The intestinal epithelial stem cell.

This article considers the role of the adult epithelial stem cell, with particular reference to the intestinal epithelial stem cell. Although the potential of adult stem cells has been revealed in a number of recent publications, the organization and control of the stem cell hierarchy in epithelial tissues is still not fully understood. The intestinal epithelium is an excellent model in which to study such hierarchies, having a distinctive polarity and high rate of cell proliferation and migration. Studies on the small intestinal crypt provide insight into the characteristics of the stem cells in normal and regenerating circumstances and demonstrate why a thorough understanding of these cells is an essential pre-requisite for stem cell based therapeutic approaches.     

On the utility of a compartmental population kinetics model of intestinal epithelial stem cell proliferation and differentiation

Background

The small intestinal epithelium is a dynamic system with specialized cell types. The various cell populations of this tissue are continually renewed and replenished from stem cells that reside in the small intestinal crypt. The cell types and their locations in the crypt and villus are well known, but the details of the kinetics of stem cell division, and precursor cell proliferation and differentiation into mature enterocytes and secretory cells are still being studied. These proliferation and differentiation events have been extensively modeled with a variety of computational approaches in the past.

Methods

A compartmental population kinetics model, incorporating experimentally measured proliferation rates for various intestinal epithelial cell types, is implemented for a previously reported scheme for the intestinal cell dynamics. A sensitivity analysis is performed to determine the effect that varying the model parameters has upon the model outputs, the steady-state cell populations.

Results

The model is unable to reproduce the experimentally known timescale of renewal of the intestinal epithelium if literature values for the proliferation rates of stem cells and transit amplifying cells are employed. Unphysically large rates of proliferation result when these parameters are allowed to vary to reproduce this timescale and the steady-state populations of terminally differentiated intestinal epithelial cells. Sensitivity analysis reveals that the strongest contributor to the steady-state populations is the transit amplifying cell proliferation rate when literature values are used, but that the differentiation rate of transit amplifying cells to secretory progenitor cells dominates when all parameters are allowed to vary.

Conclusions

A compartmental population kinetics model of proliferation and differentiation of cells of the intestinal epithelium can provide a simplifying means of understanding a complicated multistep process. However, when literature values for proliferation rates of the crypt based columnar and transit amplifying cell populations are employed in the model, it cannot reproduce the experimentally known timescale of intestinal epithelial renewal. Nevertheless, it remains a valuable conceptual tool, and its sensitivity analysis provides important clues for which events in the process are the most important in controlling the steady-state populations of specialized intestinal epithelial cells.

     

STORM: a general model to determine the number and adaptive changes of epithelial stem cells in teleost, murine and human intestinal tracts

Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0-4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0-4.1 stem cells on each crypt section of murine small intestine and 1.8-3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.     

Radiation, the ideal cytotoxic agent for studying the cell biology of tissues such as the small intestine

Epithelial tissues are highly polarized, with the proliferative compartment subdivided into units of proliferation in many instances. My interests have been in trying to understand how many cellular constituents exist, what their function is, and what the intercommunicants are that ensure appropriate steady-state cell replacement rates. Radiation has proven to be a valuable tool to induce cell death, reproductive sterilization, and regenerative proliferation in these systems, the responses to which can provide information on the number of regenerative cells (a function associated with stem cells). Such studies have helped define the epidermal proliferative units and the structurally similar units on the dorsal surface of the tongue. The radiation responses considered in conjunction with a wide range of cell kinetic, lineage tracking and somatic mutation studies together with complex mathematical modeling provide insights into the functioning of the proliferative units (crypts) of the small intestine. Comparative studies have then been undertaken with the crypts in the large bowel. In the small intestine, in which cancer rarely develops, various protective mechanisms have evolved to ensure the genetic integrity of the stem cell compartment. Stem cells in the small intestinal crypts are intolerant of genotoxic damage (including that induced by very low doses of radiation); they do not undergo cell cycle arrest and repair but commit an altruistic TP53-dependent cell suicide (apoptosis). This process is compromised in the large bowel by BCL2 expression. Recent studies have suggested a second genome protection mechanism operating in the stem cells of the small intestinal crypts that may also have a TP53 dependence. Such studies have allowed the cell lineages and genome protection mechanisms operating the small intestinal crypts to be defined.     

Live and let die in the intestinal epithelium

The intestinal epithelium is a relatively simple developmental system and a prime example of tissue renewal from a source of multipotent stem cells. Throughout adulthood, intestinal epithelial proliferation, cell-fate specification and differentiation are coupled to migration in discrete units known as crypts of Lieberkühn. Physically guided by Eph receptors and their ligands, the ephrins, stem cell progeny transit through the proliferation/differentiation switch, and Notch diversifies their subsequent fates. Wnt signalling appears to control most of these events.     

Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.     

