The Interplay between Wnt Mediated Expansion and Negative Regulation of Growth Promotes Robust Intestinal Crypt Structure and Homeostasis

The epithelium of the small intestinal crypt, which has a vital role in protecting the underlying tissue from the harsh intestinal environment, is completely renewed every 4–5 days by a small pool of stem cells at the base of each crypt. How is this renewal controlled and homeostasis maintained, particularly given the rapid nature of this process? Here, based on the recent observations from in vitro “mini gut” studies, we use a hybrid stochastic model of the crypt to investigate how exogenous niche signaling (from Wnt and BMP) combines with auto-regulation to promote homeostasis. This model builds on the sub-cellular element method to account for the three-dimensional structure of the crypt, external regulation by Wnt and BMP, internal regulation by Notch signaling, as well as regulation by internally generated diffusible signals. Results show that Paneth cell derived Wnt signals, which have been observed experimentally to sustain crypts in cultured organs, have a dramatically different influence on niche dynamics than does mesenchyme derived Wnt. While this signaling can indeed act as a redundant backup to the exogenous gradient, it introduces a positive feedback that destabilizes the niche and causes its uncontrolled expansion. We find that in this setting, BMP has a critical role in constraining this expansion, consistent with observations that its removal leads to crypt fission. Further results also point to a new hypothesis for the role of Ephrin mediated motility of Paneth cells, specifically that it is required to constrain niche expansion and maintain the crypt’s spatial structure. Combined, these provide an alternative view of crypt homeostasis where the niche is in a constant state of expansion and the spatial structure of the crypt arises as a balance between this expansion and the action of various sources of negative regulation that hold it in check

Concise Review: The Potential Use of Intestinal Stem Cells to Treat Patients With Intestinal Failure.

: Intestinal failure is a rare life-threatening condition that results in the inability to maintain normal growth and hydration status by enteral nutrition alone. Although parenteral nutrition and whole organ allogeneic transplantation have improved the survival of these patients, current therapies are associated with a high risk for morbidity and mortality. Development of methods to propagate adult human intestinal stem cells (ISCs) and pluripotent stem cells raises the possibility of using stem cell-based therapy for patients with monogenic and polygenic forms of intestinal failure. Organoids have demonstrated the capacity to proliferate indefinitely and differentiate into the various cellular lineages of the gut. Genome-editing techniques, including the overexpression of the corrected form of the defective gene, or the use of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to selectively correct the monogenic disease-causing variant within the stem cell, make autologous ISC transplantation a feasible approach. However, numerous techniques still need to be further optimized, including more robust ex vivo ISC expansion, native ISC ablation, and engraftment protocols. Large-animal models can to be used to develop such techniques and protocols and to establish the safety of autologous ISC transplantation because outcomes in such models can be extrapolated more readily to humans.The field of intestinal stem cell biology has exploded over the past 5 years with discoveries related to in vivo and in vitro stem cell identity and function. The goal of this review article is to highlight the potential use of these cells to treat various epithelial disorders of the gut and discuss the various roadblocks that will be encountered in the coming years

Wnt signalling in intestinal stem cells: lessons from mice and flies

Adult stem cells play critical roles in the basal maintenance of tissue integrity, also known as homeostasis, and in tissue regeneration following damage. The highly conserved Wnt signalling pathway is a key regulator of stem cell fate. In the gastrointestinal tract, Wnt signalling activation drives homeostasis and damage-induced repair. Additionally, deregulated Wnt signalling is a common hallmark of age-associated tissue dysfunction and cancer. Studies using mouse and fruit fly models have greatly improved our understanding of the functional contribution of the Wnt signalling pathway in adult intestinal biology. Here, we summarize the latest knowledge acquired from mouse and Drosophila research regarding canonical Wnt signalling and its key functions during stem cell driven intestinal homeostasis, regeneration, ageing and cancer

Role of NF-κB in autophagy-controlled inflammatory responses and in intestinal epithelial cell fate decisions

