Evidence For Cell Generation Controlled Proliferation In the Small Intestinal Crypt

Abstract. In this paper we discuss the hypothesis that cell proliferation is controlled by the number of generations after leaving an 'eternal' stem cell. the theory is based on a simulation of the kinetic behaviour of cells in the intestinal crypts. There is evidence of three, four and five generations of cells which are allowed to enter mitosis in the lower and upper part of the normal intestinal tract, and in some disease states, respectively. We suggest an internal proliferation control: some kind of knowledge that cells carry from generation to generation. It is an open question what sets and changes the generation counter: internal genetic information or external influences such as growth factors or chalones.
The geometric shape of the epithelial tissue in the intestinal tract can be understood as the steady state of a highly dynamic process. Age and death are determined from the beginning; cell-cell interaction or communication is not necessary and can be neglected. Our theory will be illustrated using the intestinal crypts as they are easily accessible, of a simple structure and completely described in the literature.     

Generation-dependent control mechanisms in cell proliferation and differentiation—the power of two

There is evidence that the proliferation of cells is controlled by the number of divisions after leaving a multi-potent (stem) cell. A detailed study of the growth of tissue in the small intestinal tract, more precisely the growth of crypts and villi, suggests that not only the proliferation but also the differentiation of cells obey the same biological law. We postulate a theory of a cellular internal control mechanism: the cell-generation control of differentiation and proliferation. This basic mechanism, together with external influences, determines the kinetic behaviour of the crypt-villus system.     

Optimality in the development of intestinal crypts

Intestinal crypts in mammals are comprised of long-lived stem cells and shorter-lived progenies. These two populations are maintained in specific proportions during adult life. Here, we investigate the design principles governing the dynamics of these proportions during crypt morphogenesis. Using optimal control theory, we show that a proliferation strategy known as a "bang-bang" control minimizes the time to obtain a mature crypt. This strategy consists of a surge of symmetric stem cell divisions, establishing the entire stem cell pool first, followed by a sharp transition to strictly asymmetric stem cell divisions, producing nonstem cells with a delay. We validate these predictions using lineage tracing and single-molecule fluorescence in?situ hybridization of intestinal crypts in infant mice, uncovering small crypts that are entirely composed of Lgr5-labeled stem cells, which become a minority as crypts continue to grow. Our approach can be used to uncover similar design principles in other developmental systems.     

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.     

Cloning of intestinal phospholipase A2 from intestinal epithelial RNA by differential display PCR

Differential display polymerase chain reaction (DD-PCR) is a powerful technique for comparing gene expression between cell types, or between stages of development or differentiation. Differentially expressed genes may be cloned and analysed further. Here we extend the use of DD-PCR to analyse differences in gene expression between two complex epithelia: that of the small intestine and of the large intestine. The aim of this study was to identify genes expressed preferentially in Paneth cells. Paneth cells are secretory epithelial cells putatively involved in host defense and regulation of crypt cell proliferation and are found at the base of the small intestinal crypts adjacent to the stem cell zone. Of 34 clones that were analysed, partial sequencing identified two clones related to known Paneth cell products: a homologue of secretory phospholipase A2 (clone B1) and a homologue of a neutrophil defensin (clone C5). B1 was strongly expressed in Paneth cells, as demonstrated by in-situ hybridization. B1 was also expressed at a lower level in the large intestinal epithelium. A full length B1 cDNA clone was isolated and sequenced, and shown to be highly homologous to type II secretory phospholipase A2 genes, and almost identical to the enhancing factor gene and the putative gene for the MOM-1 locus. B1 expression is limited to the intestinal tract, and we propose that it be designated intestinal phospholipase A2, or i -PLA2. The method we describe is well suited to the rapid identification of genes expressed exclusively or predominantly in Paneth cells.     

Proliferation, not apoptosis, alters epithelial cell migration in small intestine of CFTR null mice

Expression of a mutated cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to enhance proliferation within CF airways, and cells expressing a mutated CFTR have been shown to be less susceptible to apoptosis. Because the CFTR is expressed in the epithelial cells lining the gastrointestinal tract and all CF mouse models are characterized by gastrointestinal obstruction, we hypothesized that CFTR null mice would have increased epithelial cell proliferation and reduced apoptosis within the small intestine. The rate of intestinal epithelial cell migration from crypt to villus was increased in CFTR null mice relative to mice expressing the wild-type CFTR. This difference in migration could be explained by an increase in epithelial cell proliferation but not by a difference in apoptosis within the crypts of Lieberkühn. In addition, using two independent sets of CF cell lines, we found that epithelial cell susceptibility to apoptosis was unrelated to the presence of a functional CFTR. Thus increased proliferation but not alterations in apoptosis within epithelial cells might contribute to the pathophysiology of CF.     

