Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence1,2. Mutation of Apc, the most common initiating event in conventional adenomas3, activates Wnt signalling, thus conferring fitness on mutant intestinal stem cells (ISCs)4,5. Apc mutations may occur in ISCs that arise by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar6,7, it is unclear whether both generate tumours equally well in uninjured intestines. It is also unknown whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source, but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs that are not associated with adenomas. These cis elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumorigenesis.

The adaptation of intestinal helminths to vertebrates evolved strategies to attenuate host tissue damage to support reproductive needs of parasites necessary to disseminate offspring to the environment. Helminths initiate the IL-25-mediated tuft cell-ILC2 circuit that enhances barrier protection of the host although viable parasites can target and limit the pathway. We used IL-25 to create small intestinal adaptation marked by anatomic, cell compositional and immunologic changes that persisted months after induction. Small intestinal adaptation was associated with heightened resistance to barrier pathogens, including in the lung, and sustained by transcriptionally and epigenetically modified, tissue-resident, memory-effector ILC2s distinct from those described by innate 'training'; epithelial stem cells remained unaltered. Despite requiring IL-25 for induction, memory ILC2s maintained an activated state in the absence of multiple alarmins and supported mucosal resilience while avoiding adverse sensitization to chronic inflammation, revealing a pathway for deploying innate immune cells to coordinate a distributed mucosal defense.

Radiation-induced enteritis develops in cancer patients treated with radiotherapy in the abdominal and pelvic cavity, a condition that impairs their quality of life. Radiation injury depletes proliferative intestinal stem cells; in response to this, the epithelium activates a regenerative program that facilitates the healing of the intestine. However, the mechanisms that induce the activation of the intestinal regenerative program are poorly characterized.

In this study, we induced radiation-induced enteritis in mice through abdominal irradiation, mimicking clinical scenarios. Through imaging and flow cytometric analysis, we investigated the recruitment of macrophages to the small intestine during injury and healing. Additionally, we developed a coculture system for mouse and human intestinal organoids and macrophages to explore the cross talk between these cells. Then by combining in vivo ablation of macrophages, fluorescent lineage tracing, imaging, bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, human intestinal organoids, and cell trajectory analysis, we studied the macrophage induction of intestinal regeneration at the cellular and molecular level.

Our findings revealed that macrophages are recruited around the intestinal stem cell compartment upon radiation injury, promoting a fetal-like reprogramming and proliferation of epithelial cells that drives the regeneration process. In contrast, macrophage ablation led to compromised regeneration. Moreover, our single-cell RNA-seq analysis identified key secreted molecules, neuregulin 1 and osteopontin, as pivotal players in regulating this process. Additionally, characterization of human macrophage/organoid cocultures and cell trajectory inference confirmed the conservation of macrophages' role in triggering the regenerative program in primary human cells.

This study identifies macrophages as essential contributors to intestinal regeneration beyond their innate immune response. Targeting macrophages therapeutically may hold promise in enhancing regeneration and improving the quality of life for cancer survivors.

Gastric intestinal metaplasia (IM) is a precancerous stage spanning a morphological spectrum that is poorly represented by human cell line models.

We aim to establish and characterise human IM cell models to better understand IM progression along the cancer spectrum.

A large human gastric IM organoid (IMO) cohort (n=28), their clonal derivatives and normal gastric organoids (n=42) for comparison were established. Comprehensive multi-omics profiling and functional characterisation were performed.

Single-cell transcriptomes revealed IMO cells spanning a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation. Their lineage trajectories connected different cycling and quiescent stem and progenitors, highlighting differences in gastric to IM transition and the potential origin of IM from STMN1 cycling isthmus stem cells. Hybrid IMOs showed impaired differentiation potential, high lineage plasticity beyond gastric or intestinal fates and reactivation of a fetal gene programme.Cell populations in gastric IM and cancer tissues were highly similar to those derived from IMOs and exhibited a fetal signature. Genomically, IMOs showed elevated mutation burden, frequent chromosome 20 gain and epigenetic deregulation of many intestinal and gastric genes. Functionally, IMOs were FGF10 independent and showed downregulated FGFR2. Several IMOs exhibited a cell-matrix adhesion independent subpopulation that displayed chromosome 20 gain but lacked key cancer driver mutations, potentially representing the earliest neoplastic precursor of IM-induced gastric cancer.

