Intact Crypts Research Articles

IL-27 induces an IFN-like signature in murine macrophages which in turn modulate colonic epithelium.

Mucosal delivery of IL-27 has been shown to have a therapeutic benefit in murine models of inflammatory bowel disease (IBD). The IL-27 effect was associated with phosphorylated STAT1 (pSTAT1), a product of IL27 receptor signaling, in bowel tissue. To determine whether IL-27 acted directly on colonic epithelium, murine colonoids and primary intact colonic crypts were shown to be unresponsive to IL-27 in vitro and to lack detectable IL-27 receptors. On the other hand, macrophages, which are present in inflamed colon tissue, were responsive to IL-27 in vitro. IL-27 induced pSTAT1 in macrophages, the transcriptome indicated an IFN-like signature, and supernatants induced pSTAT1 in colonoids. IL-27 induced anti-viral activity in macrophages and MHC Class II induction. We conclude that the effects of mucosal delivery of IL-27 in murine IBD are in part based on the known effects of IL27 inducing immunosuppression of T cells mediated by IL-10. We also conclude that IL-27 has potent effects on macrophages in inflamed colon tissue, generating mediators that in turn act on colonic epithelium.

Calmodulin Antagonist W-7 Enhances Intermediate Conductance Ca2+- Sensitive Basolateral Potassium Channel (IKCa) Activity in Human Colonic Crypts

Intermediate conductance potassium (IKCa) channels are exquisitively Ca2+ sensitive, intracellular Ca2+ regulating channel activity by complexing with calmodulin (CaM), which is bound to the cytosolic carboxyl tail. Although CaM antagonists might be expected to decrease IKCa channel activity, the effect of W-7 in human T lymphocytes are conflicting. We therefore evaluated the effect of W-7 on basolateral IKCa channels in human colonic crypt cells. Intact crypts obtained from normal human colonic biopsies by Ca2+ chelation were used for patch clamp studies of basolateral IKCa channels in the cell-attached configuration. IKCa channel activity was studied when the bath Ca2+ concentration was changed from 1.2 mmol/L to 100 μmol/L and back to 1.2 mmol/L, as well as from 100 μmol/L to 1.2 mmol/L and back to 100 μmol/L, both in the absence and presence of 25 μmol/L W-7. Decreasing bath Ca2+ from 1.2 mmol/L to 100 μmol/L decreased IKCa channel activity reversibly in the absence of W-7, whereas there was a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. In separate experiments, increasing bath Ca2+ from 100 μmol/L to 1.2 mmol/L increased IKCa channel activity reversibly in the absence of W-7, whereas there was again a uniformly high level of channel activity at both bath Ca2+ concentrations in the presence of W-7. We, therefore, propose that W-7 has a specific stimulatory effect on basolateral IKCa channel activity, despite its ability to inhibit Ca2+/CaM-mediated, IKCa channel-dependent Cl− secretion in human colonic epithelial cells.Graphic

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca2+-and voltage-activated K+ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2- cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