FGFR3 contributes to intestinal crypt cell growth arrest

Fibroblast growth factors (FGFs) are important regulators of the dynamic development and turnover of tissues. Among FGF receptors, FGFR3 expression is confined in the intestinal crypts. We examined FGFR3-deficient mice and saw increased intestinal crypt depth but no change in villae length or in the distribution of differentiated intestinal cells, suggesting that the impact of lack of FGFR3 was limited to the progenitor cell compartment. Accordingly, enhancement of intestinal crypt proliferation was observed in FGFR3 mutant mice and interestingly, upon anti-FGFR3 antibody administration in wild type mice. Moreover, injection of FGF18, a ligand of FGFR3, in wild type mice resulted in decreased cell proliferation within the intestinal crypts. In addition, we found that ERK level of activation was increased in FGFR3-deficient intestinal epithelium. In vitro studies showed that ERK, AKT and activation was regulated by FGFs and that ERK level of activation was inversely correlated to FGFR3 level of expression in the intestinal crypt cells. Furthermore, effects of FGF18 on ERK and AKT activation paralleled FGFR3 effects on these intracellular targets. Our data indicate that FGF18 and FGFR3 are involved, possibly as partners, in the control of intestinal precursor cell proliferation.     

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.     

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.     

Site differences of Toll-like receptor expression in the mucous epithelium of rat small intestine

Toll-like receptors (TLRs) are known to recognize pathogen-associated molecular patterns and might function as receptors to detect microbes. In this study, the distribution of TLR-2, -4 and -9 were immunohistochemically investigated in the rat small intestine. As a result, TLR-2 was detected in the striated borders of villous columnar epithelial cells throughout the small intestine, except for the apices of a small number of intestinal villi. TLR-4 and -9 were detected in the striated borders of the villous columnar epithelial cells only in the duodenum. TLR-4-immunopositive minute granules were found in the apical cytoplasms of epithelial cells, subepithelial spaces and blood capillary lumina. TLR-2 and -4 were detected in the striated borders of undifferentiated epithelial cells and in the luminal substances of the intestinal crypts throughout the small intestine, but TLR-9 was not detected in the crypts throughout the small intestine. Only TLR-4 was detected in the secretory granules of Paneth cells in both the jejunal and ileal intestinal crypts. These findings suggest that duodenal TLRs might monitor indigenous bacteria proliferation in the upper alimentary tract, that TLR-2 might also monitor the proliferation of colonized indigenous bacteria throughout the small intestine, that the lack of TLR-2 at the villous apices might contribute to the settlement of indigenous bacteria, and that TLR-2 and -4 are secreted from intestinal crypts.     

Mammalian intestinal epithelial cells in primary culture: a mini-review

Epithelial cells lining the digestive tract represent a highly organized system built up by multipotent stem cells. A process of asymmetric mitosis produces a population of proliferative cells that are rapidly renewed and migrate along the crypt-villus axis, differentiating into functional mature cells before dying and exfoliating into the intestinal lumen. Isolated crypts or epithelial cells retaining high viability can be prepared within a few h after tissue sampling. After cells are cultured in serum-free media, short-term studies (16-48 h) can be conducted for endocrinology, energy metabolism, or programmed cell death. However, long-term primary culture of intestinal cells (up to 10 d) is still difficult despite progress in isolation methodologies and manipulation of the cell microenvironment. The main problem in developing primary culture is the lack of structural markers specific to the stem cell compartment. The design of a microscopic multidimensional analytic system to record the expression profiles of biomarkers all along the living intestinal crypt should improve basic knowledge of the survival and growth of adult crypt stem cells, and the selection of totipotent embryonic stem cells capable of differentiating into intestinal tissues should facilitate studies of the genomic basis of endodermal tissue differentiation.     

CONTROL OF EPITHELIAL CELL PROLIFERATION IN THE SMALL INTESTINAL CRYPT

A heat labile factor which has been shown to inhibit proliferative activity in crypt epithelium both in rat jejunum in vivo and in explants of rat jejunum maintained in organ culture has been prepared from the soluble fraction of homogenized epithelial cells isolated from rat small intestinal crypts. The factor appears to have tissue specificity, for it has no influence on epithelial cell proliferation in colonic crypts, oesophagus or skin. Extracts of rat intestinal villous cells prepared using identical techniques were without effect on proliferative activity of small intestinal crypt epithelium.
Isoprenalin, which was also found to suppress cell proliferation, did not potentiate the effect of the factor and its effects were evanescent.     

Modeling Cell Dynamics in Colon and Intestinal Crypts: The Significance of Central Stem Cells in Tumorigenesis

Colon and intestinal crypts have been widely chosen to study cell dynamics because of their fairly simple structures. In the colon and intestinal crypts, stem cells (SCs) are located at very bottom of the crypt, fully differentiated cells (FDs) are located in the top of the crypt, and transit-amplifying cells (TAs) are in the middle of the crypt between FDs and SCs. Recently, it has been discovered that there are two types of stem cells in the intestinal crypts: central stem cells (CeSCs) and border stem cells. To investigate dynamics of mutants in colon and intestinal crypts, we develop a four-compartmental stochastic model, which includes two SC compartments, and TAs and FDs compartments. We calculate the probability of the progeny of marked or mutant cells located at each of these compartments taking over the entire crypt or being washed out from the crypt. We found that the progeny of CeSCs will take over the entire crypt with a probability close to one. Interestingly, the progeny of advantageous mutant TAs and FDs will be washed out faster than disadvantageous mutants. Saliently, the model predicts that the time that the progeny of wild-type central stem cells will take over the mouse intestinal crypt is around 60 days, which is in perfect agreement with an experimental observation.     