Overall, our IMO biobank captured the heterogeneous nature of IM, revealing mechanistic insights on IM pathogenesis and progression, offering an ideal platform for studying early gastric neoplastic transformation and chemoprevention.

The gastric mucosa is a highly dynamic tissue that undergoes constant self-renewal through stem cell differentiation. Chief cells maintain a quiescent state in homeostasis but are responsible for regeneration after injury. Although the role of microbiome-host interactions in the intestine is well studied, less is known about these interactions in the stomach. Using the mouse organoid and germ-free mouse models, we show that microbiota-derived short-chain fatty acids (SCFAs) suppress the proliferation of chief cells in mice. This effect is mediated by activation of G-protein-coupled receptor 43. Most importantly, through metabolomics and transplantation studies, we show butyrate-producing Lactobacillus intestinalis modulates the proliferation of chief cells in mice. Our findings identify a mechanism by which the microbiota regulates the cell characteristics of chief cells, providing insight into the complex interplay between the host and its microbial environment and the mechanisms underlying gastric homeostasis, with potential therapeutic implications for gastric diseases.

Radiation-induced Intestinal Injury (RIII) affects quality of life in radiotherapy patients; Liangxue Guyuan Yishen Decoction (LGYD) offers protection but requires further study on its mechanism.

The aim of this study was to investigate the heterogeneity of cellular responses in the intestine at a single-cell level following radiation and LGYD treatment.

This study's design includes in vivo and in vitro assessments to evaluate LGYD's effects on intestinal cells post-radiation, targeting survival, recovery, and molecular pathways.

Mice were categorized into four groups: LGYD group, NC group, IR group, and Am group. Each group received daily drug administrations. All groups, except for the NC group, were subjected to a single whole-body irradiation at a dose rate of 70 R/min with a source-to-skin distance of 250 cm. Subsequent experiments were conducted following the irradiation, which led to severe survival impairments in the mice.

Our findings demonstrate that LGYD intervention substantially improves survival rates following lethal doses (8.5 Gy, 70R/min) of whole-body irradiation. Moreover, LGYD expedites the recovery period for intestinal injury on the fifth day after radiation by promoting repair mechanisms within intestinal tissue, with particular focus on mitigating intestinal stem cells (ISCs) damage and immune disorders. Through both in vivo and in vitro experiments, we have discovered that LGYD effectively treats RIII by activating Hes1 transcription factor activity through its key active ingredients in drug-containing serum. This activation further upregulates the downstream Stat3 and Akt gene, thereby facilitating repair processes within intestinal stem cells.

In this study, we discovered that LGYD can enhance the downstream expression and phosphorylation pathways of Stat3 and Akt by upregulating the expression of Hes1 gene following high-dose radiation exposure.

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.

In the present study, the impact of urban growth on green spaces in Kolkata Metropolitan City (KMC) was evaluated using the multi-temporal satellite observations spanning the last four decades (1990-2022). The study exhibited a rapid rise in urban areas (178.38% growth; net increase 498 sq.km), leading to a significant conversion of areas into moderate to very high built-up density zones. This urbanization has markedly altered the green-blue infrastructure, notably causing a 27% decline in urban green space (UGS) resulting a net loss of 254 sq.km. Fragmentation analysis exhibited a trend of compact, infill development in urban regions, contrasting with outgrowth, which has influenced both the cluster size and quality of UGS over the decades. The multi-indices and biophysical characterization of UGS concluded a deteriorating trend in terms of quantity (- 27.9%) and quality as well with reference. However, the existing UGS are primarily scattered and having less dense. Spatial estimation of above ground biomass (AGB) of UGS using regression analysis of field-derived AGB and L-band SAR backscatter depicted a dominance of low AGB (< 30 t/ha-1) across KMC, while the certain zones with improved UGS exhibited moderate AGB levels (50-100 t/ha-1). The fuzzy AHP-based multi-criteria analysis of urban ecological quality exhibited severe ecological deterioration in the central urban areas, moderate to high in peri-urban regions, and comparatively improved ecological conditions in the peripheral rural parts of KMC. The study also identified major native tree species for plantation strategies comprising urban afforestation, rooftop gardens, and the development of green corridors in ecologically deficient hotspot zones to improve the ecological quality within the urban landscape.

