Intestinal crypt properties fit a model that incorporates replicative ageing and deep and proximate stem cells

A model of intestinal crypt organization is suggested based on the assumption that stem cells have a finite replicative life span. The model assumes the existence in a crypt of a quiescent ('deep') stem cell and a few more actively cycling ('proximate') stem cells. Monte Carlo computer simulation of published intestinal crypt mutagenesis data is used to test the model. The results of the simulation indicate that stabilization of the crypt mutant phenotype following treatment with external mutagen is consistent with a stem cell replicative life span of about 40 divisions for mouse colon and 90-100 divisions for mouse small intestine, corresponding to a deep stem cell cycle time of about 3.9 and 8.5 weeks for colon and small intestine, respectively. Simulation of the data obtained for human colorectal crypts suggests that the proximate stem cell cycle time is about 80 h, assuming a replicative life span of 50-150 divisions, and that the deep stem cell divides approximately every 30 weeks.     

A stochastic branching model with formation of subunits applied to the growth of intestinal crypts

The intestinal epithelium is one of the most rapidly regenerating tissues in mammals. Cell production takes place in the intestinal crypts which contain about 250 cells. Only a minority of 1-60 proliferating cells are able to maintain a crypt over a long period of time. However, so far attempts to identify these stem cells were unsuccessful. Therefore, little is known about their cellular growth and selfmaintenance properties. On the other hand, the crypts appear to exhibit a life cycle which starts by fission of existing crypts and ends by fission or extinction. Data on these processes have recently become available. Here, we demonstrate how these data on the life cycle of the macroscopic crypt structure can be used to derive a quantitative model of the microscopic process of stem cell growth. The model assumptions are: (1) stem cells undergo a time independent supracritical Markovian branching process (Galton-Watson process); (2) a crypt divides if the number of stem cells exceeds a given threshold and the stem cells are distributed to both daughter crypts according to binomial statistics; (3) the size of the crypt is proportional to the stem cell number. This model combining two different stochastic branching processes describes a new class of processes whose stationary stability and asymptotic behavior are examined. This model should be applicable to various growth processes with formation of subunits (e.g. population growth with formation of colonies in biology, ecology and sociology). Comparison with crypt data shows that intestinal stem cells have a probability of over 0.8 of dividing asymmetrically and that the threshold number should be 8 or larger.     

Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of ‘field cancerization’ describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5⁺ stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

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.     

Mitotic orientation in three dimensions determined from multiple projections.

The three-dimensional orientation of mitoses in mouse small intestinal crypts of Lieberkuhn was determined from multiple projections of the mitotic figures in whole mounts of isolated intestinal crypts. We found evidence of a significant orientational bias for mitoses whose daughter cells would be added along the long axis of the crypt, and thus conform to the maintenance of the cylindrical shape of the intestinal crypt. However, we also observed many mitoses whose progeny must be rearranged if the simple cylindrical shape of the intestinal crypt is to be maintained. Our results indicate that the ultimate behavior of progeny cells and hence of local tissue form may not strictly depend on the orientation of mitosis. The methods presented may also be used in the study of mitotic orientation in other tissues.     

Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice

Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.     

Quantification of Colonic Stem Cell Mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.¹ This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 µm thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 µm apart were prepared to assess a total of >10⁴ crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts ÷ by the number of wild type (blue) crypts in a treatment group.     

Variation in crypt size and its influence on the analysis of epithelial cell proliferation in the intestinal crypt.

The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed.     

Stand by me: Fibroblasts regulation of the intestinal epithelium during development and homeostasis

The epithelium of the small intestine is composed of a single layer of cells that line two functionally distinct compartments, the villi that project into the lumen of the gut and the crypts that descend into the underlying connective tissue. Stem cells are located in crypts, where they divide and give rise to transit-amplifying cells that differentiate into secretory and absorptive epithelial cells. Most differentiated cells travel upwards from the crypt towards the villus tip, where they shed into the lumen. While some of these cell behaviors are an intrinsic property of the epithelium, it is becoming evident that tight coordination between the epithelium and the underlying fibroblasts plays a critical role in tissue morphogenesis, stem-cell niche maintenance and regionalized gene expression along the crypt-villus axis. Here, we will review the current literature describing the interaction between epithelium and fibroblasts during crypt-villus axis development and intestinal epithelium renewal during homeostasis.     

Stem-cell organization in mouse small intestine

We have investigated stem-cell organization in mouse small intestine (SI) by using a cellular marker induced by somatic mutation. In small intestinal whole mounts from heterozygous Dlb-1b/Dlb-1a mice stained with a peroxidase conjugate of Dolichos biflorus agglutinin (DBA-Px), mutations of Dlb-1b in stem cells result in loss of DBA-Px binding and so are recognizable as wholly or partly unstained crypts. The frequency of these clonal patterns can be measured during the accumulation of spontaneous mutations in untreated mice, or after treatment with ethylnitrosourea (ENU). The results show that there is a single infrequently dividing stem cell that maintains the epithelium of each crypt through a population of transit stem cells. The entire crypt epithelium is renewed approximately every 12 weeks.     

