RESUMEN
Intestinal epithelial cell activities during homeostasis and regeneration are well described, but their potential interactions with stromal cells remain unresolved. Exploring the functions of these heterogeneous intestinal mesenchymal stromal cells (iMSCs) remains challenging. This difficulty is due to the lack of specific markers for most functionally homogenous subpopulations. In recent years, however, novel clustering techniques such as single-cell RNA sequencing (scRNA-seq), fluorescence-activated cell sorting (FACS), confocal microscope, and computational remodeling of intestinal anatomy have helped identify and characterize some specific iMSC subsets. These methods help researchers learn more about the localization and functions of iMSC populations during intestinal morphogenic and homeostatic conditions. Consequently, it is imperative to understand the cellular pathways that regulate their activation and how they interact with surrounding cellular components, particularly during intestinal epithelial regeneration after mucosal injury. This review provides insights into the spatial distribution and functions of identified iMSC subtypes. It focuses on their involvement in intestinal morphogenesis, homeostasis, and regeneration. We reviewed related signaling mechanisms implicated during epithelial and subepithelial stromal cell crosstalk. Future research should focus on elucidating the molecular intermediates of these regulatory pathways to open a new frontier for potential therapeutic targets that can alleviate intestinal mucosa-related injuries.
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Arsenic is a toxicant that is ingested through drinking water and food, exposing nearly 140 million people to levels above the 10 ppb guideline concentration. Studies have shown that arsenic affects intestinal stem cells (ISCs), but the mechanisms by which arsenic alters the formation of adult cells in the small intestine are not well understood. Signals derived from intestinal stromal cells initiate and maintain differentiation. The goal of this study is to evaluate arsenic's effect on intestinal stromal cells, including PdgfrαLo trophocytes, located proximal to the ISCs, and PdgfrαHi telocytes, located proximal to the transit-amplifying region and up the villi. Adult Sox9tm2Crm-EGFP mice were exposed to 0, 33, and 100 ppb sodium arsenite in their drinking water for 13 weeks, and sections of duodenum were examined. Flow cytometry indicated that arsenic exposure dose-responsively reduced Sox9+ epithelial cells and trended toward increased Pdgfrα+ cells. The trophocyte marker, CD81, was reduced by 10-fold and 9.0-fold in the 100 ppb exposure group in male and female mice, respectively. Additionally, a significant 2.2- to 3.1-fold increase in PdgfrαLo expression was found in male mice in trophocytes and Igfbp5+ cells. PdgfrαHi protein expression, a telocyte marker, was more prevalent along the villus/crypt structure in females, whereas Gli1 expression (telocytes) was reduced in male mice exposed to arsenic. Principle coordinate analysis confirmed the sex-dependent response to arsenic exposure, with an increase in trophocyte and decrease in telocyte marker expression observed in male mice. These results imply that arsenic alters intestinal mesenchymal cells in a sex-dependent manner.
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Arsénico , Agua Potable , Humanos , Masculino , Ratones , Femenino , Animales , Arsénico/toxicidad , Intestino Delgado , Intestinos , Células del EstromaRESUMEN
Signals from the surrounding niche drive proliferation and suppress differentiation of intestinal stem cells (ISCs) at the bottom of intestinal crypts. Among sub-epithelial support cells, deep sub-cryptal CD81+ PDGFRAlo trophocytes capably sustain ISC functions ex vivo. Here, we show that mRNA and chromatin profiles of abundant CD81- PDGFRAlo mouse stromal cells resemble those of trophocytes and that both populations provide crucial canonical Wnt ligands. Mesenchymal expression of key ISC-supportive factors extends along a spatial and molecular continuum from trophocytes into peri-cryptal CD81- CD55hi cells, which mimic trophocyte activity in organoid co-cultures. Graded expression of essential niche factors is not cell-autonomous but dictated by the distance from bone morphogenetic protein (BMP)-secreting PDGFRAhi myofibroblast aggregates. BMP signaling inhibits ISC-trophic genes in PDGFRAlo cells near high crypt tiers; that suppression is relieved in stromal cells near and below the crypt base, including trophocytes. Cell distances thus underlie a self-organized and polar ISC niche.