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.

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.

Transcription factors guide tissue development by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of transcription factors. However, how transcription factors navigate chromatin features to selectively bind a small subset of all the possible genomic target loci remains poorly understood. Here we show that Cdx2-a lineage defining transcription factor that binds distinct targets in developing versus adult intestinal epithelial cells-has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic Cdx2 targets and methylated state of the CpG during development enables selective Cdx2 binding and activation of developmental enhancers and genes. In adult cells, demethylation at these enhancers prevents ectopic Cdx2 binding, instead directing Cdx2 to its canonical motif without a CpG. This shift in Cdx2 binding facilitates Ctcf and Hnf4 recruitment, establishing super-enhancers during development and homeostatic enhancers in adult cells, respectively. Induced DNA methylation in adult mouse epithelium or cultured cells recruits Cdx2 to developmental targets, promoting corecruitment of partner transcription factors. Thus, Cdx2's differential CpG motif preferences enable it to navigate distinct DNA methylation profiles, activating genes specific to appropriate developmental stages.

Hematopoiesis is the process that generates the cells of the blood and immune system from hematopoietic stem and progenitor cells (HSPCs) and represents the system with the most rapid cell turnover in a mammalian organism. HSPC differentiation trajectories, their underlying molecular mechanisms, and their dysfunctions in hematologic disorders are the focal research questions of experimental hematology. While HSPC transplantations in murine models are the traditional tool in this research field, recent advances in genome editing and next generation sequencing resulted in the development of many fundamentally new approaches for the analyses of mammalian hematopoiesis in situ and at single cell resolution. The current review will cover many recent developments in this field in murine models, from the bulk lineage tracing studies of HSPC differentiation to the barcoding of individual HSPCs with Cre-recombinase, Sleeping Beauty transposase, or CRISPR/Cas9 tools, to map hematopoietic cell fates, together with their transcriptional and epigenetic states. We also address studies of the clonal dynamics of human hematopoiesis, from the tracing of HSPC clonal behaviours based on viral integration sites in gene therapy patients to the recent analyses of unperturbed human hematopoiesis based on naturally accrued mutations in either nuclear or mitochondrial genomes. Such studies are revolutionizing our understanding of HSPC biology and hematopoiesis both under homeostatic conditions and in the response to various forms of physiological stress, reveal the mechanisms responsible for the decline of hematopoietic function with age, and in the future may advance the understanding and management of the diverse disorders of hematopoiesis.

Diet and nutritional metabolites exhibit wide-ranging effects on health and disease partly by altering tissue composition and function. With rapidly rising rates of obesity, there is particular interest in how obesogenic diets influence tissue homeostasis and risk of tumorigenesis; epidemiologically, these diets have a positive correlation with various cancers, including colorectal cancer. The gastrointestinal tract is a highly specialized, continuously renewing tissue with a fundamental role in nutrient uptake and is, in turn, influenced by diet composition and host metabolic state. Intestinal stem cells are found at the base of the intestinal crypt and can generate all mature lineages that comprise the intestinal epithelium and are uniquely influenced by host diet, metabolic by-products and energy dynamics. Similarly, tumour growth and metabolism can also be shaped by nutrient availability and host diet. In this Review, we discuss how different diets and metabolic changes influence intestinal stem cells in homeostatic and pathological conditions, as well as tumorigenesis. We also discuss how dietary changes and composition affect the intestinal epithelium and its surrounding microenvironment.