734 Colonic Biopsy Pyroptosis Predicts Clinical Response to Vedolizumab in Crohn's Disease

INTRODUCTION: Loss of intestinal barrier integrity plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD). Pyroptosis, an innate immune-mediated form of inflammatory cell death of intestinal epithelial cells (IECs) that can be quantified, is a potential cause of mucosal barrier dysfunction in IBD. We have recently shown that ileal IEC pyroptosis level was predictive of clinical response to vedolizumab in Crohn's disease (CD) patients. Ileocecal valve disease and strictures can make it difficult to obtain ileal biopsies in some CD patients. Correlation of pyroptosis levels between ileal and colon biopsies is unknown. Our aims were to determine the predictive value of pre-treatment colonic biopsy IEC pyroptosis for clinical response to vedolizumab in CD patients and to assess the correlation between colonic and ileal biopsy pyroptosis levels. METHODS: CD patients from 5 IBD centers with pre-treatment ileal and colonic biopsies obtained were enrolled. The endpoint of clinical response, defined as reduction in Harvey-Bradshaw Index of ≥5 points, was assessed ≥6 months post-treatment. Biopsies were sectioned and stained, and samples with a minimum of 10 intact crypts were analyzed by a blinded pathologist. Pyroptosis was quantified as the number of counted cells staining for activated caspase-1 normalized per 1000 cells. The primary outcome was comparison of clinical response rates to vedolizumab stratified by pre-treatment colonic IEC pyroptosis using Fischer's exact test. The secondary outcome was correlation between ileal and colonic samples using Spearman's correlation. RESULTS: Fifty-five CD patients were enrolled. Non-responder and responder groups differed significantly only with respect to prior anti-TNF exposure and immunomodulator use (Table 1). The overall clinical response to vedolizumab was 62% (34/55). Using a threshold level of 30 positive cells/1000 IECs, the clinical response rate in patients with pyroptosis below this was significantly higher compared to those above the threshold: 69% (31/45) vs. 30% (3/10), OR 5.2 (1.2, 23.0), P = 0.031. The correlation between ileal and colon biopsy pyroptosis levels was poor (rho = 0.08, P = 0.56). CONCLUSION: Pre-treatment colon pyroptosis level was predictive of clinical response to vedolizumab therapy in CD patients, independent of ileal biopsy pyroptosis levels.

Effects of watermelon powder supplementation on colitis in high-fat diet-fed and dextran sodium sulfate-treated rats

Abstract The objective of this study was to determine the effects of watermelon on colitis in rats treated with Dextran Sodium Sulfate (DSS) and high-fat diet. We hypothesized that watermelon would reduce colitis severity by maintaining the number of intact crypts and regulating cell homeostasis. Forty rats were divided into four groups: control diet, control diet + DSS, 0.33% watermelon powder diet, and 0.33% watermelon powder diet + DSS. After 4 weeks of defined diets, DSS group rats were administered 3% (w/v) DSS (40 kDa) in their drinking water for 2 days. DSS administration increased colonocyte proliferation and apoptosis while decreasing cell differentiation and the number of intact colonic crypts (P

Abstract 4990: Regenerative origin of colorectal metastasis stem cells

Abstract Metastatic cancers invariably relapse due to the emergence of resistant tumor clones capable of self-renewal, entry into and exit from quiescence, tumor re-initiation and therapy resistance. The origins of such metastasis propagating cells (MPCs), which ultimately cause cancer death, are not well-understood. To directly scrutinize MPCs in patient metastases, we established ex vivo organoid cultures from surgically resected, chemoresistant residual colorectal cancer (CRC) liver metastases. We show that the neuronal cell-adhesion molecule L1CAM, which is ectopically expressed in many cancer types and strongly associated with poor prognosis, is a marker of MPCs. L1CAM+ cells are largely quiescent in structured neoplastic glands in tumors, but when dissociated from their epithelial niche, proliferate to regenerate heterogeneous organoids or xenografts containing both L1CAM+ and L1CAM- progeny. To define the relationship between L1CAM+ MPCs and Lgr5+ intestinal stem cells, we performed single cell mRNA sequencing on ~15,000 CRC organoid-derived cells from four patients. We identified only partial overlap between Lgr5+ and L1CAM+ cells. Lgr5high cells consistent with homeostatic stem cells, have low L1CAM levels, while Lgr5low transit amplifying progenitor-like cells have high L1CAM levels. In addition, we identify a separate population of L1CAMhighLgr5- cells. The data suggest that human CRC metastases are derived from an L1CAM+ population of transit-amplifying, partially differentiated cells. L1CAM is not expressed in intact human or mouse intestinal crypts during homeostasis. However, when the intestinal epithelium is disrupted by dextran sodium sulfate-mediated colitis, L1CAM is strongly induced in cells in the middle of regenerating crypts. Intestinal epithelium specific deletion of L1CAM causes profound weight loss, poor tissue healing and reduces survival in DSS-treated mice. In turn, L1CAM knockdown/knockout in mouse or human CRC cells inhibits regeneration of organoids in vitro, subcutaneous tumors and orthotopic liver metastases in vivo. Mechanistically, L1CAM RNA expression is normally silenced in non-neuronal cells by the transcriptional repressor REST. We show that disruption of epithelial integrity by organoid dissociation or E-cadherin knockdown reduces REST binding to an L1CAM intronic enhancer, thus inducing L1CAM expression. Our results suggest that L1CAM is dispensable for epithelial homeostasis, but is required for normal and neoplastic epithelial regeneration when tissue integrity is disrupted. During cancer progression, disseminated tumor cells at the invasion front of primary tumors, in the circulation, or in isolated residual disease following therapy, induce and depend on L1CAM for survival and eventual regrowth. Thus, L1CAM represents a crucial vulnerability of disseminated and residual MPCs that could be exploited therapeutically to treat patients with metastatic cancer. Citation Format: Karuna Ganesh, Harihar Basnet, Kevin P. O'Rourke, Ashley M. Laughney, Lan He, Eduard Batlle, Scott W. Lowe, Dana Pe'er, Jinru Shia, Joan Massague. Regenerative origin of colorectal metastasis stem cells [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 4990.