Es wird vermutet, dass das Zusammenspiel von NF-κB-Signalen und Autophagie die Entzündung in verschiedenen zellulären Kontexten und als Reaktion auf unterschiedliche Stimuli reguliert. Der molekulare Mechanismus, durch den diese beiden Signalwege bei der Regulierung der Entzündungsreaktion zusammenwirken, ist jedoch noch nicht bekannt. Mithilfe biochemischer Analysen und bildgebender Verfahren haben wir zum ersten Mal die Interaktion zwischen dem autophagischen Marker LC3 und der NF-κB/p65-Untereinheit als Reaktion auf verschiedene Stressbedingungen charakterisiert. Wir konnten zeigen, dass die Anhäufung von LC3 im Zellkern nach der NF-κB-Aktivierung mit p65 interagiert, was durch die Ubiquitinierung des p65-Proteins gefördert und durch den Cargo-Rezeptor p62 erkannt wird. Zusammengenommen weisen diese Daten auf eine neue Rolle von p62 beim Transport von im Kern ubiquitiniertem p65 zu Autophagosomen hin, wo es abgebaut wird, um die entzündungsbedingte NF-κB-Hyperaktivierung zu kontrollieren. Diese Erkenntnisse sind wichtig für die Entwicklung neuer therapeutischer Strategien gegen Krankheiten, die mit einer gestörten Autophagie und einer konstitutiven NF-κB-Aktivität einhergehen. Die NF-κB-Signalübertragung spielt nicht nur eine entscheidende Rolle bei Entzündungen und der Tumorbildung, sondern ist auch für Entwicklungsprozesse wichtig. Durch die Etablierung von 3D-Organoid-Kulturen aus dem Dünndarm und unter Verwendung verschiedener Mauslinien weisen wir im zweiten Teil der Arbeit nach, dass NF-κB eine wichtige Funktion bei der Zelldifferenzierung und der Erhaltung von Stammzellen in vivo und ex-vivo spielt. Wir konnten zeigen, dass die Proliferation und das Absterben von Darmepithelzellen (IEC) bei Mäusen mit ubiquitärer Unterdrückung der NF-κB-Aktivität unverändert sind, während die Zahl der Becherzellen auf Kosten der Paneth-Zellen zunimmt. Zusammenfassend lässt sich sagen, dass unsere Ergebnisse eine neue IEC-immanente Rolle von NF-κB bei Entscheidungen über das Zellschicksal und die Differenzierung aufzeigen, die über die Regulierung der Wnt-Signale und der Sox9-Expression stromabwärts von NF-κB erfolgt. Die hier beschriebenen Erkenntnisse verbessern unser Verständnis der NF-κB-Funktionen in der Stammzellbiologie, die, wenn sie dereguliert sind, auch Auswirkungen auf die Entzündung des Darms und die Tumorentstehung haben.The interplay between NF-κB signaling and autophagy has been suggested to regulate inflammation in different cellular contexts and in response to different stimuli. However, the molecular mechanism by which these two pathways interact to regulate the inflammatory response remains elusive. By using biochemical analysis and imaging techniques, we characterized for the first time the interaction of autophagic marker LC3 and NF-κB/p65 subunit in response to different stress conditions. We demonstrated that the accumulation of LC3 within the nucleus interacts with p65 following NF-κB activation, which is promoted by ubiquitination of p65 protein and recognized by the cargo receptor p62. Together, these data identify a novel role for p62 in trafficking nuclear-ubiquitinated p65 to autophagosomes for degradation to control inflammation-driven NF-κB hyperactivation. These findings are important for developing novel therapeutic strategies against diseases involving defective autophagy and constitutive NF-κB activity. In addition to its critical role in inflammation and tumor formation, NF-κB signaling is essential in developmental processes. Establishing 3D organoid culture from the small intestine and using different mouse lines, we prove in the second part of the thesis that NF-kB plays an important function in cell differentiation and stem cell maintenance in vivo and in ex-vivo. We demonstrated that while intestinal epithelial cell (IEC) proliferation and death are unaltered in mice with ubiquitous suppression of NF-κB activity, goblet cell numbers increase at the expense of Paneth cells. In summary, our results revealed a novel IEC-intrinsic role of NF-κB in cell fate decisions and differentiation which occur via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The findings described here improve our understanding of NF-κB functions in stem cell biology which, when deregulated, also have an impact on intestinal inflammation and tumorigenesis

