Crypt Base Research Articles

Gastric Stem Cell and Cellular Origin of Cancer.

Several stem cell markers within the gastrointestinal epithelium have been identified in mice. One of the best characterized is Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) and evidence suggests that Lgr5+ cells in the gut are the origin of gastrointestinal cancers. Reserve or facultative stem or progenitor cells with the ability to convert to Lgr5+ cells following injury have also been identified. Unlike the intestine, where Lgr5+ cells at the crypt base act as active stem cells, the stomach may contain unique stem cell populations, since gastric Lgr5+ cells seem to behave as a reserve rather than active stem cells, both in the corpus and in the antral glands. Gastrointestinal stem cells are supported by a specific microenvironment, the stem cell niche, which also promotes tumorigenesis. This review focuses on stem cell markers in the gut and their supporting niche factors. It also discusses the molecular mechanisms that regulate stem cell function and tumorigenesis.

Demystifying the Differences Between Tumor-Initiating Cells and Cancer Stem Cells in Colon Cancer

Tumor-initiating cells and cancer stem cells refer to a subpopulation of self-renewing cells involved in tumor initiation and tumor maintenance, respectively. With this review, we aimed to define the functional and molecular differences between both cell types in the context of colon cancer. Recent evidence suggests that the two major stem cell populations in the normal intestinal crypt have tumor-initiating capacity and could be the cell-of-origin in colon cancer. Activation of the Wnt/β-catenin pathway (an early event in mouse intestinal carcinogenesis) in the crypt base columnar stem cells and reserve stem cells leads to adenoma formation, supporting a role in tumor initiation. On the other hand, colon cancer stem cells express several membrane markers facilitating their isolation by FACS and are associated with treatment resistance and higher metastatic potential. Tumor-initiating cells and cancer stem cells express distinct markers, display discrete biological functions, and can be studied using different molecular and cellular approaches. While cancer stem cells may be derived from tumor-initiating cells, these terms are not necessarily interchangeable and more likely reflect a specific cell state.

Asymmetric cell division-dominant neutral drift model for normal intestinal stem cell homeostasis

The normal intestinal epithelium is continuously regenerated at a rapid rate from actively cycling Lgr5-expressing intestinal stem cells (ISCs) that reside at the crypt base. Recent mathematical modeling based on several lineage-tracing studies in mice shows that the symmetric cell division-dominant neutral drift model fits well with the observed in vivo growth of ISC clones and suggests that symmetric divisions are central to ISC homeostasis. However, other studies suggest a critical role for asymmetric cell division in the maintenance of ISC homeostasis in vivo. Here, we show that the stochastic branching and Moran process models with both a symmetric and asymmetric division mode not only simulate the stochastic growth of the ISC clone in silico but also closely fit the in vivo stem cell dynamics observed in lineage-tracing studies. In addition, the proposed model with highest probability for asymmetric division is more consistent with in vivo observations reported here and by others. Our in vivo studies of mitotic spindle orientations and lineage-traced progeny pairs indicate that asymmetric cell division is a dominant mode used by ISCs under normal homeostasis. Therefore, we propose the asymmetric cell division-dominant neutral drift model for normal ISC homeostasis. NEW & NOTEWORTHY The prevailing mathematical model suggests that intestinal stem cells (ISCs) divide symmetrically. The present study provides evidence that asymmetric cell division is the major contributor to ISC maintenance and thus proposes an asymmetric cell division-dominant neutral drift model. Consistent with this model, in vivo studies of mitotic spindle orientation and lineage-traced progeny pairs indicate that asymmetric cell division is the dominant mode used by ISCs under normal homeostasis.

Interaction between smoking and ATG16L1T300A triggers Paneth cell defects in Crohn's disease.

It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets.

A morphological and molecular study of proposed early forms of traditional serrated adenoma.

