Crypt Base Research Articles

Radical and lunatic fringes modulate notch ligands to support mammalian intestinal homeostasis.

Notch signalling maintains stem cell regeneration at the mouse intestinal crypt base and balances the absorptive and secretory lineages in the upper crypt and villus. Here we report the role of Fringe family of glycosyltransferases in modulating Notch activity in the two compartments. At the crypt base, RFNG is enriched in the Paneth cells and increases cell surface expression of DLL1 and DLL4. This promotes Notch activity in the neighbouring Lgr5+ stem cells assisting their self-renewal. Expressed by various secretory cells in the upper crypt and villus, LFNG promotes DLL surface expression and suppresses the secretory lineage . Hence, in the intestinal epithelium, Fringes are present in the ligand-presenting 'sender' secretory cells and promote Notch activity in the neighbouring 'receiver' cells. Fringes thereby provide for targeted modulation of Notch activity and thus the cell fate in the stem cell zone, or the upper crypt and villus.

Sox9‐independent Control of Intestinal Paneth Cell Differentiation and Function

The genetic and signaling mechanisms controlling the generation and function of differentiated cell types are of continued interest. The intestinal Paneth cell (PC) is a post‐mitotic and dedicated secretory cell type with reported functions in supporting intestinal stem cell (ISC) maintenance in the crypt stem cell niche, and in secreting antimicrobial peptides to protect the crypt from potentially pathogenic luminal microbiota. Previous studies have reported a near‐complete loss of PCs (as defined by morphological and marker‐based criteria) in epithelium where the transcription factor Sox9 is genetically ablated, leading to the proposition that Sox9 is required for PC differentiation. Currently, however, the mechanisms through which Sox9 controls PC differentiation are not known. We have found evidence in Sox9 knockout (KO) small intestine (SI) that cells at the crypt base frequently express PC‐markers such as Lysozyme (Lyz) and Cryptdin, exhibit apical granules, and are post‐mitotic, all of which are features indicative of PC differentiation. The numbers of Lyz+ cells are highest in the distal Sox9 KO SI but are significantly reduced relative to control, and are essentially absent in the proximal SI, suggesting that regionally patterned signals may drive PC differentiation independent of Sox9. Apical granularity and expression levels of Lyz/Cryptdin are also altered, suggesting that while Sox9 KO ISCs can initiate PC differentiation, they are unable to reach full maturity. Antibody and qPCR based detection of PC gene products (including secreted antimicrobials, growth factors, and transcriptional regulators proposed to drive PC differentiation downstream of Sox9) are maintained in Sox9 KO cultured intestinal organoids and are elevated in organoids treated with pharmacological agents that drive the PC differentiation program. These results indicate that Sox9‐deficient ‘Paneth‐like’ cells are continually generated in culture and can be pushed toward PC differentiation with factors known to stimulate the PC differentiation program. We conclude that Sox9 is not required for the activation of at least sub‐sets of gene products associated with PC differentiation and function. Ongoing experiments are designed to investigate Sox9‐ dependent and independent pathways toward PC fate, and to characterize the functionality of Sox9−/− ‘Paneth‐like’ cells compared to controls.Support or Funding InformationRO1 DK091427‐05F32 DK115016‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Common Mechanism Links Activities of Butyrate in the Colon.

Two biological activities of butyrate in the colon (suppression of proliferation of colonic epithelial stem cells and inflammation) correlate with inhibition of the activity of histone deacetylases. Cellular and biochemical studies of molecules similar in structure to butyrate, but different in molecular details (functional groups, chain-length, deuteration, oxidation level, fluorination, or degree of unsaturation), demonstrated that these activities were sensitive to molecular structure, and were compatible with the hypothesis that butyrate acts by binding to the Zn2+ in the catalytic site of histone deacetylases. Structure-activity relationships drawn from a set of 36 compounds offer a starting point for the design of new compounds targeting the inhibition of histone deacetylases. The observation that butyrate was more potent than other short-chain fatty acids is compatible with the hypothesis that crypts evolved (at least in part), to separate stem cells at the base of crypts from butyrate produced by commensal bacteria.