Although the concept of cancer stem cells is still controversial, previous studies have shown that blood cancers, as well as specific types of solid cancers such as colorectal cancer, rely on stem cells during the onset of tumor growth and further tumor development. Moreover, resistance to therapeutic treatment in leukemias such as acute myeloid leukemia and in colorectal cancer can be attributed to a small population of cells with stemness properties known as minimal residual disease. In this review, we look back on the discovery of cancer stem cells and the contribution of the findings in blood cancer to a parallel discovery in solid cancers. We focus on CD44 as a stem cell marker, both in blood cancers and in several types of solid cancers, particularly of the gastrointestinal tract. This review highlights newly discovered molecular mechanisms of action of CD44 which indicate that CD44 has indeed a function in stemness, stem cell maintenance, and drug resistance. We attempt here to make the link between the functions of CD44 isoforms in stemness and their involvement in specific steps of tumor growth and metastasis.

Cell state dynamics underlying successful tissue regeneration are undercharacterized. In the intestine, damage prompts epithelial reprogramming into revival stem cells (revSCs) that reconstitute Lgr5+ intestinal stem cells (ISCs). Here single-nuclear multi-omics of mouse crypts regenerating from irradiation shows revSC chromatin accessibility overlaps with ISCs and differentiated lineages. While revSC genes themselves are accessible throughout homeostatic epithelia, damage-induced remodelling of chromatin in the crypt converges on Hippo and the transforming growth factor-beta (TGFβ) signalling pathway, which we show is transiently activated and directly induces functional revSCs. Combinatorial gene expression analysis further suggests multiple sources of revSCs, and we demonstrate TGFβ can reprogramme enterocytes, goblet and paneth cells into revSCs and show individual revSCs form organoids. Despite this, loss of TGFβ signalling yields mild regenerative defects, whereas interference in both Hippo and TGFβ leads to profound defects and death. Intestinal regeneration is thus poised for activation by a compensatory system of crypt-localized, transient morphogen cues that support epithelial reprogramming and robust intestinal repair.

Progenitors and mature cells can maintain the intestinal epithelium by dedifferentiation and facultative intestinal stem cell (fISC) function when active ISCs (aISCs) are lost to damage. Here, we modeled fISC activation in mouse intestinal organoids with doxorubicin (DXR) treatment, a chemotherapeutic known to ablate Lgr5+ aISCs in vivo. Similar fISC gene activation was observed between organoids treated with low versus high DXR, despite significantly decreased survival at the higher dose. aISCs exhibited dose-dependent loss after DXR treatment but survived at doses compatible with organoid survival. We ablated residual aISCs after DXR treatment using a Lgr52A-DTR allele and observed that aISC survival of the initial genotoxic insult is required for organoid survival following DXR treatment. These results suggest that although typical fISC genes are activated by DXR-induced injury in organoids, functional stemness remains dependent on the aISC pool. Finally, we show that human intestinal organoids require higher doses of DXR to induce loss of survival and downregulation of LGR5. Our data establish a reproducible model of DXR-induced injury in intestinal organoids and reveal differences in in vitro responses to an established in vivo damage modality.

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.

Stem cells perceive and respond to biochemical and physical signals to maintain homeostasis. Yet, it remains unclear how stem cells sense mechanical signals from their niche in vivo. In this work, we investigated the roles of PIEZO mechanosensitive channels in the intestinal stem cell (ISC) niche. We used mouse genetics and single-cell RNA sequencing analysis to assess the requirement for PIEZO channels in ISC maintenance. In vivo measurement of basement membrane stiffness showed that ISCs reside in a more rigid microenvironment at the bottom of the crypt. Three-dimensional and two-dimensional organoid systems combined with bioengineered substrates and a stretching device revealed that PIEZO channels sense extracellular mechanical stimuli to modulate ISC function. This study delineates the mechanistic cascade of PIEZO activation that coordinates ISC fate decision and maintenance.

Lineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchies in vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to two-dimensional culture systems. Studying two absorptive lineage-determining TFs, we found an expression level-independent contrasting diffusive behavior: while Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of ∼32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of ∼56%. Our results suggest that TF diffusive behavior could indicate the progression of differentiation and modulate early versus late differentiation within a lineage.

