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.     

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.     

Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone-epidermal growth factor

Abstract. There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone-epidermal growth factor (URO-EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population.
The number of mitoses and labelled cells per crypt, and thus the crypt cell production rates, were significantly decreased in the TPN group when compared to the orally fed. URO-EGF significantly increased both proliferative indices and the number of dividing cells per crypt. Crypt cell production in the small intestine was restored to those levels seen in the orally fed rats, moreover, labelling per crypt in the colon was increased to more than twice that of orally fed rats. The location of the mean labelling position and the half maximum labelling position followed the changes in crypt length in the small intestine, but to a lesser extent; thus the growth fraction was significantly increased in the TPN rats in comparison with the orally fed and the URO-EGF treated groups. Similar changes in these positions were seen in the colon, but the growth fraction was much reduced in the URO-EGF treated rats, as a consequence of the large increase in crypt length without a concomitant alteration in label distribution.     

Cell Proliferation and Ageing In Mouse Colon

The descending colon of 4 month and 2 year old mice was exposed to 1250 rad X-rays. This killed most of the epithelial cells. the surviving cells formed new crypts and surface epithelium in animals of both ages. Not all the crypts were replaced. the irradiated area contained no more than 80% of the control number of crypts per section for at least 6 weeks after irradiation. In the young mice new crypts were much larger and the labelling index (LI) was much higher than in unirradiated animals during the first week after irradiation. In the old mice the overshoot in LI and crypt size began later and continued longer than in young animals. This may be because the control of cell proliferation was much less precise in old than in young mice. The irradiation was repeated, in an attempt to age prematurely the epithelial cells by increasing the number of divisions they underwent. the overshoot in LI and cells per crypt was smaller after a second dose than after the first in both young and old mice. There was almost no overshoot after a third dose was given to young mice. Increasing the number of divisions undergone by the surviving epithelial cells did not change the timing of repopulation in young mice compared to that found in old mice. Little evidence was found for the presence of a limited proliferative lifespan in colon epithelial cells.     

AN ESTIMATION OF PROLIFERATIVE POPULATION SIZE IN STOMACH, JEJUNUM AND COLON OF DBA-2 MICE

Liquid scintillation and autoradiographic techniques have been used to provide quantitative data on the proliferative units, the crypts, of stomach, jejunum and colon of DBA-2 mice. A slight modification of the crypt squash technique has provided data suggesting that about 50% of the cells of the jejunal crypt are at any given time in the proliferative state. This value is lower in the colon while in the stomach glands only 20% of the cells are involved in cell production. The data provide estimates for cell cycle times of 26·3, 16·0 and 23·2 hr for stomach, jejunum and colon respectively.
The size and number of proliferative units have been determined for three regions of the gastrointestinal tract of mice. A review of the literature suggests that considerable strain differences may exist.     

Aspirin, age, and proximity to lymphoid nodules influence cell proliferation parameters in rat colonic crypts

Recent epidemiological studies have demonstrated a correlation between regular aspirin (acetylsalicylic acid) use and decrease risk for the development of fatal colorectal cancer. An increase in the size of the cell proliferation compartment in colorectal crypts has been correlated with an increased risk for the development of colon cancer in animals and in humans. To determine if acetylsalicylic acid acts to decrease the size of the cell proliferation compartment, young (3 month) and old (22 month) rats were treated intragastrically with: 1 the vehicle for acetylsalicylic acid delivery (0.25% wt/vol carboxymethylcellulose in 0.15 N HCI), 2 a single dose of acetylsalicylic acid (100 mg/kg), or 3 acetylsalicylic acid (30 mg/kg) given daily for 30 days. One day after the last treatment, colons were resected, fixed, sectioned and mounted on slides for immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen to assess cell proliferation parameters in the colonic crypts. The results were subjected to three way analysis of variance to assess the effects of: 1 rat age, 2 acute or chronic acetylsalicylic acid treatment, and 3 location of crypts over and away from aggregates of lymphoid nodules on the crypt proliferative parameters. Results demonstrated that: 1 acetylsalicylic acid treatment caused an overall decrease in the proliferative zone height, as measured in number of cells in the crypt column, 2 that crypts located over aggregates of lymphoid nodules had significantly higher proliferative activity than crypts located away from aggregates of lymphoid nodules, and 3 after chronic acetylsalicylic acid treatment there was a greater suppression of proliferative zone height in the crypts of old rats than in the crypts of young rats. In conclusion, acute and chronic intragastric delivery of acetylsalicylic acid caused an overall downward shift in the cell proliferation compartment of colonic crypts of young and of old rats. Whether or not acetylsalicylic acid administration will cause the same proliferative zone height response in carcinogen-treated rats is not yet established.     

Colonic cell proliferation and growth fraction in young,adult and old rats

Colonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.     

Colonic cell proliferation and growth fraction in young, adult and old rats.

