark signaling pathways associated with the two compared phenotypes as documented in available literature.

The results in this study demonstrate the usefulness of methods designed to detect differential sample variance in providing biological interpretations when biologically relevant but heterogeneous changes between two phenotypes are prevalent in specific signaling pathways. Software implementation of the developed methods is available with detailed documentation from Bioconductor package GSAR. The available methods are applicable to gene expression datasets in a normalized matrix form and could be used with other omics datasets in a normalized matrix form with available collection of feature sets.

Melatonin (MLT) is a circadian hormone that reportedly influences the development and cyclic growth of secondary hair follicles; however, the mechanism of regulation remains unknown. Here, we systematically investigated the role of MLT in hair regeneration using a hair depilation mouse model. We found that MLT supplementation significantly promoted hair regeneration in the hair depilation mouse model, whereas supplementation of MLT receptor antagonist luzindole significantly suppressed hair regeneration. By analysing gene expression dynamics between the MLT group and luzindole-treated groups, we revealed that MLT supplementation significantly up-regulated Wnt/β-catenin signalling pathway-related genes. In-depth analysis of the expression of key molecules in the Wnt/β-catenin signalling pathway revealed that MLT up-regulated the Wnt/β-catenin signalling pathway in dermal papillae (DP), whereas these effects were facilitated through mediating Wnt ligand expression levels in the hair follicle stem cells (HFSCs). Using a DP-HFSCs co-culture system, we verified that MLT activated Wnt/β-catenin signalling in DPs when co-cultured with HFSCs, whereas supplementation of DP cells with MLT alone failed to activate Wnt/β-catenin signalling. In summary, our work identified a critical role for MLT in promoting hair regeneration and will have potential implications for future hair loss treatment in humans.

Genetically lean and obese individuals have distinct intestinal microbiota and function. However, the underlying mechanisms of the microbiome heterogeneity and its regulation on epithelial function such as intestinal stem cell (ISC) fate remain unclear. Employing pigs of genetically distinct breeds (obese Meishan and lean Yorkshire), this study reveals transcriptome-wide variations in microbial ecology of the jejunum, characterized by enrichment of active Lactobacillus species, notably the predominant Lactobacillus amylovorus (L. amylovorus), and lactate metabolism network in obese breeds. The L. amylovorus-dominant heterogeneity is paralleled with epithelial functionality difference as reflected by highly expressed GPR81, more proliferative ISCs and activated Wnt/β-catenin signaling. Experiments using in-house developed porcine jejunal organoids prove that live L. amylovorus and its metabolite lactate promote intestinal organoid growth. Mechanistically, L. amylovorus and lactate activate Wnt/β-catenin signaling in a GPR81-dependent manner to promote ISC-mediated epithelial proliferation. However, heat-killed L. amylovorus fail to cause these changes. These findings uncover a previously underrepresented role of L. amylovorus in regulating jejunal stem cells via Lactobacillus-lactate-GPR81 axis, a key mechanism bridging breed-driven intestinal microbiome heterogeneity with ISC fate. Thus, results from this study provide new insights into the role of gut microbiome and stem cell interactions in maintaining intestinal homeostasis.

Intestinal barriers play a crucial role in human physiology, both in homeostatic and pathological conditions. Disruption of the intestinal barrier is a significant factor in the pathogenesis of gastrointestinal inflammatory diseases, such as inflammatory bowel disease. The profound influence of the gut microbiota on intestinal diseases has sparked considerable interest in manipulating it through dietary interventions, probiotics, and fecal microbiota transplantation as potential approaches to enhance the integrity of the intestinal barrier. Numerous studies have underscored the protective effects of specific microbiota and their associated metabolites. In recent years, an increasing body of research has demonstrated that Akkermansia muciniphila (A. muciniphila, Am) plays a beneficial role in various diseases, including diabetes, obesity, aging, cancer, and metabolic syndrome. It is gaining popularity as a regulator that influences the intestinal flora and intestinal barrier and is recognized as a 'new generation of probiotics'. Consequently, it may represent a potential target and promising therapy option for intestinal diseases. This article systematically summarizes the role of Am in the gut. Specifically, we carefully discuss key scientific issues that need resolution in the future regarding beneficial bacteria represented by Am, which may provide insights for the application of drugs targeting Am in clinical treatment.

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

Chlorogenic acids are plant secondary metabolites, chemically-polyphenols with similar biological activity, formed through the esterification of quinic acid and hydrocinnamic acid moieties. They are best known for their high concentration in coffee and other dietary sources and the antioxidant properties that they exhibit. Both chlorogenic acids and plant extracts containing significant amounts of the compounds show promising in vitro activity against colorectal cancer. With coffee being the most popular drink in the world, and colorectal cancer at an unfortunate peak in incidence and mortality, the mechanisms through which the anti-tumorigenic effect of chlorogenic acids could be functionalized for CRC prevention seem appealing to study. Therefore, this review aims to enable a better understanding of the modes of action of chlorogenic acids in combating carcinogenesis, with a focus on cell cycle arrest, the induction of apoptosis, and the modulation of Wnt, Pi3K/Akt, and MAPK signal transduction pathways, alongside the reduction in the number of inflammatory cytokines and chemokines and the counterintuitive beneficial elevation of oxidative stress.

Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome includes valve leaflet thickening and progressive valve degeneration. However, the underlying mechanisms by which the disease progresses remain undefined.

Mice with Fibrillin 1 gene variant Fbn1C1039G/+ recapitulate histopathologic features of Marfan syndrome, and Wnt (Wingless-related integration site) signaling activity was detected in TCF/Lef-lacZ (T-cell factor/lymphoid enhancer factor-β-galactosidase) reporter mice. Single-cell RNA sequencing was performed from mitral valves of wild-type and Fbn1C1039G/+ mice at 1 month of age. Inhibition of Wnt signaling was achieved by conditional induction of the secreted Wnt inhibitor Dkk1 (Dickkopf-1) expression in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ; Fbn1C1039G/+ mice. Dietary doxycycline was administered for 1 month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for ECM (extracellular matrix) and immune cells were performed.

Wnt signaling is activated early in mitral valve disease progression, before immune cell infiltration in Fbn1C1039G/+ mice. Single-cell transcriptomics revealed similar mitral valve cell heterogeneity between wild-type and Fbn1C1039G/+ mice at 1 month of age. Wnt pathway genes were predominantly expressed in valve interstitial cells and valve endothelial cells of Fbn1C1039G/+ mice. Inhibition of Wnt signaling in Fbn1C1039G/+ mice at 1 month of age prevented the initiation of MVD as indicated by improved ECM remodeling and reduced valve leaflet thickness with decreased infiltrating macrophages. However, later, Wnt inhibition starting at 2 months did not prevent the progression of MVD.

Wnt signaling is involved in the initiation of mitral valve abnormalities and inflammation but is not responsible for later-stage valve disease progression once it has been initiated. Thus, Wnt signaling contributes to MVD progression in a time-dependent manner and provides a promising therapeutic target for the early treatment of congenital MVD in Marfan syndrome.

Organoids, which are 3D multicellular constructs, have garnered significant attention in recent years. Existing organoid culture methods predominantly utilize natural and synthetic polymeric hydrogels. This study explored the potential of a composite hydrogel mainly consisting of calcium silicate (CS) nanowires and methacrylated gelatin (GelMA) as a substrate for organoid formation and functionalization, specifically for intestinal and liver organoids. Furthermore, the research delved into the mechanisms by which CS nanowires promote the structure formation and development of organoids. It was discovered that CS nanowires can influence the stiffness of the hydrogel, thereby regulating the expression of the mechanosensory factor yes-associated protein (YAP). Additionally, the bioactive ions released by CS nanowires in the culture medium could accelerate Wnt/β-catenin signaling, further stimulating organoid development. Moreover, bioactive ions were found to enhance the nutrient absorption and ATP metabolic activity of intestinal organoids. Overall, the CS/GelMA composite hydrogel proves to be a promising substrate for organoid formation and development. This research suggested that inorganic biomaterials hold significant potential in organoid research, offering bioactivities, biosafety, and cost-effectiveness.

Maintenance of the intestinal epithelium requires constant self-renewal and regeneration. Tight regulation of proliferation and differentiation of intestinal stem cells within the crypt region is critical to maintaining homeostasis. The transcriptional co-factors β-catenin and YAP are required for proliferation during normal homeostasis as well as intestinal regeneration after injury: aberrant signaling activity results in over proliferation and tumorigenesis. Although both YAP and β-catenin activity are controlled along canonical pathways, it is becoming increasingly clear that non-canonical regulation of these transcriptional regulators plays a role in fine tuning their activity. We have shown previously that MAMDC4 (Endotubin, AEGP), an integral membrane protein present in endosomes, regulates both YAP and β-catenin activity in kidney epithelial cells and in the developing intestinal epithelium. Here we show that MAMDC4 interacts with members of the signalosome and mediates cross-talk between YAP and β-catenin. Interestingly, this cross-talk occurs through a non-canonical pathway involving interactions between AMOT:YAP and AMOT:β-catenin.

Genetically modified intestinal organoids are being explored as potential surrogates of immortalized cell lines and gene-engineered animals. However, genetic manipulation of intestinal organoids is time-consuming, and the efficiency is far beyond satisfactory. To ensure the yield of the genetically modified organoids, large quantity of starting materials is required, and the procedure usually takes more than 10 days. Two major obstacles that restrict the genetic delivery efficiency are the three-dimensional culture condition and that the genetic delivery is carried out in cell suspensions. In the present study, we introduce a novel highly efficient strategy for building genetically modified intestinal organoids in which genetic delivery was performed in freshly established monolayer primary intestinal epithelial cells under two-dimensional conditions and subsequentially transformed into three-dimensional organoids. The total procedure can be finished within 10 hr while displaying much higher efficiency than the traditional methods. Furthermore, this strategy allowed for the selection of transgenic cells in monolayer conditions before establishing high-purity genetically modified intestinal organoids.