Traditional serrated adenoma (TSA) is the least common subtype of serrated colorectal polyp. Large protuberant lesions are easily recognised; however, the origins of TSAs are not known, and early forms have not been described. Some large TSAs present with a flat 'shoulder' component surrounding the central protuberant component. We hypothesised that small polyps with the same histology as these shoulder regions may represent early TSAs. Thus the primary aim of the study is to describe the histology of these presumptive early TSAs. We collected 70 small (<10 mm) polyps that may represent early TSAs on the basis of typical TSA cytology covering the luminal surface. We also identified 12 large TSAs with a shoulder component resembling these small polyps. The study polyp patients had a mean age of 58 years, and 54% were female; the polyps had a mean diameter of 4.1 mm and were predominantly distal (71%). Morphologically, slit-like serrations were present in 81%, ectopic crypt formations were present in 67%, and a villous component was present in 47%. These histological features were similar to those of the 12 shoulder lesions. Immunohistochemical stains showed an absence of β-catenin nuclear expression in 96% of the small polyps, retained expression of MLH1 in 100%, and Ki67 positivity restricted to the crypt bases and ectopic crypt formations. BRAF and KRAS mutations were identified in 47% and 31% of the polyps, respectively. BRAF-mutated polyps were more likely than KRAS-mutated polyps to arise in a precursor polyp (82% versus 18%, P < 0.001), and were more likely to have slit-like serrations (100% versus 73%, P = 0.003). These morphological, immunohistochemical and molecular findings are similar to what has been reported in large TSAs, and support the hypothesis that these polyps represent early forms of TSA.

Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice.

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3−/− embryos died prior to blastocyst implantation, whereas Rnpc3+/− mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3lox/lox) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.

Hypothesis: Caco-2 cell rotational 3D mechanogenomic turing patterns have clinical implications to colon crypts.

Colon crypts are recognized as a mechanical and biochemical Turing patterning model. Colon epithelial Caco‐2 cell monolayer demonstrated 2D Turing patterns via force analysis of apical tight junction live cell imaging which illuminated actomyosin meshwork linking the actomyosin network of individual cells. Actomyosin forces act in a mechanobiological manner that alters cell/nucleus/tissue morphology. We observed the rotational motion of the nucleus in Caco‐2 cells that appears to be driven by actomyosin during the formation of a differentiated confluent epithelium. Single‐ to multi‐cell ring/torus‐shaped genomes were observed prior to complex fractal Turing patterns extending from a rotating torus centre in a spiral pattern consistent with a gene morphogen motif. These features may contribute to the well‐described differentiation from stem cells at the crypt base to the luminal colon epithelium along the crypt axis. This observation may be useful to study the role of mechanogenomic processes and the underlying molecular mechanisms as determinants of cellular and tissue architecture in space and time, which is the focal point of the 4D nucleome initiative. Mathematical and bioengineer modelling of gene circuits and cell shapes may provide a powerful algorithm that will contribute to future precision medicine relevant to a number of common medical disorders.

Two-photon microscopy of Paneth cells in the small intestine of live mice

Paneth cells are one of the principal epithelial cell types in the small intestine, located at the base of intestinal crypts. Paneth cells play key roles in intestinal host-microbe homeostasis via granule secretion, and their dysfunction is implicated in pathogenesis of several diseases including Crohn’s disease. Despite their physiological importance, study of Paneth cells has been hampered by the limited accessibility and lack of labeling methods. In this study, we developed a simple in vivo imaging method of Paneth cells in the intact mouse small intestine by using moxifloxacin and two-photon microscopy (TPM). Moxifloxacin, an FDA-approved antibiotic, was used for labeling cells and its fluorescence was strongly observed in Paneth cell granules by TPM. Moxifloxacin labeling of Paneth cell granules was confirmed by molecular counterstaining. Comparison of Paneth cells in wild type, genetically obese (ob/ob), and germ-free (GF) mice showed different granule distribution. Furthermore, Paneth cell degranulation was observed in vivo. Our study suggests that TPM with moxifloxacin labeling can serve as a useful tool for studying Paneth cell biology and related diseases.

LncGata6 maintains stemness of intestinal stem cells and promotes intestinal tumorigenesis.