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Mucosa Intestinal , Nicho de Células Madre , Animales , Ratones , Mucosa Intestinal/metabolismo , Intestinos , Transducción de Señal , Diferenciación Celular , Proliferación CelularRESUMEN
Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.
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Intestinos , Nicho de Células Madre , Humanos , Ratones , Animales , Nicho de Células Madre/fisiología , Mucosa Intestinal/metabolismo , Diferenciación Celular , Proteínas Morfogenéticas Óseas/metabolismo , Músculo LisoRESUMEN
The intestinal tract is lined by a single layer of epithelium that is one of the fastest regenerating tissues in the body and which therefore requires a very active and exquisitely controlled stem cell population. Rapid renewal of the epithelium is necessary to provide a continuous physical barrier from the intestinal luminal microenvironment that contains abundant microorganisms, whilst also ensuring an efficient surface for the absorption of dietary components. Specialised epithelial cell populations are important for the maintenance of intestinal homeostasis and are derived from adult intestinal stem cells (ISCs). Actively cycling ISCs divide by a neutral drift mechanism yielding either ISCs or transit-amplifying epithelial cells, the latter of which differentiate to become either absorptive lineages or to produce secretory factors that contribute further to intestinal barrier maintenance or signal to other cellular compartments. The mechanisms controlling ISC abundance, longevity and activity are regulated by several different cell populations and signalling pathways in the intestinal lamina propria which together form the ISC niche. However, the complexity of the ISC niche and communication mechanisms between its different components are only now starting to be unravelled with the assistance of intestinal organoid/enteroid/colonoid and single-cell imaging and sequencing technologies. This review explores the interaction between well-established and emerging ISC niche components, their impact on the intestinal epithelium in health and in the context of intestinal injury and highlights future directions and implications for this rapidly developing field.
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Intestinos/citología , Nicho de Células Madre , Animales , Transducción de SeñalRESUMEN
BACKGROUND: SAMP1/YitFcsJ (SAMP1) mice spontaneously develop terminal ileitis resembling human Crohn disease. SAMP1 mice have exhibited alteration of epithelial cell lineage distribution and an overall proliferation of the crypt cell population; however, it has not been evaluated whether epithelial differentiation is impaired because of dysfunction of intestinal stem cells (ISCs) or their niche factors. METHODS: Using the intestine of SAMP1 mice aged 10 to 14 weeks, morphometric alterations in the crypt-villus architecture, ISCs, crypt cells, and differentiated cells; organoid formation capacity of intestinal crypts; and niche signaling pathways were analyzed and compared with those of age-matched control AKR/J (AKR) mice. RESULTS: The ileum of SAMP1 mice showed increased depth of intestinal crypts and decreased surface area of the villi compared with those in the ileum of AKR mice. The number of ISCs in the ileal crypts did not differ between SAMP1 and AKR mice; however, the number of Paneth cells decreased and the number of transient amplifying cells increased. The organoid formation rate of the ileal crypts of SAMP1 mice decreased significantly compared with that of AKR mice. The performance of RNA sequencing for intestinal crypts found that the expression of ISC niche factors, such as Wnt3, Dll1, and Dll4, was decreased significantly in the ileal crypts of SAMP1 mice compared with those of AKR mice. Among the ISC niche signals, the Notch signaling-related genes tended to be downregulated. In particular, immunocytochemistry revealed that the expression of Paneth cell-expressing Notch ligand Dll4 was significantly decreased in the intestinal tissue and organoids of SAMP1 mice compared with those of AKR mice. CONCLUSIONS: Depletion of niche factors for ISCs contributes to the alteration of epithelial differentiation in SAMP1 mice.