The intestine and liver share a unique regenerative property that sets them apart from other mammalian visceral organs. The intestinal epithelium exhibits rapid renewal, making it one of the fastest renewing tissues in humans. Under physiological conditions, intestinal stem cells within each intestinal crypt continuously differentiate into the different types of intestinal epithelial cells to maintain intestinal homeostasis. However, when exposed to tissue damage or stressful conditions such as inflammation, intestinal epithelial cells in the gastrointestinal tract exhibit plasticity, allowing fully differentiated cells to regain their stem cell properties. Likewise, hepatic epithelial cells possess a remarkable regenerative capacity to restore lost liver mass through proliferation-mediated liver regeneration. When the proliferation-mediated regenerative capacity is impaired, hepatocytes and biliary epithelial cells (BECs) can undergo plasticity-mediated regeneration and replenish each other. The transition of mammalian liver progenitor cells to hepatocytes/BECs can be observed under tightly controlled experimental conditions such as severe hepatocyte injury accompanied by the loss of regenerative capacity. In this review, we will discuss the mechanism by which cellular plasticity contributes to the regeneration process and the potential therapeutic implications of understanding and harnessing cellular plasticity in the gut and liver.

Spermatogenesis is characterized by the seminiferous epithelial cycle, a periodic pattern of germ cell differentiation with a wave-like progression along the length of seminiferous tubules. While key signaling and metabolic components of the cycle are known, the transcriptional changes across the cycle and the correlations between germ cell and somatic lineages remain undefined. Here, we use spatial transcriptomics via RNA SeqFISH+ to profile 2,638 genes in 216,090 cells in mouse testis and identify a periodic transcriptional pattern across tubules that precisely recapitulates the seminiferous epithelial cycle, enabling us to map cells to specific timepoints along the developmental cycle. Analyzing gene expression in somatic cells reveals that Sertoli cells exhibit a cyclic transcriptional profile closely synchronized with germ cell development while other somatic cells do not demonstrate such synchronization. Remarkably, in mouse testis with drug-induced ablation of germ cells, Sertoli cells independently maintain their cyclic transcriptional dynamics. By analyzing expression data, we identify an innate retinoic acid cycle, a network of transcription factors with cyclic activation, and signaling from germ cells that could interact with this network. Together, this work leverages spatial geometries for mapping the temporal dynamics and reveals a regulatory mechanism in spermatogenesis where Sertoli cells oscillate and coordinate with the cyclical progression of germ cell development.

The adult mammary gland is maintained by lineage-restricted progenitor cells through pregnancy, lactation, involution, and menopause. Injury resolution, transplantation-associated mammary gland reconstitution, and tumorigenesis are unique exceptions, wherein mammary basal cells gain the ability to reprogram to a luminal state. Here, we leverage newly developed cell-identity reporter mouse strains, and time-resolved single-cell epigenetic and transcriptomic analyses to decipher the molecular programs underlying basal-to-luminal fate switching in vivo. We demonstrate that basal cells rapidly reprogram toward plastic cycling intermediates that appear to hijack molecular programs we find in bipotent fetal mammary stem cells and puberty-associatiated cap cells. Loss of basal-cell specifiers early in dedifferentiation coincides with activation of Notch and BMP, among others. Pharmacologic blockade of each pathway disrupts basal-to-luminal transdifferentiation. Our studies provide a comprehensive map and resource for understanding the coordinated molecular changes enabling terminally differentiated epithelial cells to transition between cell lineages and highlights the stunning rapidity by which epigenetic reprogramming can occur in response to disruption of tissue structure.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24 h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

Metabolism serves not only as the organism's energy source but also yields metabolites crucial for maintaining tissue homeostasis and overall health. Intestinal stem cells (ISCs) maintain intestinal homeostasis through continuous self-renewal and differentiation divisions. The intricate relationship between metabolic pathways and intestinal homeostasis underscores their crucial interplay. Metabolic pathways have been shown to directly regulate ISC self-renewal and influence ISC fate decisions under homeostatic conditions, but the cellular and molecular mechanisms remain incompletely understood. Understanding the intricate involvement of various pathways in maintaining intestinal homeostasis holds promise for devising innovative strategies to address intestinal diseases. Here, we provide a comprehensive review of recent advances in the regulation of intestinal homeostasis. We describe the regulation of intestinal homeostasis from multiple perspectives, including the regulation of intestinal epithelial cells, the regulation of the tissue microenvironment, and the key role of nutrient metabolism. We highlight the regulation of intestinal homeostasis and ISC by nutrient metabolism. This review provides a multifaceted perspective on how intestinal homeostasis is regulated and provides ideas for intestinal diseases and repair of intestinal damage.