The intestinal epithelium harbours remarkable self-renewal capacity that is driven by Lgr5+ intestinal stem cells (ISCs) at the crypt base. However, the molecular mechanism controlling Lgr5+ ISC stemness is incompletely understood. We show that a Gata6 long noncoding RNA (lncGata6) is highly expressed in ISCs. LncGata6 knockout or conditional knockout in ISCs impairs the stemness of ISCs and epithelial regeneration. Mechanistically, lncGata6 recruits the NURF complex onto the Ehf promoter to induce its transcription, which promotes the expression of Lgr4/5 to enhance Wnt signalling activation. Moreover, the human orthologue lncGATA6 is highly expressed in the cancer stem cells of colorectal cancer and promotes tumour initiation and progression. Antisense oligonucleotides against lncGATA6 exhibit strong therapeutic efficacy on colorectal cancer. Thus, targeting lncGATA6 will have potential clinical applications in colorectal cancer treatment as an ideal therapeutic target.

The localization and distribution of cells labeled by a somatic stem cell-recognizing antibody (A3) in rat colon development; possible presence of a new cell type forming the intestinal stem cell niche.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma (MFH)-derived cloned cells, recognizes somatic stem cells (bone-marrow/hair follicle stem cells). We investigated the distribution of cells immunoreactive to A3 in the developing rat intestine (particularly, the colon), focusing on the ontogenic kinetics of A3-positive cells. In the rat intestine, A3 labeled spindle-shaped stromal cells localized in the submucosa and labeled endothelial cells of capillaries in the lamina propria forming villi in the early development stage. With development progression, A3-positive cells were exclusively localized around the crypts of the colon. Double immunofluorescence revealed that A3-positive cells around the crypts reacted to vimentin (for mesenchymal cells) and Thy-1 (for mesenchymal stromal cells) but not to α-SMA (for mesenchymal myofibroblastic cells) or CD34 (for hematopoietic stem cells), indicating that A3-positive cells around the crypts may have characteristics of immature mesenchymal cells. In addition, A3 labeled a few epithelial cells at the base of colon crypts. Furthermore, immunoelectron microscopy revealed that A3-positive cells lay inside myofibroblasts adjacent to the epithelium of the crypts. A3-positive cells were regarded as a new type of immature mesenchymal cells around the crypts. Collectively, A3-positive cells might take part in the stem cell niche in the colon, which is formed through epithelial-mesenchymal interaction.

The intestinal epithelial response to damage.

The constant renewal of the intestinal epithelium is fueled by intestinal stem cells (ISCs) lying at the base of crypts, and these ISCs continuously give rise to transit-amplifying progenitor cells during homeostasis. Upon injury and loss of ISCs, the epithelium has the ability to regenerate by the dedifferentiation of progenitor cells that then regain stemness and repopulate the pool of ISCs. Epithelial cells receive cues from immune cells, mesenchymal cells and the microbiome to maintain homeostasis. This review focuses on the response of the epithelium to damage and the interplay between the different intestinal compartments.

Wnt signaling suppresses MAPK-driven proliferation of intestinal stem cells

Intestinal homeostasis depends on a slowly proliferating stem cell compartment in crypt cells, followed by rapid proliferation of committed progenitor cells in the transit amplifying (TA) compartment. The balance between proliferation and differentiation in intestinal stem cells (ISCs) is regulated by Wnt/β-catenin signaling, although the mechanism remains unclear. We previously targeted PORCN, an enzyme essential for all Wnt secretion, and demonstrated that stromal production of Wnts was required for intestinal homeostasis. Here, a PORCN inhibitor was used to acutely suppress Wnt signaling. Unexpectedly, the treatment induced an initial burst of proliferation in the stem cell compartment of the small intestine, due to conversion of ISCs into TA cells with a loss of intrinsic ISC self-renewal. This process involved MAPK pathway activation, as the proliferating cells in the base of the intestinal crypt contained phosphorylated ERK1/2, and a MEK inhibitor attenuated the proliferation of ISCs and their differentiation into TA cells. These findings suggest a role for Wnt signaling in suppressing the MAPK pathway at the crypt base to maintain a pool of ISCs. The interaction between Wnt and MAPK pathways in vivo has potential therapeutic applications in cancer and regenerative medicine.