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Enfermedad de Crohn , Células Epiteliales/citología , Ileítis , Nicho de Células Madre , Animales , Diferenciación Celular , Enfermedad de Crohn/patología , Ileítis/patología , Mucosa Intestinal/citología , Ratones , Ratones EndogámicosRESUMEN
OBJECTIVES: Enterochromaffin (EC) cells have been associated with functional gastrointestinal disorders such as IBS. Recently, we found that glial cell-derived neurotrophic factor (GDNF)-rearranged during transfection (RET) localized in EC cells in human colonic epithelia. Here, we examine the role of GDNF-RET in the pathophysiology of diarrhoea-predominant irritable bowel syndrome (IBS-D). MATERIALS AND METHODS: GDNF was assessed by ELISA and immunohistochemistry in biopsies from IBS-D patients and healthy controls. Stress was induced by using a wrap-restraint stress (WRS) procedure to serve as an acute stress-induced IBS model. The function of GDNF-RET axis to intestinal stem cell (ISC) homeostasis, and EC cell numbers were assessed in vivo and in vitro. RESULTS: GDNF-RET was expressed in EC cells in human colon. GDNF was significantly increased in IBS-D patients. WRS mice showed increased GDNF-RET levels in colon. WRS induced visceral hypersensitivity by expanding of ISC and differentiation of EC cell via GDNF-RET. Furthermore, GDNF-treated mice recapitulated the phenotype of WRS mice. In vitro, GDNF treatment amplified Wnt signal and increased serotonin levels in colonic organoids in a dose-dependent manner. CONCLUSIONS: We identified GDNF-RET was presented in colonic epithelium of patients with IBS-D. GDNF-RET played important roles in regulating ISC and EC cell differentiation. Our findings, thus, provide RET inhibitor as new therapeutic targets for treatment of patients with IBS-D.
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Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Intestinos/fisiopatología , Síndrome del Colon Irritable/fisiopatología , Proteínas Proto-Oncogénicas c-ret/metabolismo , Adulto , Animales , Células Enterocromafines/metabolismo , Células Enterocromafines/patología , Femenino , Homeostasis , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Síndrome del Colon Irritable/metabolismo , Síndrome del Colon Irritable/patología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Estrés FisiológicoRESUMEN
Intestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn's disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNFΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, "inflammation-free" SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells.
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Enfermedad de Crohn/patología , Ileítis/patología , Organoides/patología , Nicho de Células Madre , Animales , Medios de Cultivo Condicionados/farmacología , Dexametasona/farmacología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Inflamación/patología , Mucosa Intestinal/metabolismo , Intestino Delgado/patología , Masculino , Ratones Endogámicos C57BL , Organoides/efectos de los fármacos , Organoides/ultraestructura , Transducción de Señal/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/patología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Wnt3A/farmacologíaRESUMEN
Intestinal stem cells (ISCs) are confined to crypt bottoms and their progeny differentiate near crypt-villus junctions. Wnt and bone morphogenic protein (BMP) gradients drive this polarity, and colorectal cancer fundamentally reflects disruption of this homeostatic signaling. However, sub-epithelial sources of crucial agonists and antagonists that organize this BMP gradient remain obscure. Here, we couple whole-mount high-resolution microscopy with ensemble and single-cell RNA sequencing (RNA-seq) to identify three distinct PDGFRA+ mesenchymal cell types. PDGFRA(hi) telocytes are especially abundant at the villus base and provide a BMP reservoir, and we identified a CD81+ PDGFRA(lo) population present just below crypts that secretes the BMP antagonist Gremlin1. These cells, referred to as trophocytes, are sufficient to expand ISCs in vitro without additional trophic support and contribute to ISC maintenance in vivo. This study reveals intestinal mesenchymal structure at fine anatomic, molecular, and functional detail and the cellular basis for a signaling gradient necessary for tissue self-renewal.
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Intestinos , Transducción de Señal , Proliferación Celular , Mucosa Intestinal , Células MadreRESUMEN
The gut absorbs dietary nutrients and provides a barrier to xenobiotics and microbiome metabolites. To cope with toxin exposures, the intestinal epithelium is one of the most rapidly proliferating tissues in the body. The stem cell niche supplies essential signaling factors including Wnt proteins secreted by subepithelial myofibroblasts. Unexpectedly, therapeutically effective doses of orally administered PORCN inhibitors that block all Wnt secretion do not affect intestinal homeostasis. We find that intestinal myofibroblasts are intrinsically resistant to multiple xenobiotics, including PORCN inhibitors and the anthracycline antibiotic doxorubicin. These myofibroblasts have high expression of a subset of drug transporters; knockout of Mrp1/Abcc1 enhances drug sensitivity. Tamoxifen administration to Rosa26CreERT2;mT/mG mice visually highlights the drug-resistant intestinal stromal compartment and identifies small populations of drug-resistant cells in lung, kidney, and pancreatic islets. Xenobiotic resistance of the Wnt-producing myofibroblasts can protect the intestinal stem cell niche in the face of an unpredictable environment.