Group 2 innate lymphocytes (ILC2s) are prevalent in small intestine but engagement of type 2 immunity during basal processes are incompletely described. Thymic stromal lymphopoietin (TSLP), a cytokine implicated in ILC2 activation, was constitutively expressed in villus telocytes and crypt-associated trophocytes, specialized fibroblasts that sustain epithelial identity. Feeding increased TSLP and induced ILC2 type 2 cytokines that were attenuated by deletion of TSLP in PDGFRα + stromal cells or TSLP receptor on ILC2s. Mouse and human telocytes expressed receptors for glucagon-like peptide-2 (GLP-2), which is released by enteroendocrine cells (EECs) after eating. GLP-2 induced intestinal TSLP, TSLP-dependent ILC2 cytokine production, and tuft cell hyperplasia. The telocyte-alarmin relay couples EEC nutrient detection with amplification of a tuft cell chemosensory circuit that diversifies surveillance of ingested cargo.

Intestinal telocyte TSLP relays signals from enteroendocrine cells to ILC2s to amplify the tuft cell circuit in response to feeding.

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.

Total-body irradiation (TBI) with gamma rays can damage organisms in various unexpected ways and trigger several organ dysfunction syndromes, such as acute radiation syndrome (ARS). Hematopoietic cells and enterocytes are particularly sensitive to radiation due to their self-renewal ability and rapid division, which leads to hematopoietic ARS (H-ARS) and gastrointestinal ARS (GI-ARS). We previously showed that a lipid-based small molecule, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), improved 30-day survival and alleviated H-ARS symptoms in BALB/c mice after a lethal dose (LD70/30) of gamma-ray TBI. In this study, we investigated the mitigating effects of PLAG on radiation-induced GI damage that occurs under the same conditions as H-ARS in BALB/c mice. Our study showed that PLAG facilitated the structural restoration of intestinal tissues by increasing villus height, crypt depth, crypt number, mucin-producing goblet cells, and proliferating cell nuclear antigen (PCNA)-positive crypt cells. PLAG significantly improved intestinal absorptive capacity and reduced intestinal injury-induced bacterial translocation. In addition, PLAG effectively inhibited radiation-induced necroptosis signaling activation in the intestinal crypt cells, which was responsible for sustained tissue damage and the release of high mobility group box 1 (HMGB1), a typical damage-associated molecular pattern. Overall, our findings support the radiation-mitigating potential of PLAG against GI-ARS after accidental radiation exposure.

Colorectal cancer (CRC), one of the most common tumors in the world, is generally proposed to be generated from intestinal stem cells (ISCs). Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive ISCs are located at the bottom of the crypt and harbor self-renewal and differentiation capacities, serving as the resource of all intestinal epithelial cells and CRC cells as well. Here we review recent progress in ISCs both in non-tumoral and tumoral contexts. We summarize the molecular mechanisms of ISC self-renewal, differentiation, and plasticity for intestinal homeostasis and regeneration. We also discuss the function of ISCs in colorectal tumorigenesis as cancer stem cells and summarize fate dynamic, competition, niche regulation, and remote environmental regulation of ISCs for CRC initiation and propagation.