Abstract 3910: Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement

Abstract At the base of normal intestinal crypts, stem cells maintain the highly regenerative gut epithelium. These intestinal stem cells are well characterized for their high expression of the G coupled protein receptor LGR5 (also known as GPR49). Together with R-spondins (potent Wnt signaling modulators) and stem cell growth factors, LGR5 forms part of a signaling cascade responsible for the regulation of cellular proliferation. Key mutations in the APC or BRAF pathways of intestinal stem cells lead to lesions and metastatic colorectal cancer if not diagnosed and resected at an early stage. The majority of primary and metastatic tumors arising from these mutations overexpress LGR5. It has been reported that metastatic colorectal cancer (CRC) patients expressing higher levels of LGR5 in tumor biopsies had increased rates of relapse. BNC101 is a first-in-class anti-LGR5 humanized monoclonal antibody. Biacore analysis of BNC101 has demonstrated a high affinity to LGR5 (Kd=16nM) with no cross-reactivity to LGR4 or LGR6 receptors. Immunoprecipitation studies have also shown that BNC101 has no off-target binding. The murine equivalent of BNC101 has demonstrated antitumor activity in multiple CRC patient-derived xenografts. Flow cytometry studies on CRC cell lines have revealed that LGR5 is located intracellularly with a small fraction present on the cell surface. To better understand its mechanism of action, BNC101 was conjugated to Alexa Fluor 647 and incubated with human CRC cell lines. It was shown that BNC101 interacts with membrane-bound LGR5 and is internalized within 5 minutes. Incubation of the cells for 24 hours at clinically relevant concentrations led to accumulation of fluorophore-conjugated BNC101 within the cell. This intracellular accumulation of BNC101 was further demonstrated with receptor recycling kinetic studies whereby only partial receptor recycling occurred, which may be attributed to the large intracellular LGR5 pool. CHO cell lines overexpressing LGR5 with a GFP tag were used to determine co-localization of BNC101 with its LGR5 ligand. BNC101 is currently in a safety and dose escalation phase I clinical trial in patients with recurrent metastatic CRC. We were able to demonstrate BNC101 target engagement with LGR5 for the first time in tumor biopsies following treatment. Tumor biopsies were analyzed by mass spectrometry together with Matrix Assisted Laser Desorption/Ionization; co-localization of BNC101 with LGR5 was observed, providing us with evidence for the first time that BNC101 infiltrates the patient tumor and engages with the overexpressed LGR5 receptor. Citation Format: Daniel J. Inglis, John Licari, Kristen R. Georgiou, Nicole L. Wittwer, Ross W. Hamilton, Donna M. Beaumont, Michaela A. Scherer, Tina C. Lavranos. Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3910.

Abstract 4082: Contrasting role for claudin-2 in colitis-associated cancer (CAC) and spontaneous colon tumorigenesis (CRC) by regulating mucosal healing

Abstract A link between chronic inflammation and colon cancer has long been appreciated. Accordingly, patients with ulcerative colitis are at higher risk for developing colitis-associated cancer (CAC). Mucosal barrier dysregulation allies with inflammation and colon cancer. Accordingly, claudin-2, a tight junction protein, is upregulated in ulcerative colitis, CAC and spontaneous colon cancer (CRC). Remarkably, claudin-2 expression is restricted to the colon crypt base, among undifferentiated and proliferative colonocytes, and its increased expression is responsible for the regulation of paracellular permeability, differentiation and proliferation. However, causal role of claudin-2 in CAC and CRC remains unclear. Considering the recent data from our laboratory and of others, that in vivo genetic manipulation of claudin-2 has inverse effects on colitis, we hypothesized an adaptive role for increased claudin-2 expression in colitis by promoting mucosal healing. We further postulated that mucosal healing can manifest in contrasting phenotypes for the CAC and CRC. To examine, Villin-claudin-2 transgenic (Cldn-2) mice were subjected to CAC by exposing them to AOM/DSS-induced cancer or interbred with the APCmin mice, the mouse model of spontaneous intestinal cancer. It was intriguing that the inflammation-driven colon cancer (AOM/DSS induced colon cancer) was significantly suppressed in Cldn-2 mice compared to WT littermates (p&amp;lt;0.001). These mice also showed immune suppressive milieu ideal for spontaneous colon tumorigenesis. Accordingly, we found contrasting disparity in results from APCmin/Cldn-2 mice, generated by the cross between Cldn-2 and APCmin mice. These mice showed significant increase in intestinal tumor burden (248+40%, p&amp;lt;0.001) and colonic tumor burden [Incidence (172.50±18%, p&amp;lt;0.001) and size (521.5±34%, p&amp;lt;0.001)] compared to APCmin mice. H&amp;E analysis confirmed aggressive phenotype of APCmin-Cldn-2 mice tumors (vs APCmin mice tumors). APCmin/Cldn-2 mice also survived less than the APCmin mice. Further, inflammation induction by subjecting these mice to Dextran Sodium Sulfate (DSS) resulted in precocious colonic tumorigenesis only in APCmin mice, rejecting a role for the pro-inflammatory signaling in colon cancer progression in APCmin/Cldn-2 mice. High throughput transcriptome and oncogenic array analyses further suggested modifications of the key inflammatory and oncogenic signaling, in supporting these disparate phenotypes. Taken together, our data provide strong support for the key significance for the context and diverse immunogenic signaling in regulating CAC versus CRC. These studies also suggest that claudin-2 manipulated mice can provide the much needed mouse modelling to help identify key immunogenic signaling supportive of the colon tumorigenesis for therapeutic gains. Citation Format: Rizwan Ahmad, Balawant Kumar, Narendra Kumar, Mary K. Washington, Punita Dhawan, Amar B. Singh. Contrasting role for claudin-2 in colitis-associated cancer (CAC) and spontaneous colon tumorigenesis (CRC) by regulating mucosal healing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4082.