Colonic epithelial proliferation was investigated in three groups of rats, aged 3, 60 and 121 weeks. As reported in previous work, the crypts were markedly longer in the young rats, and the number of labelled cells per crypt was significantly greater. There was an upward movement of the marker positions derived from the distribution of labelled cells within the crypt of the young rats. This was a consequence of the increased crypt length, so that the growth fraction, as expressed as a percentage of crypt length, was the same. The proliferative changes between the young rats and the other aged rats were therefore effected by altering the size of the crypts, while maintaining the kinetic organisation. There was no evidence of any proliferative changes or changes in the growth fraction when the colons of the old rats were compared with those of the 60 week old rats.     

Epithelial cell kinetics in the descending colon of the rat

The influence of experimental bypass on the epithelial cell kinetics in the rat descending colon was studied. It was found that the number of cells per crypt was markedly reduced at 6 weeks after bypass. The percentage of labelled crypt cells, 1 h after 3HTdR, and the distribution of labelled cells in the crypt was normal. Also the life span of the epithelial cells was the same in control and bypassed colon. The response of crypt cell proliferation to ischaemia-induced cell loss in the bypassed descending colon was similar to the one previously described for normal descending colon. This indicates that the absence of the normal luminal contents does not result in a different response of colonic crypts to induced cell loss. Furthermore, it was found that the number of cells per crypt and the proliferative activity did not change in the transverse colon after temporary ischaemia of the bypassed descending colon. This indicates that the increase in crypt cell proliferation after ischaemia-induced cell loss is a local response.     

Colon Cryptogenesis: Asymmetric Budding

The process of crypt formation and the roles of Wnt and cell-cell adhesion signaling in cryptogenesis are not well described; but are important to the understanding of both normal and cancer colon crypt biology. A quantitative 3D-microscopy and image analysis technique is used to study the frequency, morphology and molecular topography associated with crypt formation. Measurements along the colon reveal the details of crypt formation and some key underlying biochemical signals regulating normal colon biology. Our measurements revealed an asymmetrical crypt budding process, contrary to the previously reported symmetrical fission of crypts. 3D immunofluorescence analyses reveals heterogeneity in the subcellular distribution of E-cadherin and β-catenin in distinct crypt populations. This heterogeneity was also found in asymmetrical budding crypts. Singular crypt formation (i.e. no multiple new crypts forming from one parent crypt) were observed in crypts isolated from the normal colon mucosa, suggestive of a singular constraint mechanism to prevent aberrant crypt production. The technique presented improves our understanding of cryptogenesis and suggests that excess colon crypt formation occurs when Wnt signaling is perturbed (e.g. by truncation of adenomatous polyposis coli, APC protein) in most colon cancers.     

PROLIFERATION KINETICS IN EPITHELIUM OF GUINEA-PIG COLON

Autoradiographs of crypts of guinea-pig colon labelled with ³H-thymidine were quantitatively analysed. The analysis involved: (a) comparison of proliferation parameters in crypts situated in two well-defined regions of colonic folds, i.e. in the top and the basal parts of the fold, and (b) comparison of proliferation parameters in the lower and in the upper parts of an individual crypt. The results provide evidence that: (1) Crypts situated in the top region of folds are significantly longer than those situated in the basal region. (2) The duration of cell cycle and its phases is similar in the crypts of the two regions examined. (3) The rate of ³H-thymidine uptake is higher in the middle than in the initial and final parts of the S phase, and its pattern is the same for the crypts of the two regions of the fold as well as for the parts of the individual crypt. (4) The proliferating pool size is larger in crypts situated in the top than in the basal region of the fold. (5) The proliferating pool size is larger in the lower parts of crypts from each region than in the upper parts. Control of cellular proliferation in the crypt epithelium is considered to operate separately at the level of the individual crypt and at the level of a group of crypts situated in a given region of the colonic wall.     

IGF-II promotes mesoderm formation

IGF-II is abundant in the nascent mesoderm of the gastrulating mouse embryo. Its function at this developmental stage is unknown. We investigated it by following the in vitro and in vivo differentiation of several androgenetic, biparental, parthenogenetic, and androgenetic Igf2 -/- murine ES cell lines; these cells differed in endogenous IGF-II levels because Igf2 is paternally expressed in the mouse embryo in most tissues. The expression of mesoderm markers and the subsequent formation of muscle structures were correlated with endogenous IGF-II level during teratoma formation and during in vitro differentiation. In addition, the absence of Igf2 in androgenetic Igf2 -/- ES cells led to a severe impairment of mesoderm development, demonstrating the dependence of the preferential mesoderm development of androgenetic ES cells upon Igf2 activity, among the numerous known imprinted genes. The addition of exogenous IGF-II to in vitro differentiation culture medium led to a specific increase in the expression of mesoderm markers. Thus, we propose a novel model in which the binding of IGF-II to its principal signaling receptor, IGF1R, at the surface of mesoderm precursor cells increases the formation of mesoderm cells.     

