Revealing human intestinal stem cell and crypt dynamics.
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ABSTRACT:
SUBMITTER: Baker AM
PROVIDER: S-EPMC4904973 | biostudies-other | 2014 Jul-Sep
REPOSITORIES: biostudies-other
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Project description:Intestine is a common site of adverse drug effects in clinical trials; thus, improved in vitro models for preclinical screening of pharmaceutical compounds are sought. A planar, self-renewing human intestinal monolayer platform based on primary adult gastrointestinal stem cells, termed the 2D crypt model, has been developed to screen for the effects of various compounds on the intestinal epithelium. The 2D crypt platform is based on a standard 12-well plate format and consists of cell culture inserts with a collagen film overlaying an impermeable film patterned with an array of micron-scale holes. This two-chamber format enables a gradient of growth factors to be applied such that the tissue self-organizes into spatially segregated stem and differentiated cell compartments. The patterned monolayer mimics a gut epithelium in possessing a stem cell niche, migrating proliferative and differentiated cells. Once established, the 2D crypts replicate many aspects of in vivo physiology, including cell migration, maturation, and apoptotic cell death. The planar geometry of the system simplifies dosing, sampling, and imaging during assay. An immunofluorescence-based assay was established to quantitatively assess cell density, proliferation, migration, viability, and the abundance and localization of postmitotic lineages as a function of time. The model was used to perform a small-scale screen of compounds, including signaling molecules, endogenous hormones/cytokines, and microbial metabolites, on tissue homeostasis. Hit compounds that significantly impacted proliferation and/or differentiation were readily identified. The 2D crypt platform represents a significant innovation in the development of microphysiological systems for emulating the gut epithelium for compound screens.
| S-EPMC9337237 | biostudies-literature
Project description:Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.
| S-EPMC4893006 | biostudies-literature
Project description:Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems.
| S-EPMC3145618 | biostudies-literature
Project description:Intestinal crypt progenitor/stem (ICPS) cell apoptosis and vascular endothelial cell apoptosis are responsible for the initiation and development of ionizing radiation (IR)-evoked gastrointestinal syndrome. The signaling mechanisms underlying IR-induced ICPS cell apoptosis remain largely unclear. Our findings provide evidence that ?-arrestin-2 (?arr2)-mediated ICPS cell apoptosis is crucial for IR-stimulated intestinal injury. ?Arr2-deficient mice exhibited decreased ICPS cell and intestinal Lgr5(+) (leucine-rich repeat-containing G-protein-coupled receptor 5-positive) stem cell apoptosis, promoted crypt proliferation and reproduction, and protracted survival following lethal doses of radiation. Radioprotection in the ICPS cells isolated from ?arr2-deficient mice depended on prolonged nuclear factor-?B (NF-?B) activation via direct interaction of ?arr2 with I?B? and subsequent inhibition of p53-upregulated modulator of apoptosis (PUMA)-mediated mitochondrial dysfunction. Unexpectedly, ?arr2 deficiency had little effect on IR-induced intestinal vascular endothelial cell apoptosis in mice. Consistently, ?arr2 knockdown also provided significant radioresistance by manipulating NF-?B/PUMA signaling in Lgr5(+) cells in vitro. Collectively, these observations show that targeting the ?arr2/NF-?B/PUMA novel pathway is a potential radiomitigator for limiting the damaging effect of radiotherapy on the gastrointestinal system. Significance statement: acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiotherapy. The issue of whether the critical factor for the initiation of radiation-induced intestinal injury is intestinal stem cell apoptosis or endothelial cell apoptosis remains unresolved. ?Arrs have recently been found to be multifunctional adaptor of apoptosis. Here, we found that ?-arrestin-2 (?arr2) deficiency was associated with decreased radiation-induced ICPS cell apoptosis, which prolonged survival in abdominally irradiated mice. Moreover, ?arr2 deficiency-mediated intestinal progenitor/stem cell radioprotection relied on protracted NF-?B activation and subsequent suppression of PUMA induction. Our results suggest that ICPS cell apoptosis is the factor involved in the initiation and development of radiation-induced gastrointestinal syndrome. ?Arr2 is a potential target for lessening radiation-induced ICPS cell apoptosis.
| S-EPMC5072429 | biostudies-literature
Project description:Gastrointestinal (GI) mucosal damage is a devastating adverse effect of radiation therapy. We have recently reported that expression of Dclk1, a Tuft cell and tumor stem cell (TSC) marker, 24h after high dose total-body gamma-IR (TBI) can be used as a surrogate marker for crypt survival. Dietary pectin has been demonstrated to possess chemopreventive properties, whereas its radioprotective property has not been studied. The aim of this study was to determine the effects of dietary pectin on ionizing radiation (IR)-induced intestinal stem cell (ISC) deletion, crypt and overall survival following lethal TBI. C57BL/6 mice received a 6% pectin diet and 0.5% pectin drinking water (pre-IR mice received pectin one week before TBI until death; post-IR mice received pectin after TBI until death). Animals were exposed to TBI (14 Gy) and euthanized at 24 and 84h post-IR to assess ISC deletion and crypt survival respectively. Animals were also subjected to overall survival studies following TBI. In pre-IR treatment group, we observed a three-fold increase in ISC/crypt survival, a two-fold increase in Dclk1+ stem cells, increased overall survival (median 10d vs. 7d), and increased expression of Dclk1, Msi1, Lgr5, Bmi1, and Notch1 (in small intestine) post-TBI in pectin treated mice compared to controls. We also observed increased survival of mice treated with pectin (post-IR) compared to controls. Dietary pectin is a radioprotective agent; prevents IR-induced deletion of potential reserve ISCs; facilitates crypt regeneration; and ultimately promotes overall survival. Given the anti-cancer activity of pectin, our data support a potential role for dietary pectin as an agent that can be administered to patients receiving radiation therapy to protect against radiation-induces mucositis.