Colorectal Cancer Through Simulation and Experiment

Colorectal cancer has continued to generate a huge amount of research interest over several decades, forming a canonical example of tumourigenesis since its use in Fearon and Vogelstein’s linear model of genetic mutation. Over time, the field has witnessed a transition from solely experimental work to the inclusion of mathematical biology and computer-based modelling. The fusion of these disciplines has the potential to provide valuable insights into oncologic processes, but also presents the challenge of uniting many diverse perspectives. Furthermore, the cancer cell phenotype defined by the ‘Hallmarks of Cancer’ has been extended in recent times and provides an excellent basis for future research. We present a timely summary of the literature relating to colorectal cancer, addressing the traditional experimental findings, summarising the key mathematical and computational approaches, and emphasising the role of the Hallmarks in current and future developments. We conclude with a discussion of interdisciplinary work, outlining areas of experimental interest which would benefit from the insight that mathematical and computational modelling can provide

Deciphering the complex signalling systems that regulate intestinal epithelial cell death processes and shedding

Intestinal epithelial cells play a fundamental role in maintaining homeostasis. Shedding of intestinal cells in a controlled manner is critical to maintenance of barrier function. Barrier function is maintained during this shedding process by a redistribution of tight junctional proteins to facilitate closure of the gap left by the shedding cell. However, despite the obvious importance of epithelial cell shedding to gut health a central question is how the extrusion of epithelial cells is achieved, enabling barrier integrity to be maintained in the healthy gut and restored during inflammation remains largely unanswered. Recent studies have provided evidence that excessive epithelial cell shedding and loss of epithelial barrier integrity is triggered by exposure to lipopolysaccharide (LPS) or tumour necrosis factor (TNF). Subsequent studies have provided evidence of the involvement of specific cellular components and signalling mechanisms as well as the functionality of microbiota that can be either detrimental or beneficial for intestinal barrier integrity. This review, will focus on the evidence and decipher how the signalling systems through which the mucosal immune system and microbiota can regulate epithelial cell shedding and how these mechanisms interact to preserve the viability of the epithelium

Characterisation of paracrine Wnt regulators in colorectal cancer microenvironment

Aberrant Wnt activation is commonly found in colorectal cancer (CRC), especially in cells adjacent to the tumour microenvironment (TME). This indicates the crucial interaction between tumour cells and their niche. Comprehensive transcriptomic analyses by the Cancer Genome Atlas project show genes that are differentially upregulated in tumour stromal population. Of these, a cluster of genes includes markers of cancer-associated fibroblasts (CAFs) that are associated with poor survival. I hypothesise that a subset of CRCs is dependent on paracrine Wnt activation from the surrounding CAFs for tumour progression. PERIOSTIN (POSTN) is a matricellular protein that is often upregulated in CAFs and is associated with poor CRC prognosis. Human primary fibroblasts established from CRC tissues are found to retain transcriptomic and functional profiles that are distinct between normal and cancer-associated populations in vitro, including POSTN upregulation in CAFs. In vitro gain-of-function studies show that POSTN potentiates Wnt activation in Wnt ligand-dependent manner, and promotes Wnt receptor stabilisation via yet unclear mechanism(s) interacting with Wnt components on cell membrane. To better study paracrine Wnt activation, a 3D model is developed in which intestinal epithelial cells reside on top of fibroblasts. Co-culture of normal small intestinal epithelial cells with normal fibroblasts show an organised, differentiated crypt/villi-like structure of the epithelial layer, whereas disorganised multi-layered epithelial cells with altered polarity are observed with CAFs. Moreover, the contribution of CAF-derived POSTN in tumour progression is assessed utilising the 3D model system and in vivo xenograft formation approaches. This project demonstrates POSTN is upregulated in CRC and acts as a potential CAF-derived Wnt regulator, providing insight into a novel Wnt regulation that may be at play in CRC tumour progression. The tissue-engineered 3D co-culture system will be a useful tool to study different cell type interactions without sacrificing biomimetic organisation by providing a simple and malleable system

Elucidating the Complex Signaling Events Driving Intestinal Stem Cell Plasticity Following Injury