Igf2r improves the survival and transmission ratio of Igf2 transgenic mice

Mammals with excess insulin-like growth factor 2 (IGFII) during embryogenesis have developmental defects that can lead to perinatal lethality. In adults, higher levels of IGFII increase the risk of cancer and may accelerate the development of atherosclerosis. IGFII can be increased as a consequence of genetic abnormalities and polymorphisms, and through epigenetic mechanisms. Decreasing IGFII levels thus can benefit human health. Degradation of IGFII is mediated by the insulin-like growth factor type 2 receptor (IGF2R). The growth-stimulatory effects of IGFII, and their attenuation by the IGF2R, are considered important for the evolution of IGFII/IGF2R interaction and imprinting. The IGFII/IGF2R interactions during development have been previously examined in mice carrying knock-out alleles of these genes or their regulators. Here we tested the ability of the IGF2R to ameliorate the negative effects of IGFII on development and survival in crosses between Igf2 and Igf2r transgenic mice, which may be a better model for natural variations in the levels of these genes' products. A fraction of hemizygous Igf2 transgenic mice die in the perinatal period, some with cleft palates, with an ensuing reduction in the frequency of transgenic mice among the surviving offspring. The Igf2r transgene lowers the frequency of cleft palate and increases the percentage of Igf2 transgenic mice among the live offspring. These findings draw attention to the fact that Igf2-associated lethality selects for the retention of IGFII/IGF2R binding in present day mammals; it may have played a similar role in the acquisition of IGFII/IGF2R binding in ancient mammals.     

The effect of estrogen on epithelial cell proliferation and differentiation in the crypts of the descending colon of the mouse: a radioautographic study

The regulatory role of estrogen on cell population kinetics in the descending colon was studied in intact female and ovariectomized mice. In the colonic crypts from intact mice, the crypt size (the number of epithelial cells per crypt column) and the proliferative activity of epithelial cells fluctuated slightly during the estrous cycle. Peak cellularity per crypt column was exhibted during estrus and early diestrus, whereas peaks in labeling index were seen during estrus and late metestrus. While the population size of mucous cells showed a minimal variation, the number of proliferative vacuolated cells per crypt column varied inversely with that of differentiated columnar cells during estrous cycle. The vacuolated cells were increased in number in the preovulatory phase and the columnar cells in the postovulatory phase. Three weeks after bilateral ovariectomy, the colonic crypt appeared to reach a new steady state, which was characterized by a small crypt size, a decrease in the number of differentiated cells, an increase in the relative number of proliferative cells and a relative increase in the proliferative activity of the crypt as compared to intact mice. When ovariectomized mice were treated with estrogen, the number of 3H-thymidine-labeled cells in the crypt was decreased as compared to untreated ovariectomized mice, the decrease being greater after a single injection than after multiple injections of estrogen, and the vacuolated-columnar cell line being affected more than mucous cell line. Meanwhile, the crypt size as well as the population size of differentiated cells in the crypt failed to return to normal after estrogen treatments. Thus, estrogen did not promote differentiation of epithelial cells in the crypt.     

A cytokinetic study of small-intestinal and colonic mucosa after resection of 70% of the small intestine

Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied. In the distal ileum there is a significant reduction in cell cycle time (Tc) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in Tc at these sites, but there does appear to be a significant reduction in Tc on the part of the cells in the stem-cell zone at the crypt base. In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

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.     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

Cell survival and proliferation are modified by insulin-like growth factor 2 between days 9 and 10 of mouse gestation

The size of mammalian species involves the interaction of multiple genetic modifiers that control the timing and extent of growth mechanisms. Disruption of the paternal allele of the imprinted embryonic gene coding for insulin-like growth factor 2 (IGF2, Igf2(+m/-p)), results in viable mice that are 60% the weight of wild-type littermates. Differences in weight are first detected at embryonic day (E) 11, and the growth deficit is maintained throughout life. We report the mechanisms that account for this unusual phenotype. In order to quantify growth, we used novel methods to generate single cell suspensions of post-implantation mouse embryos. We were then able to quantify cell number, cell proliferation and cell death between E8.5 and E11.5 using flow cytometry. Determination of total embryo cell number also allowed us to time litters by a method other than by plugging. Wild-type and Igf2(+m/-p) embryos accumulated similar total cell numbers up to E9.25, but cell number began to diverge by around E9.5, with significant differences by E11 (75% of wild type). A relative increase in pyknotic nuclei, sub-GI cytometry counts and caspase activity, all indicative of cell death, occurred in Igf2(+m/-p) embryos at E9.25, reverting to wild-type levels by E9.75. This was followed at E9.75 by a significant reduction in the proportion of cells in S phase, quantified by S-phase cytometry counts and BrdU labelling. No significant differences in cell size were detected. We conclude that the majority of the cell number differences between wild-type and Igf2(+m/-p) mice can be accounted for by modification of cell survival and proliferation during the period (E9 to E10) of post-implantation development.