ERBB3 Positively Correlates with Intestinal Stem Cell Markers but Marks a Distinct Non Proliferative Cell Population in Colorectal Cancer

Several studies have suggested ERBB3/HER3 may be a useful prognostic marker for colorectal cancer. Tumours with an intestinal stem cell signature have also been shown to be more aggressive. Here, we investigate whether ERBB3 is associated with intestinal stem cell markers in colorectal cancer and if cancer stem cells within tumours are marked by expression of ERBB3. Expression of ERBB3 and intestinal stem cell markers (LGR5, EPHB2, CD44s and CD44v6) was assessed by qRT-PCR in primary colorectal tumours (stages 0 to IV) and matched normal tissues from 53 patients. The localisation of ERBB3, EPHB2 and KI-67 within tumours was investigated using co-immunofluorescence. Expression of ERBB3 and intestinal stem cell markers were significantly elevated in adenomas and colorectal tumours compared to normal tissue. Positive correlations were found between ERBB3 and intestinal stem cell markers. However, co-immunofluorescence analysis showed that ERBB3 and EPHB2 marked specific cell populations that were mutually exclusive within tumours with distinct proliferative potentials, the majority of ERBB3+ve cells being non-proliferative. This pattern resembles cellular organisation within normal colonic epithelium where EPHB2 labelled proliferative cells reside at the crypt base and ERBB3+ve cells mark differentiated cells at the top of crypts. Our results show that ERBB3 and intestinal stem cell markers correlate in colorectal cancers. ERBB3 localises to differentiated cell populations within tumours that are non-proliferative and distinct from cancer stem cells. These data support the concept that tumours contain discrete stem, proliferative and differentiation compartments similar to that present in normal crypts.     

Crypt cell development in newborn rat small intestine

Three monoclonal antibodies were prepared against luminal membranes from small intestinal cells of 3-d-old rats (YBB 1/27, YBB 3/10) and crypt cell membranes from adult rats (CC 4/80). The antibodies were shown to define specific stages of development of the intestinal crypt cells. The YBB 1/27 antigen was first detected at the luminal membrane of the epithelial cells in fetal intestine at day 20 of gestation; it was confined to the crypt cells and lower villus cells between 1 and 20-22 d after birth, and could not be detected in any region of the intestine in older animals. The YBB 3/10 antigen, identified as a set of high Mr proteins, was localized over the entire surface membrane of fetal intestinal cells and of crypt and villus cells after birth; after weaning (20-22 d after birth) it gradually disappeared from the villus cells and became confined to the region of the crypts. The CC 4/80 antigen, identified as a protein (or a set of related proteins) of molecular mass 28-34 kD, was shown to appear in the crypt cells 10-14 d after birth. Its distribution changed after weaning, when it disappeared from the crypts, and was localized in the absorptive lower villus cells. This change in pattern could, in part, be prematurely elicited by cortisone injection in younger animals. These results have demonstrated the presence of specific surface membrane components on the intestinal crypt cells, and suggested that fetal antigens may be retained in these cells after birth.     

Modulation of stemness in a human normal intestinal epithelial crypt cell line by activation of the WNT signaling pathway

The small intestine consists of two histological compartments composed of the crypts and the villi. The function of the adult small intestinal epithelium is mediated by four different types of mature cells: enterocytes, goblet, enteroendocrine and Paneth. Undifferentiated cells reside in the crypts and produce these four types of mature cells. The niche-related Wnt and Bmp signaling pathways have been suggested to be involved in the regulation and maintenance of the stem cell microenvironment. In our laboratory, we isolated the first normal human intestinal epithelial crypt (HIEC) cell model from the human fetal intestine and in this study we investigated the expression of a panel of intestinal stem cell markers in HIEC cells under normal culture parameters as well as under conditions that mimic the stem cell microenvironment. The results showed that short term stimulation of HIEC cells with R-spondin 1 and Wnt-3a±SB-216763, a glycogen synthase kinase 3β (GSK3β) inhibitor, induced β-catenin/TCF activity and expression of the WNT target genes, cyclin D2 and LGR5. Treatment of HIEC cells with noggin, an antagonist of BMP signaling, abolished SMAD2/5/8 phosphorylation. Inducing a switch from inactive WNT/active BMP toward active WNT/inactive BMP pathways was sufficient to trigger a robust intestinal primordial stem-like cell signature with predominant LGR5, PHLDA1, PROM1, SMOC2 and OLFM4 expression. These findings demonstrate that even fully established cultures of intestinal cells can be prompted toward a CBC stem cell-like phenotype. This model should be useful for studying the regulation of human intestinal stem cell self-renewal and differentiation.     

Development of the pattern of cell renewal in the crypt-villus unit of chimaeric mouse small intestine

We have previously shown that the epithelium of each adult intestinal crypt in chimaeric mice is derived from a single progenitor cell. Whether the crypts are monoclonal from the outset-that is, are formed by the proliferation of a single cell-or whether their formation is initiated by several cells was not known. Here we report that many crypts contain cells of both chimaeric genotypes in the neonatal period indicating a polyclonal origin at this stage of morphogenesis. The cellular organization of the early neonatal crypt is therefore different from that of the adult crypt, which includes a zone of 'anchored' stem cells above the crypt base. Within 2 weeks, however, the crypt progenitor cell and its descendants displace all other cells from the crypt and the crypt attains monoclonality. The distribution of enterocytes on chimaeric villi in the neonate shows a mottled pattern of mosaicism which is progressively replaced by coherent sheets of cells from the crypts, and within two weeks the orderly adult clonal pattern is established.     

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.     

Circadian variation in migration velocity in small intestinal epithelium

The variation in migration rates of cells within the small intestinal epithelium was studied over a 24-hr period at 3-hr intervals (migration of cells was studied independently for the crypts and the villi using the changing distributions of [3H]TdR labelled cells as an indicator of cell migration). Clear changes in the rates of cell movement were observed during a 24-hr period for both crypt and villus epithelium. The rates of cell migration in these two compartments did not correlate well with the exception of samples taken at 18.00 hours. At this time of day there appeared to be no cell movement at all in either crypts or villi. There was not a good correlation between the migration velocity throughout the day and the changes in the number of mitoses. It is proposed that mitotic rates do not directly govern migration rates but that the converse may be true. Further, the lack of correlation between crypt and villus migration rates at any time of day suggest that the mechanisms controlling all movement in these two regions of small intestinal epithelium may be different.