Establishment of human intestinal organoid culture and gene delivery system

Objective To establish human intestinal organoid culture in vitro and effective gene delivery system mediating by lentivirus into organoid, it will be essential to develop colorectal cancer research and gene therapy. Methods (1) The biological specimen of ileum or colon by surgical resection is taken 3 to 5 cm and separated mucous membrane from muscular layer. It is digested and separated by ethylene diamine tetraacetic acid Chelation Buffer for collecting intestinal crypts. The intestinal crypts is resuspended with in Matrigel. The sample is seeded on pre-warmed96-well plate, after solidification, which is added with culture medium including growth factors. The organoid status can be observed in everyday, the medium be exchanged in each 2 or 3 days, and the organoid structure be passaged in each 6 or 7 days. (2) Packaging high-titer lentivirus carrying with reporter gene, green fluorescent protein (GFP), infect into intestinal organoid, and the expression quantity of fluorescent is observed in organoid. Results We could culture mini-gut organoid with budding spherical structures using sporadic intestinal crypts ex vivo, and the livability of both groups of crypt/organoid are greater than 70%. Intestinal organoid can express a protein marker about absorptive cell which is located in normal intestinal epithelium. Reporter gene mediating by lentivirus vector can be stably expressed in organoids. Conclusion This new intestinal organoid culture model not only is used to research gene functions in maintaining intestinal homeostasis. Key words: Intestinal organoid; Crypt base columnar cells; Lentivirus; Gene-editing; Gene delivery system

Mouse Intestinal Krt15+ Crypt Cells Are Radio-Resistant and Tumor Initiating.

SummaryTwo principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer.

Local Mesenchymal Stem Cell Therapy in Experimentally Induced Colitis in the Rat.

BackgroundMultipotent mesenchymal stem cells (MSCs) have been used in inflammatory bowel diseases because of their immunomodulatory and regenerative properties. We investigated their local use in an experimental model of colitis in the rat.Materials and MethodsColitis was induced into 20 Wistar rats with local TNBS instillation. Allogeneic stem cells were derived from rat adipose tissue and labeled with PKH2 linker dye with creation of a control and a second group treated by a local injection into the rectal wall of 2×106 allogeneic adipose tissue-derived stem cells (ADSCs). The thicknesses of different components of the rectum were measured with comparisons made in different parts of the colon of the Hunter inflammatory score. PKH2-dyed ADSCs were detected by fluorescence microscopy.Results and ConclusionsTotal colitis was induced in 19/20 rats with homing of fluorescent ADSCs. to the crypt base and perivascular space of the submucosa. There were no differences in component rectal wall thicknesses with a higher Hunter score in the treated group compared with the controls, in the rectum (3.8±2.74 vs. 1.5±2.37, respectively; p=0.017) and in right colon (2.5±1.08 vs. 0.20±0.42, respectively; p=0.0001). Local colonic injection of allogeneic adipose stem cells. in experimental colitis is feasible and safe. There is demonstrable homing of cells in chemically-induced colitis both to the treated region and parts of the colon distant to the MSC treatment site. Such cells readily proliferate in vitro and could potentially be a source for future treatment of resistant disease.