Signaling events governing intestinal stem cell (ISC) homeostasis maintain the delicate balance of active self-renewal and passive differentiation to replenish intestinal epithelial cells (IEC) every 3-5 days. However, under certain contexts, ISC function is irreversibly compromised—requiring committed IEC lineages to dedifferentiate and regain “stemness”. In the current studies, we examine the signaling events driving epithelial cell responses to injury to expose pathologic failures in the healing response. Our specific goal is to tease out the cellular contexts that promote dedifferentiation to design effective therapeutics for disease conditions compromising ISC function. First, we generated a novel transgenic animal using the epithelial-specific Villin promoter to constitutively ablate the critical negative regulator in the Phosphotidyl-inositol 3 Kinase (PI3K) signaling pathway, p85α, in IECs. At baseline, removal of p85α led to increased activation of PI3K/Akt, as seen through elevations in phosphorylated PTEN, Akt, and GSK3β. The consequence of the persistent activation was a shifting of cells out of the stem cell niche, into more differentiated the proliferative progenitor pool, as seen by a decrease in Lgr5+ cells per crypt and an increase in Axin2+ transcripts by in situ hybridization. Further investigations revealed PI3K activation led to a bolstered secretory lineage, increased total number and size of Paneth cells within the crypt through reduced Notch signaling. Subsequent studies mechanistically clarify mitochondrial bioenergentics in p85-deficient epithelial cells demonstrate enhanced State III respiration to drive production of ROS. ROS activates p38-MAPK to mount observed differentiation pressure. Using whole-body irradiation to target ISCs, we revealed that p85-deficient crypts underwent restitution at a faster pace than their control counterparts. The consequence of the enhanced regeneration was an increased organism survival after lethal irradiation. Transcript flux revealed that p85-deficient crypts increased Wnt and PI3K signaling targets, specifically Lgr5, Axin2 and Survivin earlier in the regenerative process, suggesting the increased secretory progenitor pool could enact dedifferentiation mechanisms to replenish ISCs. Using human samples of radiation-induced intestinal injury, we demonstrate the requirement of Wnt-target Survivin protein expression in IEC survival, suggesting a conserved mechanism and possible avenue for future therapeutic intervention. Next, using isolated crypt epithelial cells from patients with inflammatory bowel disease (IBD), we establish that steroid-treated (prednisone) IBD patients harbor aberrant Wnt/β-catenin and NFκB signaling in IECs despite clinical improvement. At the molecular level, human IECs had significantly blunted cytosolic accumulation of Axin2 protein and subsequently decreased nuclear localization of downstream transcriptional activator, p-β-cateninSer552. Using a validated murine model of IBD, Dextran Sodium Sulfate-induced colitis (DSS colitis), we appreciated conserved blunting of inflammation-induced Wnt activation following dexamethasone administration. Studies in isolated murine ISC cultures revealed that the blunting of Wnt activation occurred in the absence of inflammatory stimulus—suggesting steroids directly affect ISC activation by interfering with β-catenin transcriptional activity. By tracking β-catenin-directed TCF/LEF transcriptional activity with a stably-transfected luciferase construct, we identified the signaling disruption occurred downstream of the β-catenin destruction complex, and resulted in limited ISC activation and mobilization in response to injury. Appropriately, primary ISC cultures from mice demonstrated that at high doses, steroid therapy inappropriately limits ISC activation sufficient Wnt ligands in culture. This leads to a depleted ISC pool and preventing mucosal restitution after injury

A compact fiber-optic probe-based singlet oxygen luminescence detection system

This paper presents a novel compact fiberoptic basedsinglet oxygen near-infrared luminescence probe coupledto an InGaAs/InP single photon avalanche diode (SPAD)detector. Patterned time gating of the single-photon de-tector is used to limit unwanted dark counts and eliminatethe strong photosensitizer luminescence background.Singlet oxygen luminescence detection at 1270 nm is con-firmed through spectral filtering and lifetime fitting forRose Bengal in water, and Photofrin in methanol as mod-el photosensitizers. The overall performance, measuredby the signal-to-noise ratio, improves by a factor of 50over a previous system that used a fiberoptic-coupledsuperconducting nanowire single-photon detector. Theeffect of adding light scattering to the photosensitizer isalso examined as a first step towards applications in tissuein vivo