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Engineered Glycocalyx Regulates Stem Cell Proliferation in Murine Crypt Organoids

At the base of the intestinal crypt, long-lived Lgr5+ stem cells are intercalated by Paneth cells that provide essential niche signals for stem cell maintenance. This unique epithelial anatomy makes the intestinal crypt one of the most accessible models for the study of adult stem cell biology. The glycosylation patterns of this compartment are poorly characterized, and the impact of glycans on stem cell differentiation remains largely unexplored. We find that Paneth cells, but not Lgr5+ stem cells, express abundant terminal N-acetyllactosamine (LacNAc). Employing an enzymatic method to edit glycans in cultured crypt organoids, we assess the functional role of LacNAc in the intestinal crypt. We discover that blocking access to LacNAc on Paneth cells leads to hyperproliferation of the neighboring Lgr5+ stem cells, which is accompanied by the downregulation of genes that are known as negative regulators of proliferation.

Ultrastructural and Immunohistochemical Study on the Lamina Propria Cells Beneath Paneth Cells in the Rat Ileum

Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell-derived cellular processes which extended into the LP and the LP cells. The results showed that various cells-including blood capillary, subepithelial stromal cell, and nerve fiber-were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 μm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34+ CD31- αSMA- stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFRαhi αSMA+ stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFRαhi αSMA- stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34+ CD31- αSMA- PDGFRα-/lo phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. Anat Rec, 301:1074-1085, 2018. © 2018 Wiley Periodicals, Inc.

Loss of Paneth Cell Autophagy Causes Acute Susceptibility to Toxoplasma gondii-Mediated Inflammation

The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by IFN-γ- and intestinal microbiota-mediated inflammation, Paneth cell loss, and bacterial dysbiosis. Paneth cells are a prominent secretory epithelial cell type that resides at the base of intestinal crypts and releases antimicrobial peptides. We demonstrate that the microbiota triggers basal Paneth cell-specific autophagy via induction of IFN-γ, a known trigger of autophagy, to maintain intestinal homeostasis. Deletion of the autophagy protein Atg5 specifically in Paneth cells results in exaggerated intestinal inflammation characterized by complete destruction of the intestinal crypts resembling that seen in pan-epithelial Atg5-deficient mice. Additionally, lack of functional autophagy in Paneth cells within intestinal organoids and T.gondii-infected mice causes increased sensitivity to the proinflammatory cytokine TNF along with increased intestinal permeability, leading to exaggerated microbiota- and IFN-γ-dependent intestinal immunopathology. Thus, Atg5 expression in Paneth cells is essential for tissue protection against cytokine-mediated immunopathology during acute gastrointestinal infection.

The tankyrase inhibitor G007-LK inhibits small intestine LGR5+ stem cell proliferation without altering tissue morphology

BackgroundThe WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages.ResultsWe treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible.ConclusionWe show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.

Coexpression of CD44 and Ki-67 in colon`s neoplast

the evaluation of Ki-67 and CD44 expression in the 'serrated' polyps of the colon and comparison them with adenocarcinomas and tubular and tubule-villous adenomas of the colon. The study is including 49 'serrated' polyps, 34 tubular (AT) and tubulo-villous (ATV) adenomas and 32 adenocarcinomas of the colon. Antibodies CD44 and Ki-67 were used as immunohistochemical markers in this study. A statistically significant difference (p<0.01) was observed between traditional serrated adenomas (TSA) from hyperplastic polyps (HP) and sessile serrated adenomas (SSA) in the Ki-67 level and the localization of the Ki-67 and CD44 reaction: surface areas of the crypts (upper third) in TSA and base of crypts (lower third) in HP and SSA. There was no difference between HP and SSA (p>0.05), neither by marker localization, nor by their level. In all 'serrated' polyps of the colon, the Ki-67 reaction was nuclear; CD44 - membrane (except for 1 TSA). we are the first ones who suggested to evaluate not the overall level of reactions of CD44 and Ki-67, but particular level for each third part of crypts. The similarities of TSA, AT and ATV and between HP and SSA are shown as well as the principal statistical difference between these two groups. The cytoplasmic reaction of CD44 in adenocarcinomas and the membrane reaction of CD44 in 98% of the 'serrated' polyps of the colon are described. For the first time coexpression of CD44 and Ki-67 on particulate thirds of crypts in neoplasms of the colon is shown and the potential reasons for this phenomenon are discussed.

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche.

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices.