Paneth Cells during Viral Infection and Pathogenesis.

Paneth cells are major secretory cells located in the crypts of Lieberkühn in the small intestine. Our understanding of the diverse roles that Paneth cells play in homeostasis and disease has grown substantially since their discovery over a hundred years ago. Classically, Paneth cells have been characterized as a significant source of antimicrobial peptides and proteins important in host defense and shaping the composition of the commensal microbiota. More recently, Paneth cells have been shown to supply key developmental and homeostatic signals to intestinal stem cells in the crypt base. Paneth cell dysfunction leading to dysbiosis and a compromised epithelial barrier have been implicated in the etiology of Crohn’s disease and susceptibility to enteric bacterial infection. Our understanding of the impact of Paneth cells on viral infection is incomplete. Enteric α-defensins, produced by Paneth cells, can directly alter viral infection. In addition, α-defensins and other antimicrobial Paneth cell products may modulate viral infection indirectly by impacting the microbiome. Here, we discuss recent insights into Paneth cell biology, models to study their function, and the impact, both direct and indirect, of Paneth cells on enteric viral infection.

The Muc2 mucin coats murine Paneth cell granules and facilitates their content release and dispersion.

Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal crypts. Unlike intestinal goblet cells, which secrete the mucin Muc2, Paneth cells are best known for producing an array of antimicrobial factors. We unexpectedly identified Muc2 staining localized around Paneth cell granules. Electron microscopy (EM) confirmed an electron lucent halo around these granules, which was lost in Paneth cells from Muc2-deficient (-/-) mice. EM and immunostaining for lysozyme revealed that Muc2-/- Paneth cells contained larger, more densely packed granules within their cytoplasm, and we detected defects in the transcription of key antimicrobial genes in the ileal tissues of Muc2-/- mice. Enteroids derived from the small intestine of wild-type and Muc2-/- mice revealed phenotypic differences in Paneth cells similar to those seen in vivo. Moreover, lysozyme-containing granule release from Muc2-/- enteroid Paneth cells was shown to be impaired. Surprisingly, Paneth cells within human ileal and duodenal tissues were found to be Muc2 negative. Thus Muc2 plays an important role in murine Paneth cells, suggesting links in function with goblet cells; however human Paneth cells lack Muc2, highlighting that caution should be applied when linking murine to human Paneth cell functions. NEW & NOTEWORTHY We demonstrate for the first time that murine Paneth cell granules possess a halo comprised of the mucin Muc2. The presence of Muc2 exerts an impact on Paneth cell granule size and number and facilitates the release and dispersal of antimicrobials into the mucus layer. Interestingly, despite the importance of Muc2 in murine Paneth cell function, our analysis of Muc2 in human intestinal tissues revealed no trace of Muc2 expression by human Paneth cells.

Colorectal cancer: genetic abnormalities, tumor progression, tumor heterogeneity, clonal evolution and tumor-initiating cells.

Colon cancer is the third most common cancer worldwide. Most colorectal cancer occurrences are sporadic, not related to genetic predisposition or family history; however, 20–30% of patients with colorectal cancer have a family history of colorectal cancer and 5% of these tumors arise in the setting of a Mendelian inheritance syndrome. In many patients, the development of a colorectal cancer is preceded by a benign neoplastic lesion: either an adenomatous polyp or a serrated polyp. Studies carried out in the last years have characterized the main molecular alterations occurring in colorectal cancers, showing that the tumor of each patient displays from two to eight driver mutations. The ensemble of molecular studies, including gene expression studies, has led to two proposed classifications of colorectal cancers, with the identification of four/five non-overlapping groups. The homeostasis of the rapidly renewing intestinal epithelium is ensured by few stem cells present at the level of the base of intestinal crypts. Various experimental evidence suggests that colorectal cancers may derive from the malignant transformation of intestinal stem cells or of intestinal cells that acquire stem cell properties following malignant transformation. Colon cancer stem cells seem to be involved in tumor chemoresistance, radioresistance and relapse.