Mutation induced clonal markers from polymorphic loci: application to stem cell organisation in the mouse intestine

Marked clones of cells can be induced somatically in intestinal epithelium. The markers are induced by mutation affecting single alleles of polymorphic genetic loci such as Dlb-1 or G6PD whose product can be detected histochemically. Analysis of mutated clones allows the organisation of intestinal stem cells to be investigated. In both small and large bowel the crypts, which contain the intestinal stem cell population, are populated with the progeny of a single cell several times during the life of the mouse. This process could occur by stochastic or hierarchical renewal of cells within the stem cell population.     

Isolation of hamster intestinal epithelial cells using hypoosmotic media and PVP

Vibration of hamster small intestinal segments in hypotonic media containing PVP is a rapid method for obtaining quantitative yields of viable intestinal epithelial cells. This preparation of epithelial cells offers a unique system for the study of epithelial cell function in vitro. The method for cell separation combines hypoosmotic swelling of cells, which separates them at the desmosomes, with mechanical agitation which releases the cells from the lamina propria. No chemical agents known to affect cell proteins and cell surfaces are employed in this procedure. Only a short time is elapsed between in vivo and in vitro conditions, i.e., a preparation time of approximately 75 minutes. Although the technique yields a pure population of epithelial cells, the cells are of different morphologies, are removed from different areas of the crypts and villi, and therefore presumably have different functions. Examination of the intestinal tissue remaining after several vibration intervals by light and scanning electron microscopy indicates that the sequences of release of cells is removal of: (1) cells from the villus based, (2) cells from the lower one-half to two-thirds of the villi, (3) cells from the villus tips (and some crypts), and (4) cells from the crypts. When pools of a + b cells are compared to pools of c + d cells, it is found that villus cells can be characterized by: (1) processes, such as monosaccharide absorption, associated with the brush border, and (2) synthesis of components (e. g., glycoproteins) of the brush border. Surprisingly, disaccharide hydrolytic activity is found in cells which transport monosaccharides poorly. The subpopulations of cells synthesize proteins equally.     

c-Myc is required for the formation of intestinal crypts but dispensable for homeostasis of the adult intestinal epithelium

In self-renewing tissues such as the skin epidermis and the bone marrow, Myc proteins control differentiation of stem cells and proliferation of progenitor cell types. In the epithelium of the small intestine, we show that c-Myc and N-Myc are expressed in a differential manner. Whereas c-Myc is expressed in the proliferating transient-amplifying compartment of the crypts, N-Myc is restricted to the differentiated villus epithelium and a single cell located near the crypt base. c-Myc has been implicated as a critical target of the canonical Wnt pathway, which is essential for formation and maintenance of the intestinal mucosa. To genetically assess the role of c-Myc during development and homeostasis of the mammalian intestine we induced deletion of the c-myc(flox) allele in the villi and intestinal stem cell-bearing crypts of juvenile and adult mice, via tamoxifen-induced activation of the CreER(T2) recombinase, driven by the villin promoter. Absence of c-Myc activity in the juvenile mucosa at the onset of crypt morphogenesis leads to a failure to form normal numbers of crypts in the small intestine. However, all mice recover from this insult to form and maintain a normal epithelium in the absence of c-Myc activity and without apparent compensation by N-Myc or L-Myc. This study provides genetic and molecular evidence that proliferation and expansion of progenitors necessary to maintain the adult intestinal epithelium can unexpectedly occur in a Myc-independent manner.     

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo

Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.     

The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.

Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in an increased survival of animals exposed to a lethal dose of irradiation. The mechanisms underlying these effects are not clear. The present experiments were designed to investigate the nature of any proliferative changes induced in the crypts of the small intestine by protracted exposure to KGF. Tritiated thymidine or bromodeoxyuridine labeling showed statistically significant increases in labeling in the stem cell zone of the crypt, with a concomitant reduction in labeling in the upper regions of the crypt corresponding to the late-dividing transit population. The increase in labeling in the lower regions of the crypt was also observed with Ki-67 staining, but the reduction in the upper regions of the crypt seen with tritiated thymidine was not observed with Ki-67. Metaphase arrest data suggest that the rate of progression through the cell cycle is essentially the same in KGF-treated animals as in controls, but there is a statistically significant increase in the number of mitotic events per crypt. Double labeling studies suggest that, at certain times of the day, there is a greater influx into S phase than efflux. The data overall indicate that KGF induces some complex proliferative changes in the intestinal crypts and are consistent with the hypothesis that the radioprotection may be afforded, at least in part, by a KGF-induced increase in stem cell numbers and/or increases in the number of stem cells in the S phase of the cell cycle. This alteration in the homeostasis of the crypt is compensated for by a foreshortening of the dividing transit lineage.