Bone Cells and Mast Cells Express MMP-9 During Tooth Eruption In Vivo

Context: Tooth development takes place inside a bony crypt in the alveolar bones. Tooth eruption requires, therefore, the removal of bone and lamina propria to establish the eruptive pathway. Matrix metalloproteinases (MMPs) belong to a family of enzymes responsible for the degradation of the extracellular matrix (ECM). MMP-9 is a member of this family and is secreted by a variety of cells, including mast cells. Both have been identified as possible collaborators for tissue remodeling during tooth eruption. Aims: The aim of this study is to analyze the bone remodeling, MMP-9 expression, and the prevalence of mast cells throughout the eruptive process of rat molars. Materials and Methods: Twenty-eight rats (4–17 days old) were used in this study. Histomorphometric analyses were performed to determine the bone area in the basal and coronal regions of the bony crypt and the prevalence of mast cells at the lamina propria along the eruptive process. Immunohistochemistry was performed to detect MMP-9 expression in the lamina propria and alveolar bone. Results: Increased bone apposition at the base of the bony crypt was observed at 14 days and increased resorption of the coronal bone was detected after 9 days. An elevated number of mast cells was observed in 9-day-old rats, and MMP-9 was strongly expressed in mast cells and osteoclasts. Conclusion: Taken together, we suggest that mast cells may contribute to the recruitment of osteoclast precursors during tooth eruption and that MMP-9 is possibly involved in the removal of the ECM during the eruptive process.

Identification of Malignancy-Associated Changes in Histologically Normal Tumor-Adjacent Epithelium of Patients with HPV-Positive Oropharyngeal Cancer

The incidence of HPV-positive oropharyngeal cancer (HPV+ OPC) is increasing, thus presenting new challenges for disease detection and management. Noninvasive methods involving brush biopsies of diseased tissues were recently reported as insufficient for tumor detection in HPV+ OPC patients, likely due to differences between the site of tumor initiation at the base of involuted crypts and the site of brush biopsy at the crypt surface. We hypothesized that histologically normal surface epithelial cells in the oropharynx contain changes in nuclear morphology that arise due to tumor proximity. We analyzed the nuclear phenotype of matched tumor, tumor-adjacent normal, and contralateral normal tissues from biopsies of nine HPV+ OPC patients. Measurements of 89 nuclear features were used to train a random forest-based classifier to discriminate between normal and tumor nuclei. We then extracted voting scores from the trained classifier, which classify nuclei on a continuous scale from zero (“normal-like”) to one (“tumor-like”). In each case, the average score of the adjacent normal nuclei was intermediate between the tumor and contralateral normal nuclei. These results provide evidence for the existence of phenotypic changes in histologically normal, tumor-adjacent surface epithelial cells, which could be used as brush biopsy-based biomarkers for HPV+ OPC detection.

Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5-and Bmi1-Expressing Intestinal Cell Populations.

Background & AimsContinual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein–coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function.MethodsWe used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC–green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed.ResultsTwo novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established.ConclusionsThese data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.

Overexpression of insulin receptor substrate-4 is correlated with clinical staging in colorectal cancer patients.

Insulin receptor substrate-4 (IRS-4), a poorly studied member of the IRS family, may play an important role in colorectal cancer (CRC) tumourigenesis. The aim of this pilot study was to elucidate the potential role of IRS-4 in colorectal carcinogenesis by evaluating IRS-4 expression in different types of colorectal tumours (n = 20) and comparing its expression to normal mucosa (n = 20). Tissue samples were collected from 18 patients with CRC and 2 with precancerous lesions (tubulovillous adenomas), all of whom were undergoing potentially curative surgery. IRS-4 expression was evaluated using immunohistochemical staining and compared to clinicopathological features. In normal colonic crypts, the subcellular localization of IRS-4 varied from the crypt base compartment to the surface epithelium. Nuclear IRS-4 staining decreased while non-nuclear IRS-4 increased as cells approached the top of the crypt. In the patients studied, colorectal tumours showed a significant (p<0.001) increase of IRS-4 expression compared with adjacent normal tissue. Furthermore, nuclear IRS-4 intensity of CRC patients was significantly (p<0.0001) higher in colonic tumoural tissue than in paired normal specimens. Tumour expression of IRS-4 in CRC patients was positively associated with T (p<0.0001) and N (p<0.05), of TNM (tumour and nodes and metastasis) staging system. Taken together, these results suggest that increase of IRS-4 expression may be involved to some extent in colorectal cancer.

Paneth cells in intestinal physiology and pathophysiology.