Tu1389 Increased Basolateral Small Conductance Potassium Channel (KCNQ1/ KCNE3) Activity in Ulcerative Colitis

Introduction and Aims: Basolateral K+ channels keep colonic cells in a hyperpolarized state to ensure transcellular ionic movements. Intermediate conductance K+ (IK) channels dominate the basolateral membrane conductance in human colonic cells, but their expression and activity are markedly decreased in ulcerative colitis (UC), thus contributing to defective Na+ and water absorption across inflamed mucosa (J Pathol 2012;226:463-470). This study is the first to characterize basolateral small conductance (KCNQ1/KCNE3) K+ channels in normal human colon, and explore their activity in UC. Methods: Intact crypts were isolated from distal colonic biopsies taken while investigating patients with functional diarrhea (normals) and patients with UC not receiving steroids. Expression of KCNQ1 and KCNE3 mRNAs was determined by qPCR. Patch clamp recording was used to study (i) basolateral KCNQ1/KCNE3 channel activity in normal and UC crypt cells and (ii) in normal crypts cells, the effects of forskolin (an activator of adenylate cyclase) and inflammatory mediators present at high concentrations in UC. Results: qPCR product bands were of the expected size (KCNQ1 160 bp, KCNE3 119 bp, β-actin 148 bp). Product identity was confirmed by automated sequencing and melting curve analysis, and KCNQ1 and KCNE3 mRNA expression was similar in normal and UC patients. In normal crypts (n=34), usually single channels (conductance 6.8 ± 0.5 pS) were seen in 36% of patches, whereas in UC crypts (n=9), two or more channels were seen in 74% of patches, the difference in channel prevalence between the two groups being significant (P<0.001). Overall channel activity was 10-fold greater in UC than control patients (P<0.001). These channels constituted a greater calculated basolateral conductance (GSK) in UC (5.85 ± 0.62 mS.cm-2) than in control patients (0.28 ± 0.04 mS.cm-2, P<0.001), but this increase in GSK did not compensate for the decrease in basolateral membrane conductance (from 22 mS.cm-2 to 5 mS.cm-2) previously attributed to decreased IK channel expression/activity in UC (J Pathol 2012;226:463-470). In control crypts, 10μM forskolin and 1μM thromboxane A2 stimulated channel activity 30-fold (n=8, P<0.005) and 10-fold (n=5, P<0.05) respectively, while 100nM PGE2, 10nM IL-1β and 1μM LTD4 had no effect. Conclusions: Crypt cells in normal human colon express cAMP-activated KCNQ1/ KCNE3 channels which make a far smaller contribution to overall basolateral conductance than IK channels. In active UC, KCNQ1/KCNE3 channel activity is significantly increased, but this fails to compensate for the depolarizing effect of decreased IK channel expression/ activity. This may explain, at least in part, why defective electrogenic Na+ absorption rather than increased electrogenic Cl secretion is the dominant pathophysiological mechanism of diarrhea in active UC (J Pathol 2012;226:463-470).

A human colonic crypt culture system to study regulation of stem cell-driven tissue renewal and physiological function.