| S-EPMC4536042 | biostudies-literature
Project description:The Notch signaling pathway regulates intestinal epithelial cell homeostasis, including stem cell maintenance, progenitor cell proliferation and differentiation. Notch1 and Notch2 receptors are expressed in the epithelium, but individual contributions to these functions are unclear. We used genetic deletion to define receptor roles on stem cell function, cell proliferation/differentiation, and repair after injury. Loss of Notch1 induced a transient secretory cell hyperplasia that spontaneously resolved over time. In contrast, deletion of Notch2 had no secretory cell effect. Compound deletions of Notch1 and Notch2 resulted in a more severe secretory cell hyperplasia than deletion of Notch1 alone. Furthermore, only double deletion of Notch1 and Notch2 decreased cell proliferation, suggesting a low threshold for maintenance of proliferation compared to differentiation. Stem cells were affected by deletion of Notch1, with reduced expression of Olfm4 and fewer LGR5(+) stem cells. Deletion of Notch2 had no apparent affect on stem cell homeostasis. However, we observed impaired crypt regeneration after radiation in both Notch1- and Notch2-deleted intestine, suggesting that higher Notch activity is required post-injury. These findings suggest that Notch1 is the primary receptor regulating intestinal stem cell function and that Notch1 and Notch2 together regulate epithelial cell proliferation, cell fate determination, and post-injury regeneration.
| S-EPMC4433599 | biostudies-literature
Project description:Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.
| S-EPMC4478798 | biostudies-literature
Project description:The data presented in this article are related to the research article entitled "Distribution of muscarinic acetylcholine receptor subtypes in the murine small intestine" (E.D. Muise, N. Gandotra, J.J. Tackett, M.C. Bamdad, R.A. Cowles, 2016) [1]. We recently demonstrated that neuronal serotonin stimulates intestinal crypt cell division, and induces villus growth and crypt depth (E.R. Gross, M.D. Gershon, K.G. Margolis, Z.V. Gertsberg, Z. Li, R.A. Cowles, 2012; M.D. Gershon, 2013) [2], [3]. Scopolamine, a nonspecific muscarinic receptor antagonist, inhibited serotonin-induced intestinal mucosal growth [2]. Here we provide data regarding the localization of muscarinic acetylcholine receptor 2 to the intestinal crypt stem cell compartment.
| S-EPMC5196237 | biostudies-literature
Project description:The rapid turnover of the mammalian intestinal epithelium is supported by stem cells located around the base of the crypt. In addition to the Lgr5 marker, intestinal stem cells have been associated with other markers that are expressed heterogeneously within the crypt base region. Previous quantitative clonal fate analyses have led to the proposal that homeostasis occurs as the consequence of neutral competition between dividing stem cells. However, the short-term behaviour of individual Lgr5(+) cells positioned at different locations within the crypt base compartment has not been resolved. Here we establish the short-term dynamics of intestinal stem cells using the novel approach of continuous intravital imaging of Lgr5- Confetti mice. We find that Lgr5(+) cells in the upper part of the niche (termed 'border cells') can be passively displaced into the transit-amplifying domain, after the division of proximate cells, implying that the determination of stem-cell fate can be uncoupled from division. Through quantitative analysis of individual clonal lineages, we show that stem cells at the crypt base, termed 'central cells', experience a survival advantage over border stem cells. However, through the transfer of stem cells between the border and central regions, all Lgr5(+) cells are endowed with long-term self-renewal potential. These findings establish a novel paradigm for stem-cell maintenance in which a dynamically heterogeneous cell population is able to function long term as a single stem-cell pool.
| S-EPMC3964820 | biostudies-literature
Project description:Background & aimsIntestinal epithelial homeostasis is maintained by active-cycling and slow-cycling stem cells confined within an instructive crypt-based niche. Exquisite regulating of these stem cell populations along the proliferation-to-differentiation axis maintains a homeostatic balance to prevent hyperproliferation and cancer. Although recent studies focus on how secreted ligands from mesenchymal and epithelial populations regulate intestinal stem cells (ISCs), it remains unclear what role cell adhesion plays in shaping the regulatory niche. Previously we have shown that the cell adhesion molecule and cancer stem cell marker, CD166/ALCAM (activated leukocyte cell adhesion molecule), is highly expressed by both active-cycling Lgr5+ ISCs and adjacent Paneth cells within the crypt base, supporting the hypothesis that CD166 functions to mediate ISC maintenance and signal coordination.MethodsHere we tested this hypothesis by analyzing a CD166-/- mouse combined with immunohistochemical, flow cytometry, gene expression, and enteroid culture.ResultsWe found that animals lacking CD166 expression harbored fewer active-cycling Lgr5+ ISCs. Homeostasis was maintained by expansion of the transit-amplifying compartment and not by slow-cycling Bmi1+ ISC stimulation. Loss of active-cycling ISCs was coupled with deregulated Paneth cell homeostasis, manifested as increased numbers of immature Paneth progenitors due to decreased terminal differentiation, linked to defective Wnt signaling. CD166-/- Paneth cells expressed reduced Wnt3 ligand expression and depleted nuclear ?-catenin.ConclusionsThese data support a function for CD166 as an important cell adhesion molecule that shapes the signaling microenvironment by mediating ISC-niche cell interactions. Furthermore, loss of CD166 expression results in decreased ISC and Paneth cell homeostasis and an altered Wnt microenvironment.
| S-EPMC5404029 | biostudies-literature