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Aciltransferasas/fisiología , Proliferación Celular/efectos de los fármacos , Resistencia a Múltiples Medicamentos , Mucosa Intestinal/efectos de los fármacos , Proteínas de la Membrana/fisiología , Miofibroblastos/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Aciltransferasas/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Hormonales/farmacología , Broncodilatadores/farmacología , Células Cultivadas , Doxorrubicina/farmacología , Femenino , Homeostasis , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Miofibroblastos/citología , Miofibroblastos/metabolismo , Propionatos/farmacología , Quinolinas/farmacología , Transducción de Señal , Tamoxifeno/farmacología , Proteínas Wnt/metabolismoRESUMEN
Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells.
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Receptores de la Familia Eph/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Efrina-B1/metabolismo , Efrina-B2/metabolismo , Humanos , Intestinos/citología , Cinética , Masculino , Ratones Endogámicos C57BL , Fosforilación , Proteolisis , Transducción de Señal , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismoRESUMEN
The field of gastrointestinal epithelial stem cells is a rapidly developing area of adult stem cell research. The discovery of Lgr5(+) intestinal stem cells has enabled us to study many hidden aspects of the biology of gastrointestinal adult stem cells. Marked by Lgr5 and Troy, several novel endodermal stem cells have been identified in the gastrointestinal tract. A precise working model of stem cell propagation, dynamics, and plasticity has been revealed by a genetic labeling method, termed lineage tracing. This chapter introduces the reidentification of crypt base columnar cells as Lgr5(+) stem cells in the intestine. Subsequently, it will discuss dynamic clonal evolution and cellular plasticity in the intestinal stem cell zone, as well as in stem cell zones of stomach glands.
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Evolución Clonal , Tracto Gastrointestinal/citología , Mucosa Intestinal/citología , Células Madre/citología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Biomarcadores de Tumor/genética , Linaje de la Célula/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Tracto Gastrointestinal/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Células Madre/metabolismoRESUMEN
BACKGROUND & AIMS: Intestinal epithelial stem cells that express Lgr5 and/or Bmi1 continuously replicate and generate differentiated cells throughout life1. Previously, Paneth cells were suggested to constitute an epithelium-intrinsic niche that regulates the behavior of these stem cells2. However, ablating Paneth cells has no effect on maintenance of functional stem cells3-5. Here, we demonstrate definitively that a small subset of mesenchymal, subepithelial cells expressing the winged-helix transcription factor Foxl1 are a critical component of the intestinal stem cell niche. METHODS: We genetically ablated Foxl1+ mesenchymal cells in adult mice using two separate models by expressing either the human or simian diphtheria toxin receptor (DTR) under Foxl1 promoter control. CONCLUSIONS: Killing Foxl1+ cells by diphtheria toxin administration led to an abrupt cessation of proliferation of both epithelial stem- and transit-amplifying progenitor-cell populations that was associated with a loss of active Wnt signaling to the intestinal epithelium. Therefore, Foxl1-expressing mesenchymal cells constitute the fundamental niche for intestinal stem cells.
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A heterogeneous set of intestinal stem cells markers has been described in intestinal glands but the ultrastructural identity of intestinal stem cells remains unknown. By using electron microscopy, this study demonstrated the presence of cells with stem morphology in the intestinal glands of mice of different ages. These putative intestinal stem cells have large, euchromatic, irregular shaped nucleus, large, visible nucleolus, few ER cisternae and mitochondria. Their morphology is distinct from the morphology of any other intestinal gland cell. Stem cells located at the base of intestinal glands undergo mitosis. This study enhances the hypothesis of a gland (crypt) base columnar cell that gives rise to all the intestinal lineages.