The intestinal epithelium is one of the most rapidly renewing tissues in the human body and fulfils vital physiological roles such as barrier function and transport of nutrients and fluid. Investigation of gut epithelial physiology in health and disease has been hampered by the lack of ex vivo models of the native human intestinal epithelium. Recently, remarkable progress has been made in defining intestinal stem cells and in generating intestinal organoid cultures. In parallel, we have developed a 3D culture system of the native human colonic epithelium that recapitulates the topological hierarchy of stem cell-driven tissue renewal and permits the physiological study of native polarized epithelial cells. Here we describe methods to establish 3D cultures of intact human colonic crypts and conduct real-time imaging of intestinal tissue renewal, cellular signalling, and physiological function, in conjunction with manipulation of gene expression by lentiviral or adenoviral transduction. Visualization of mRNA- and protein-expression patterns in cultured human colonic crypts, and cross-validation with crypts derived from fixed mucosal biopsies, is also described. Alongside studies using intestinal organoids, the near-native human colonic crypt culture model will help to bridge the gap that exists between investigation of colon cancer cell lines and/or animal (tissue) studies, and progression to clinical trials. To this end, the near native human colonic crypt model provides a platform to aid the development of novel strategies for the prevention of inflammatory bowel disease and cancer.

Rice prolamin extract ameliorates acute murine colitis by inhibiting nuclear factor-kappa B and modulating intestinal apoptosis and cell proliferation.

We investigated the impact of rice prolamin extract (RPE) on lipopolysaccharide (LPS)-induced nuclear factor (NF)-κB signalling in intestinal epithelial cells and macrophages, and determined the therapeutic efficacy of RPE in acute murine colitis. The effect of RPE on LPS-induced NF-κB signalling and proinflammatory gene expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, immunofluorescence and electrophoretic mobility shift assay (EMSA). The in-vivo efficacy of RPE was assessed in mice with 3% dextran sulphate sodium (DSS)-induced colitis. Apoptotic and cellular proliferative activities were evaluated by immunostaining with cleaved caspase-3 and proliferating cell nuclear antigen (PCNA) antibodies. RPE inhibited LPS-induced expression of monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and LPS-induced NF-κB signalling in intestinal epithelial cells and macrophages. RPE-fed, DSS-exposed mice showed less weight loss, longer colon length and lower histological score compared to control diet-fed, DSS-exposed mice. Immunostaining analysis revealed a significant decrease of cleaved caspase-3 positive cells in RPE-fed, DSS-exposed mice compared to DSS-exposed mice. Also, the number of PCNA-positive cells within intact colonic crypts decreased significantly in RPE-fed, DSS-exposed mice compared to control diet-fed, DSS-exposed mice. DSS-induced NF-κB signalling was inhibited by RPE. RPE ameliorates intestinal inflammation by inhibiting NF-κB activation and modulating intestinal apoptosis and cell proliferation in an acute murine colitis.

Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived

In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic epithelium.

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Separation of Intact Intestinal Epithelium from Mesenchyme

Current protocols for separating adult intestinal epithelial cells from the underlying muscular and mesenchymal tissues typically involve extended incubations, harsh mechanical treatment, and exposure to either proteases or chelating agents. The drawbacks of these approaches include fragmentation, contamination with other cell types, reduced viability, and under-representation of crypt cells. Here we describe a gentle procedure that allows harvesting of pure, fully viable sheets of murine intestinal epithelium, with intact crypts and villi, without enzymes or EDTA. The mesenchyme retains intact villus core projections, is virtually free from epithelial cells, and can be cultured in vitro.

Evidence that two distinct crypt cell types secrete chloride and potassium in human colon

BackgroundHuman colon may secrete substantial amounts of water secondary to chloride (Cl−) and/or potassium (K+) secretion in a variety of diarrhoeal diseases. Ion secretion occurs via Cl− and K+ channels,...

Abstract 3711: Human colon mucosal crypts in culture: a model to assess colon chemoprevention.