The crosstalk and balance regulation of Wnt-Notch have been known to be essential for cell fate decision and tissue regeneration, however, how this balance is maintained and how the Wnt-Notch pathways are connected with cell cycle regulation is still not clear. By analyzing the molecular alterations in mouse model with accelerated aging phenotypes due to loss of p21 function in a Werner syndrome background, we observed that Wnt3 and β-Catenin were down-regulated, while Notch1 and Hes1 were up-regulated. This disruption in Wnt-Notch signaling was accompanied by the loss of intestinal stem cell compartment, increase in Bmi1 positive cells, loss of Olfm4/Lgr5 positive cells, and reduced secretory Paneth cells and goblet cells in the intestinal crypts of p21TKO mice. BrdU incorporation, cleaved caspase 3, and Tunel assay results revealed the fast turnover of intestinal epithelia, which may result in abnormal stem cell mobilization and exhaustion of the stem cell reservoir in the intestinal crypts. We further identified shift of DREAM complex towards MMB complex due to the loss of p21 as the cause for faster turnover of intestinal epithelia. Importantly, we identified the E2F1 as the transcriptional regulator for Notch1, which linked the p21-DREAM/MMB/Rb-E2F1 pathway with Wnt-Notch pathway. The overexpression of p21 rescued the DREAM pathway, as well as the imbalance of Wnt-Notch pathway. In summary, our data identify p21 as an important factor in maintaining sequential mobilization, proliferation, and homeostasis of intestinal stem cells.

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.

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.

Significance: Intestinal stem cells (ISCs) are crucial for the continuous renewal and regeneration of the small intestinal epithelium. ISC fate decisions are strictly controlled by metabolism. Mitochondria act as the central hubs of energetic metabolism and dynamically remodel their morphology to perform required metabolic functions. Mitochondrial dysfunction is closely associated with a variety of gastrointestinal diseases. Recent Advances: In recent years, several studies have reported that mitochondria are potential therapeutic targets for regulating ISC function to alleviate intestinal diseases. However, how mitochondrial quality control mediates ISCs under physiological conditions and protects against intestinal injury remains to be comprehensively reviewed. Critical Issues: In this review, we summarize the available studies about how mitochondrial metabolism, redox state, dynamics, autophagy, and proteostasis impact ISC proliferation, differentiation, and regeneration, respectively. Future Directions: We propose that remodeling the function of mitochondria in ISCs may be a promising potential future direction for the treatment of intestinal diseases. This review may provide new strategies for therapeutically targeting the mitochondria of ISCs in intestinal diseases.

Epigenetic clocks, built on DNA methylation patterns of bulk tissues, are powerful age predictors, but their biological basis remains incompletely understood. Here, we conducted a comparative analysis of epigenetic age in murine muscle, epithelial, and blood cell types across lifespan. Strikingly, our results show that cellular subpopulations within these tissues, including adult stem and progenitor cells as well as their differentiated progeny, exhibit different epigenetic ages. Accordingly, we experimentally demonstrate that clocks can be skewed by age-associated changes in tissue composition. Mechanistically, we provide evidence that the observed variation in epigenetic age among adult stem cells correlates with their proliferative state, and, fittingly, forced proliferation of stem cells leads to increases in epigenetic age. Collectively, our analyses elucidate the impact of cell type composition, differentiation state, and replicative potential on epigenetic age, which has implications for the interpretation of existing clocks and should inform the development of more sensitive clocks.

Wuliangye strong aroma baijiu (hereafter, Wuliangye baijiu) is a traditional Chinese grain liquor containing short-chain fatty acids, ethyl caproate, ethyl lactate, other trace components, and a large proportion of ethanol. The effects of Wuliangye baijiu on intestinal stem cells and intestinal epithelial development have not been elucidated. Here, the role of Wuliangye baijiu in intestinal epithelial regeneration and gut microbiota modulation was investigated by administering a Lieber-DeCarli chronic ethanol liquid diet in a mouse model to mimic long-term (8 weeks') light/moderate alcohol consumption (1.6 g kg-1 day-1) in healthy human adults.

Wuliangye baijiu promoted colonic crypt proliferation in mice. According to immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses, compared with the ethanol-only treatment, Wuliangye baijiu increased the number of intestinal stem cells and goblet cells and the expression of enteroendocrine cell differentiation markers in the mouse colon. Furthermore, gut microbiota analysis showed an increase in the relative abundance of microbiota related to intestinal homeostasis following Wuliangye baijiu administration. Notably, increased abundance of Bacteroidota, Faecalibaculum, Lachnospiraceae, and Blautia may play an essential role in promoting stem-cell-mediated intestinal epithelial development and maintaining intestinal homeostasis.