Wnt control of stem cells and differentiation in the intestinal epithelium

The intestinal epithelium represents a very attractive experimental model for the study of integrated key cellular processes such as proliferation and differentiation. The tissue is subjected to a rapid and perpetual self-renewal along the crypt-villus axis. Renewal requires division of multipotent stem cells, still to be morphologically identified and isolated, followed by transit amplification, and differentiation of daughter cells into specialized absorptive and secretory cells. Our understanding of the crucial role played by the Wnt/beta-catenin signaling pathway in controlling the fine balance between cell proliferation and differentiation in the gut has been significantly enhanced in recent years. Mutations in some of its components irreversibly lead to carcinogenesis in humans and in mice. Here, we discuss recent advances related to the Wnt/beta-catenin signaling pathway in regulating intestinal stem cells, homeostasis, and cancer. We emphasize how Wnt signaling is able to maintain a stem cell/progenitor phenotype in normal intestinal crypts, and to impose a very similar phenotype onto colorectal adenomas.     

EphB receptors coordinate migration and proliferation in the intestinal stem cell niche

More than 10(10) cells are generated every day in the human intestine. Wnt proteins are key regulators of proliferation and are known endogenous mitogens for intestinal progenitor cells. The positioning of cells within the stem cell niche in the intestinal epithelium is controlled by B subclass ephrins through their interaction with EphB receptors. We report that EphB receptors, in addition to directing cell migration, regulate proliferation in the intestine. EphB signaling promotes cell-cycle reentry of progenitor cells and accounts for approximately 50% of the mitogenic activity in the adult mouse small intestine and colon. These data establish EphB receptors as key coordinators of migration and proliferation in the intestinal stem cell niche.     

Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed “enteroids” when derived from small intestine and “colonoids” when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.     

Intestinal stem cell function in Drosophila and mice

Epithelial cells of the digestive tracts of most animals are short-lived, and are constantly replenished by the progeny of long-lived, resident intestinal stem cells. Proper regulation of intestinal stem cell maintenance, proliferation and differentiation is critical for maintaining gut homeostasis. Here we review recent genetic studies of stem cell-mediated homeostatic growth in the Drosophila midgut and the mouse small intestine, highlighting similarities and differences in the mechanisms that control stem cell proliferation and differentiation.     

Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

Somatic cells have been proposed to be limited in the number of cell divisions they can undergo. This is thought to be a mechanism by which stem cells retain their integrity preventing disease. However, we have recently discovered intestinal crypt stem cells that persist for the lifetime of a mouse, yet divide every day. We now demonstrate biochemically that primary isolated Lgr5+ve stem cells contain significant telomerase activity. Telomerase activity rapidly decreases in the undifferentiated progeny of these stem cells and is entirely lost in differentiated villus cells. Conversely, asymmetric segregation of chromosomes has been proposed as a mechanism for stem cells to protect their genomes against damage. We determined the average cell cycle length of Lgr5+ve stem cells at 21.5 h and find that Lgr5+ve intestinal stem cells randomly segregate newly synthesized DNA strands, opposing the 'immortal strand' hypothesis.     

Lifespan extension by preserving proliferative homeostasis in Drosophila

Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs) and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK) signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan.     

Thyroid Hormone Regulation of Adult Intestinal Stem Cell Development: Mechanisms and Evolutionary Conservations

The adult mammalian intestine has long been used as a model to study adult stem cell function and tissue renewal as the intestinal epithelium is constantly undergoing self-renewal throughout adult life. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells located in the crypt. The development of this self-renewal system is, however, poorly understood. A number of studies suggest that the formation/maturation of the adult intestine is conserved in vertebrates and depends on endogenous thyroid hormone (T3). In amphibians such as Xenopus laevis, the process takes place during metamorphosis, which is totally dependent upon T3 and resembles postembryonic development in mammals when T3 levels are also high. During metamorphosis, the larval epithelial cells in the tadpole intestine undergo apoptosis and concurrently, adult epithelial stem/progenitor cells are formed de novo, which subsequently lead to the formation of a trough-crest axis of the epithelial fold in the frog, resembling the crypt-villus axis in the adult mammalian intestine. Here we will review some recent molecular and genetic studies that support the conservation of the development of the adult intestinal stem cells in vertebrates. We will discuss the mechanisms by which T3 regulates this process via its nuclear receptors.     

An enteroendocrine cell-based model for a quiescent intestinal stem cell niche

We have shown that the kinetics of conversion of intestinal crypt cell populations to a partially or wholly mutant phenotype are consistent with a model in which each crypt contains an infrequently dividing 'deep' stem cell that is the progenitor of several more frequently dividing 'proximate' stem cells. An assumption of our model is that each deep stem cell exists in a growth inhibitory niche. We have used information from the literature to develop a model for a quiescent intestinal stem cell niche. This niche is postulated to be primarily defined by an enteroendocrine cell type that maintains stem cell quiescence by secretion of growth inhibitory peptides such as somatostatin and guanylin/uroguanylin. Consistent with this model, there is evidence that the proteins postulated as defining a growth-inhibitory stem cell niche can act as intestinal tumour suppressors. Confirmation that a growth-inhibitory niche does exist would have important implications for our understanding of intestinal homeostasis and tumorigenesis.