Abstract Introduction: In order to advance a primary cell culture model of human colonic carcinogenesis we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomatous polyps. Methods: Fresh biopsy samples were transported in culture medium containing 1.5mM Ca2+ and antimicrobials. The mucosa/submucosa was surgically separated from the muscularis propria and incubated in 10mM dithiothreitol followed by 8mM ethylenediaminetetraacetic acid. Crypts were agitated free, washed with slow spins, and immediately embedded in a 3-dimensional environment at a density of 50 crypts/50μl Matrigel. After 3 hours, media was replaced to remove non-viable cellular products. Serum-free medium, containing low Ca2+ (0.15mM), was used to maintain crypt cultures. Cell proliferation and differentiation were assessed with markers Ki67 and E-cadherin by confocal microscopy. The fluorescence signal was quantified for the bottom 1/3 of the crypts (35 cells from base on one crypt edge) using morphometric imaging software. In parallel studies, abnormal crypt-like structures were isolated from adenomatous polyps/adenocarcinoma and maintained in a similar culture system. Results: At day-1 in culture, Ki67 expression was strong in the proliferating cells at the crypt base, with E-cadherin staining increasing towards the crypt apex. By day-3, E-cadherin expression became much more prominent and Ki67 decreased dramatically. This culture system preserved the in vivo phenotype of the crypts and allowed for the colonic epithelial cells to follow the physiological sequence of progression from proliferation to differentiation. Adenomatous tissue in culture at day-1 showed expression for Ki67 and E-cadherin but lacked spacial organization. Conclusion: Intact colonic crypts from normal and abnormal human mucosa can be viably maintained in 3-D culture for 72 hours. This primary human in vitro 3-D model may be used to model efficacy of colonic cancer chemopreventives. Epithelial Cell Proliferation and Differentiation in Cultured Human Crypts (fold changes) Markers Zero-time 24 hours 48 hours 72 hours Ki67 1.00 0.36 0.18 0.00 E-cadherin 1.00 2.00 2.89 3.22 Citation Format: Michael K. Dame, Yan Jiang, Danielle Kim Turgeon, Henry Appelman, Muhammad N. Aslam, Kelly Copley, Durga Attili, Dean E. Brenner, James Varani. Human colon mucosal crypts in culture: a model to assess colon chemoprevention. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3711. doi:10.1158/1538-7445.AM2013-3711

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens.

Immunolabelling of Thin Slices of Mouse Descending Colon and Jejunum

This protocol describes a method for efficient immunolabelling of thin tissue slices containing a few rows of intact intestinal crypts, which yields large numbers of them being oriented favorably for recording stacks of optical sections aligned with the crypt long axis (Bellis et al., 2012). The latter can then be used for cell positional analysis, 3D-reconstruction and -analysis. The simple epithelium lining the small intestine is organized into contiguous crypts of Lieberk&#252;hn (Potten, 1998; Barker et al., 2012; De Mey and Freund, 2013) several of which making up a crypt/villus unit. Each crypt is a multicellular proliferation unit with a tight hierarchical organization. Under steady state conditions, the epithelium is continuously and rapidly renewed, driven by divisions of multipotent intestinal SCs near the crypt base and cell removal from the villus tip. Techniques for analyzing the organization of the crypts play an important role in the field. Maximal efficiency is obtained by using optical sections obtained from confocal scanning and/or Nomarski optics passing through the center of the longitudinal crypt axis to view the crypt as two cell columns of hierarchical lineage starting from cells positioned at or near the crypt base. This enables positional analysis of certain cellular capacities like performing DNA synthesis, undergoing mitosis and apoptosis (Caldwell et al., 2007; Fleming et al., 2007; Quyn et al., 2010), responding to injury (Potten et al., 1997), or expressing genes (Barker et al., 2012; Bjerknes et al., 2012; Itzkovitz et al., 2012). Our protocol has allowed us to demonstrate that some divisions are asymmetric with respect to cell fate and the occurrence of oriented cell division (OCD) in 80% of the proliferating cells in the upper stem cell and transit amplifying zones. It has further revealed planar cell polarities which are important for crypt homeostasis and stem cell biology and alterations in apparently normal crypts and microadenomas of mice carrying germline Apc mutations shedding new light on the first stages of progression towards colorectal cancer.