The evolutionarily conserved Wnt signaling pathway plays a central role in development and adult tissue homeostasis across species. Wnt proteins are secreted, lipid-modified signaling molecules that activate the canonical (β-catenin dependent) and non-canonical (β-catenin independent) Wnt signaling pathways. Cellular behaviors such as proliferation, differentiation, maturation, and proper body-axis specification are carried out by the canonical pathway, which is the best characterized of the known Wnt signaling paths. Wnt signaling has emerged as an important factor in stem cell biology and is known to affect the self-renewal of stem cells in various tissues. This includes but is not limited to embryonic, hematopoietic, mesenchymal, gut, neural, and epidermal stem cells. Wnt signaling has also been implicated in tumor cells that exhibit stem cell-like properties. Wnt signaling is crucial for bone formation and presents a potential target for the development of therapeutics for bone disorders. Not surprisingly, aberrant Wnt signaling is also associated with a wide variety of diseases, including cancer. Mutations of Wnt pathway members in cancer can lead to unchecked cell proliferation, epithelial-mesenchymal transition, and metastasis. Altogether, advances in the understanding of dysregulated Wnt signaling in disease have paved the way for the development of novel therapeutics that target components of the Wnt pathway. Beginning with a brief overview of the mechanisms of canonical and non-canonical Wnt, this review aims to summarize the current knowledge of Wnt signaling in stem cells, aberrations to the Wnt pathway associated with diseases, and novel therapeutics targeting the Wnt pathway in preclinical and clinical studies.

Wnt/β-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies.

Using our antibody-based platform of Wnt/β-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures.

A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo.

Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/β-catenin pathway modulation for the treatment of pulmonary fibrosis.

The epithelium lining the intestinal tract serves a multifaceted role. It plays a crucial role in nutrient absorption and immune regulation and also acts as a protective barrier, separating underlying tissues from the gut lumen content. Disruptions in the delicate balance of the gut epithelium trigger inflammatory responses, aggravate conditions such as inflammatory bowel disease, and potentially lead to more severe complications such as colorectal cancer. Maintaining intestinal epithelial homeostasis is vital for overall health, and there is growing interest in identifying nutraceuticals that can strengthen the intestinal epithelium. α-Ketoglutarate, a metabolite of the tricarboxylic acid cycle, displays a variety of bioactive effects, including functioning as an antioxidant, a necessary cofactor for epigenetic modification, and exerting anti-inflammatory effects. This article presents a comprehensive overview of studies investigating the potential of α-ketoglutarate supplementation in preventing dysfunction of the intestinal epithelium.

Colorectal mucinous adenocarcinoma (MAC) is one of the most lethal histological types of colorectal cancer, and its mechanism of development is not well understood. In this study, we aimed to clarify the molecular characteristics of MAC via in silico analysis using The Cancer Genome Atlas database. The expression of genes on chromosome 20q (Chr20q) was negatively associated with the expression of MUC2, which is a key molecule that can be used to distinguish between MAC and nonmucinous adenocarcinoma (NMAC). This was consistent with a significant difference in copy number alteration of Chr20q between the two histological types. We further identified 475 differentially expressed genes (DEGs) between MAC and NMAC, and some of the Chr20q genes among the DEGs are considered to be pivotal genes used to define MAC. Both in vitro and in vivo analysis showed that simultaneous knockdown of POFUT1 and PLAGL2, both of which are located on Chr20q, promoted MUC2 expression. Moreover, these genes were highly expressed in NMAC but not in MAC according to the results of immunohistological studies using human samples. In conclusion, POFUT1 and PLAGL2 are considered to be important for defining MAC, and these genes are associated with MUC2 expression.

As the economy rapidly develops, chemicals are widely produced and used. This has exacerbated the problems associated with environmental pollution, raising the need for efficient toxicological evaluation techniques to investigate the toxic effects and mechanisms of toxicity of environmental pollutants. The progress in the techniques of cell culture in three dimensions has resulted in the creation of models that are more relevant in terms of biology and physiology. This enables researchers to study organ development, toxicology, and drug screening. Adult stem cells (ASCs) and induced pluripotent stem cells (iPSCs) can be obtained from various mammalian tissues, including cancerous and healthy tissues. Such stem cells exhibit a significant level of tissue memory and ability to self-assemble. When cultivated in 3D in vitro environments, the resulting organoids demonstrate a remarkable capacity to recapitulate the cellular composition and function of organs in vivo. Recently, many tumors' tissue-derived organoids have been widely used in research on tumor pathogenesis, drug development, precision medicine, and other fields, including those derived from colon cancer, cholangiocarcinoma, liver cancer, and gastric cancer. However, the application of organoid models for evaluating the toxicity of environmental pollutants is still in its infancy. This review introduces the characteristics of the toxicity responses of organoid models upon exposure to pollutants from the perspectives of organoid characteristics, tissue types, and their applications in toxicology; discusses the feasibility of using organoid models in evaluating the toxicity of pollutants; and provides a reference for future toxicological studies on environmental pollutants based on organoid models.