In summary, these findings suggest that Wuliangye baijiu can be used to regulate intestinal stem cell proliferation and differentiation in mice and to alter gut microbiota distributions, thereby promoting intestinal homeostasis. This research elucidates the mechanism by which Wuliangye baijiu promotes intestinal health. © 2024 Society of Chemical Industry.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestines without a cure. Current therapies suppress inflammation to prevent further intestinal damage. However, healing already damaged intestinal epithelia is still an unmet medical need. Under physiological conditions, Lgr5+ intestinal stem cells (ISCs) in the intestinal crypts replenish the epithelia every 3-5 days. Therefore, understanding the regulation of Lgr5+ ISCs is essential. Previous data suggest vitamin D signaling is essential to maintain normal Lgr5+ ISC function in vivo. Our recent data indicate that to execute its functions in the intestines optimally, 1,25(OH)2D requires high concentrations that, if present systemically, can cause hypercalcemia (i.e., blood calcium levels significantly higher than physiological levels), leading to severe consequences. Using 5-bromo-2'-deoxyuridine (BrdU) to label the actively proliferating ISCs, our previous data suggested that de novo synthesized locally high 1,25(OH)2D concentrations effectively enhanced the migration and differentiation of ISCs without causing hypercalcemia. However, although sparse in the crypts, other proliferating cells other than Lgr5+ ISCs could also be labeled with BrdU. This current study used high-purity Lgr5+ ISC lines and a mouse strain, in which Lgr5+ ISCs and their progeny could be specifically tracked, to investigate the effects of de novo synthesized locally high 1,25(OH)2D concentrations on Lgr5+ ISC function. Our data showed that 1,25(OH)2D at concentrations significantly higher than physiological levels augmented Lgr5+ ISC differentiation in vitro. In vivo, de novo synthesized locally high 1,25(OH)2D concentrations significantly elevated local 1α-hydroxylase expression, robustly suppressed experimental colitis, and promoted Lgr5+ ISC differentiation. For the first time, this study definitively demonstrated 1,25(OH)2D's role in Lgr5+ ISCs, underpinning 1,25(OH)2D's promise in IBD therapy.

Tissue-specific adult stem cells are pivotal in maintaining tissue homeostasis, especially in the rapidly renewing intestinal epithelium. At the heart of this process are leucine-rich repeat-containing G protein-coupled receptor 5-expressing crypt base columnar cells (CBCs) that differentiate into various intestinal epithelial cells. However, while these CBCs are vital for tissue turnover, they are vulnerable to cytotoxic agents. Recent advances indicate that alternative stem cell sources drive the epithelial regeneration post-injury. Techniques like lineage tracing and single-cell RNA sequencing, combined with in vitro organoid systems, highlight the remarkable cellular adaptability of the intestinal epithelium during repair. These regenerative responses are mediated by the reactivation of conserved stem cells, predominantly quiescent stem cells and revival stem cells. With focus on these cells, this review unpacks underlying mechanisms governing intestinal regeneration and explores their potential clinical applications.

Adult tissues set the scene for a continuous battle between cells, where a comparison of cellular fitness results in the elimination of weaker "loser" cells. This phenomenon, named cell competition, is beneficial for tissue integrity and homeostasis. In fact, cell competition plays a crucial role in tumor suppression, through elimination of early malignant cells, as part of Epithelial Defense Against Cancer. However, it is increasingly apparent that cell competition doubles as a tumor-promoting mechanism. The comparative nature of cell competition means that mutational background, proliferation rate and polarity all factor in to determine the outcome of these processes. In this review, we explore the intricate and context-dependent involvement of cell competition in homeostasis and regeneration, as well as during initiation and progression of primary and metastasized colorectal cancer. We provide a comprehensive overview of molecular and cellular mechanisms governing cell competition and its parallels with regeneration.