Metabolic trajectory of cellular differentiation in small intestine by Phasor Fluorescence Lifetime Microscopy of NADH

There is a lack of fast and high resolution methods to measure metabolic activity of single cells in their native environment. Here we develop a straightforward, non-invasive and sensitive method to measure metabolic phenotype of single cells in a live tissue. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cellular metabolic fingerprints related to different rates of oxidative phosphorylation and glycolysis. For the first time we measure a three dimensional metabolic gradient in the small intestine (SI) epithelia that appears tightly associated with epithelial cell proliferation, differentiation and the Wnt gradient. The highest free/bound NADH ratios are measured at the base of the crypt within the highly proliferative stem cells, indicating high levels of glycolysis. For the first time mouse small intestinal stem cells in intact live crypts are identified within the tissue by their metabolic fingerprint.

Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells

Chemoprotective effects of nutritional compounds are usually studied in cell lines. Studies using primary human colon cells have been limited due to the lack of established methods regarding their culture. We therefore optimized isolation and culture of non-transformed human epithelial cells from individual donors to enrich viable cells and sufficient amounts of intact RNA. Isolated epithelial cells were seeded in different coated cell culture dishes combined with several media (2–24h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24h) derived from indigestible carbohydrates. In none of the coated (fibronectin, laminin) cell culture dishes isolated epithelial cells did attach. The number of these cells remaining in suspension, decreased already after 2h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24h (91±4%) and were suitable to gain a sufficient quantity of RNA. The use of colon crypts with an appropriate cell culture medium could double the lifespan of intestinal epithelial cells from 12 up to 24h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon.

Basolateral membrane Cl conductance in guinea pig distal colon activates during electrogenic K secretion and Cl secretion

Epinephrine (epi) activates transient Cl secretion (sec) and sustained K sec across isolated distal colonic mucosa of guinea pig. The Ca-activated Cl channel inhibitor CaCCinh-A01 [30μM] reduced electrogenic K sec, detected as short-circuit current (Isc), consistent with basolateral Cl channels providing an exit pathway for Cl entering via Na/K/2Cl-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl channel inhibitor, also reduced K secretory Isc (IC50 = 9.4μM). Epi activated whole-cell Cl current in isolated intact colonic crypts. This epi-activated Cl current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K sec, CaCCinh-A01 augmented epi-activated Cl secretory Isc as well as PGE2-activated Cl sec. Synergistic Cl sec activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of Tmem16A, a Ca-activated Cl channel, was supported by RT-PCR of Tmem16A-mRNA, immuno-blot with Tmem16A-antibodies, and immuno-reactivity in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons involved in channel activation. Inhibition of K sec and augmentation of Cl sec by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes with basolateral membrane Cl channels that contribute to production of electrogenic K sec and limiting Cl sec. Maximal Cl sec occurs only for synergistic activation mechanisms that inactivate these basolateral membrane Cl channels. [NIH DK65845]

Activation of the basolateral membrane Cl−conductance essential for electrogenic K+secretion suppresses electrogenic Cl−secretion

Adrenaline activates transient Cl(-) secretion and sustained K(+) secretion across isolated distal colonic mucosa of guinea-pigs. The Ca(2+)-activated Cl(-) channel inhibitor CaCCinh-A01 (30 μm) significantly reduced electrogenic K(+) secretion, detected as short-circuit current (I(sc)). This inhibition supported the cell model for K(+) secretion in which basolateral membrane Cl(-) channels provide an exit pathway for Cl(-) entering the cell via Na(+)-K(+)-2Cl(-) cotransporters. CaCCinh-A01 inhibited both I(sc) and transepithelial conductance in a concentration-dependent manner (IC(50) = 6.3 μm). Another Cl(-) channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I(sc) (IC(50) = 9.4 μm). Adrenaline activated whole-cell Cl(-) current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl(-) current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K(+) secretion, CaCCinh-A01 augmented the electrogenic Cl(-) secretion activated by adrenaline as well as that activated by prostaglandin E(2). Synergistic Cl(-) secretion activated by cholinergic/prostaglandin E(2) stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca(2+)-activated Cl(-) channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K(+) secretion and augmentation of Cl(-) secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl(-) channels contributes to the production of electrogenic K(+) secretion and limits the rate of Cl(-) secretion. Maximal physiological Cl(-) secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl(-) channels.