Small intestinal mucosa is characterised by villus forming connective tissues with highly specialised surface lining epithelial cells essentially contributing to the establishment of the intestinal border. In order to perform these diverse functions, spatially distinct compartments of epithelial differentiation are found along the crypt-villus axis, including Paneth cells as a highly specialised cell type. Paneth cells locate in crypts and assist undifferentiated columnar cells, called crypt base columnar cells, and rapidly amplifying cells in the regeneration of absorptive and secretory cell types. There is some evidence that Paneth cells are involved in the configuration and function of the stem cell zone as well as intestinal morphogenesis and crypt fission. However, the flow of Paneth cells to crypt bottoms requires strong Wnt signalling guided by EphB3 and partially antagonised by Notch. In addition, mature Paneth cells are essential for the production and secretion of antimicrobial peptides including α-defensins/cryptdins. These antimicrobials are physiologically involved in shaping the composition of the microbiome. The autophagy related 16-like 1 (ATG16L1) is a genetic risk factor and is involved in the exocytosis pathway of Paneth cells as well as a linker molecule to PPAR signalling and lipid metabolism. There is evidence that injuries of Paneth cells are involved in the etiopathogenesis of different intestinal diseases. The review provides an overview of the key points of Paneth cell activities in intestinal physiology and pathophysiology.

Abstract IA6: Lgr5 stem cell-based organoids in human disease

Abstract The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of human stomach, liver, pancreas, prostate and kidney. Using CRISPR/Cas9 technology, genes can be efficiently modified in organoids of various origins. Citation Format: Hans Clevers. Lgr5 stem cell-based organoids in human disease [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA6.

Multicolor lineage tracing reveals clonal architecture and dynamics in colon cancer

Colon cancers are composed of phenotypically heterogeneous tumor cell subpopulations with variable expression of putative stem cell and differentiation antigens. While in normal colonic mucosa, clonal repopulation occurs along differentiation gradients from crypt base toward crypt apex, the clonal architecture of colon cancer and the relevance of tumor cell subpopulations for clonal outgrowth are poorly understood. Using a multicolor lineage tracing approach in colon cancer xenografts that reflect primary colon cancer architecture, we here demonstrate that clonal outgrowth is mainly driven by tumor cells located at the leading tumor edge with clonal axis formation toward the tumor center. While our findings are compatible with lineage outgrowth in a cancer stem cell model, they suggest that in colorectal cancer tumor cell position may be more important for clonal outgrowth than tumor cell phenotype.

GUCY2C maintains intestinal LGR5+ stem cells by opposing ER stress.

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life, lineage plasticity, and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context, the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis, including proliferation, lineage commitment, and DNA damage repair. However, a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c–/–) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced in Gucy2c–/– mice, associated with loss of active Lgr5+ cells but a reciprocal increase in reserve Bmi1+ cells. GUCY2C was expressed in crypt base Lgr5+ cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress, typically absent from ISCs, was elevated throughout the crypt base in Gucy2c–/– mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs, an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs in Gucy2c–/–mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs.

Subtle Deregulation of the Wnt-Signaling Pathway Through Loss of Apc2 Reduces the Fitness of Intestinal Stem Cells

The importance of the Wnt‐signaling pathway on the regulation and maintenance of the intestinal stem cell (ISC) population is well recognized. However, our current knowledge base is founded on models using systems of gross deregulation of the Wnt‐signaling pathway. Given the importance of this signaling pathway on intestinal homeostasis, there is a need to explore the role of more subtle alterations in Wnt‐signaling levels within this tissue. Herein, we have used a model of Apc2 loss to meet this aim. Apc2 is a homolog of Apc which can also form a destruction complex capable of binding β‐catenin, albeit less efficiently than Apc. We show that systemic loss of Apc2 results in an increase in the number of cells displaying nuclear β‐catenin at the base of the intestinal crypt. This subsequently impacts the expression levels of several ISC markers and the fitness of ISCs as assessed by organoid formation efficiency. This work provides the first evidence that the function and fitness of ISCs can be altered by even minor misregulation of the Wnt‐signaling pathway. Our data highlights the importance of correct maintenance of this crucial signaling pathway in the maintenance and function of the ISC population. Stem Cells 2018;36:114–122

ZBP-89 function in colonic stem cells and during butyrate-induced senescence.

ZBP-89 (Zfp148, ZNF148) is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP-89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a Zfp148CreERT2 transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the Zfp148 locus in the colon using the Cdx2NLSCreERT2 transgene, reduced the size and number of polyps formed in the Apc-deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the Apc deletion, only when Zfp148 was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed ZNF148 gene expression, inducing CDKN2a (p16Ink4a) gene expression. Collectively, Zfp148 mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the CDKN2A promoter.