Isolation of rat intestinal crypt cells

Abstract. A technique is presented which yields single cells and intact crypts in suspension from unfixed rat intestinal mucosal epithelium. Everted lengths of intestine were digested by 27 mM sodium citrate in phosphate-buffered saline (pH = 7.3) at 37°C. Mucosal cells were dislodged by vibratory stress (hand vortexing) following incubation for prescribed intervals at 37°C in 1.5 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM dithiothreitol (dtt). Alkaline phosphatase determinations, phase microscopy, and in vivo and in vitro evaluations of tritiated thymidine ([³H]TdR) incorporation were performed on isolated intestinal cells. Data indicate that cells were sequentially derived from villus tip to crypt base as judged by cellular morphology, alkaline phosphatase activity/mg protein and radioactivity per μg protein. Upon completion of the intestinal cell isolation assay, scanning electron microscopy of the remaining intestine revealed that approximately 95% of the crypt openings were vacant; the villi were totally denuded; the supporting structures, including the lamina propria, appeared intact. In vitro radiolabelling of intestinal cell fractions enriched with crypts revealed a linear incorporation of [³H]TdR from 0–60 min which was strongly influenced by the presence of foetal calf serum (FCS). Measurements of the compensatory response of the mucosa to resection of 70% of the small bowel indicated that the mucosal cell separation is capable of detecting alterations in crypt cell proliferation. Previously, such alterations were monitored by other methods utilizing microdissection procedures or stathmokinetic agents.     

Modulation of in vitro thymidine incorporation into crypt cells from the murine small intestine

A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro. The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [3H]TdR incorporation in vitro. Lymphocyte culture supernatants suppressed [3H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12-O-tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [3H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [3H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [3H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli.     

Modulation of in vitro thymidine incorporation into crypt cells from the murine small intestine

Abstract. A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro . The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [³H]TdR incorporation in vitro . Lymphocyte culture supernatants suppressed [³H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12- O -tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [³H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [³H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [³H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli.     

A METHOD FOR QUANTITATION OF PROLIFERATIVE INTESTINAL MUCOSAL CELLS ON A WEIGHT BASIS: SOME VALUES FOR C57BL/6

A method for determining the number of intestinal mucosal crypts, S cells, and total proliferative cells, on a weight basis has been presented. The number of crypts was obtained (following injection of tritiated thymidine) by dividing the disintegrations per minute (dpm) per mg intestine by the dpm per crypt. Multiplication of the number of crypts per mg by the number of labeled cells per crypt (determined radioautographically) resulted in the number of S cells per mg intestine. Division of the number of S cells per mg by the fraction of proliferative cells in S (obtained by cell cycle analysis) resulted in the number of proliferative cells per mg intestine. Values for duodenum, jejunum, and ileum of male C57BL/6 mice are given.     

Bringing target-matched PI3King from the bench to the clinic

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.     

Mosaic analysis of small intestinal development using the<Emphasis Type="Italic"> spf</Emphasis><Superscript><Emphasis Type="Italic">ash</Emphasis></Superscript>-heterozygous female mouse

Mosaic analysis using the spf(ash)-heterozygous female mouse was performed to clarify the cell lineage and cell behavior during small intestinal development with special attention given to the villus and crypt formation. The spf(ash) mutation, located on the X-chromosome, causes ornithine transcarbamylase (OTC) deficiency, which leads to mosaic expression of this enzyme in the small intestine of the heterozygous female mouse. In the small intestine in heterozygous fetuses, very small patches, which were aggregates of OTC-positive cells or negative cells, with no definite orientation to the villus structures were observed. In the neonatal small intestine, the intervillus region (the presumptive crypts) was polyclonal, and the majority of crypts were comprised exclusively cells of either genotype in 2-week-old small intestine. These results suggest that extensive migration and cell mixing of small intestinal epithelial cells, which have no definite correlation with the villus formation, occur in fetal stages of development, and that the crypt morphogenesis commences after birth independently of the monoclonality of the epithelial cells. Our data with the mosaic mice also reconfirmed the monoclonality of the adult small intestinal crypts demonstrated in mouse aggregation chimeras.     

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.     

Double labelling with bromodeoxyuridine and [3H]-thymidine of proliferative cells in small intestinal epithelium in steady state and after irradiation

The simultaneous immunohistochemical detection of bromodeoxyuridine (BrdU) and [3H]-thymidine ([3H]TdR), by conventional autoradiography, was performed on the mouse small intestine (ileum). Proliferation was studied under normal conditions as well as after 3 Gy of gamma-rays. The BrdU method in conjunction with [3H]TdR autoradiography appears to be reliable and useful for the study of cell kinetics especially in disturbed states, on condition that [3H]TdR is delivered to the animals before BrdU. It has been found that cells in the crypt are delayed by irradiation in their progression through the cell cycle predominantly in late S phase. The cells at the bottom of the crypt are more affected than the more differentiated but proliferating cells in the upper part of the crypt.     

A method for the isolation and culture of human colonic crypts in collagen gels

Summary Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 ±1.2×10⁴ ( ± SD) crypts/cm² of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.     

Effects of puromycin, cycloheximide and noradrenaline on cell migration within the crypts and on the villi of the small intestine. A model to explain cell movement in both regions

The normal process of cell migration, occurring as part of the replacement scheme within the small intestinal epithelium, was investigated extensively. The effects of puromycin, cycloheximide and noradrenaline on the movement of tritiated thymidine [( 3H]TdR) prelabelled crypt or villus cells have been studied. These studies have led to the formulation of a model for the mechanism of cell migration, postulating that the crypts and villi behave as separate units, with regard to cell migration, in addition to their distinct structural and functional properties. It is proposed that crypt cell migration is an active process requiring protein synthesis and protein glycosylation, whilst movement of villus epithelial cells is passive, depending on the continued contraction of smooth muscle cells in the lamina propria.     

Cell position dependence of labelling thymidine nucleotides using the de novo and salvage pathways in the crypt of small intestine

About twice as much tritiated thymidine ([3H]TdR) is taken up by cells at the bottom of the crypt of the small intestine as by the rapidly cycling mid-crypt cells. However, the uptake of tritiated deoxyuridine ([3H]UdR) is even throughout the crypt. Exogenous thymidine is incorporated about four times and eight times more efficiently than deoxyuridine by the cells in the mid-crypt and cells at the bottom of the crypt, respectively. However all S phase cells in the crypt appear to be capable of using either precursors, i.e. either the de novo or salvage pathway. Since methotrexate (1 or 5 mg/kg) inhibits (at 5 mg/kg completely) the uptake of [3H]UdR, but has no effect on [3H]TdR uptake, the de novo and salvage pathways appear to be independent. Within the precision of the methods used in the experiments the 3 hr inhibition of the de novo pathway of deoxythymidylic acid (dTMP) synthesis by methotrexate does not produce any increase in utilization of the salvage pathway measured by incorporation of [3H]TdR into DNA. The increased efficiency of thymidine utilization by crypt base cells is not attributable to differences in accessibility of thymidine; differences in the rate of DNA synthesis or the size of the nuclei. It appears that crypt base cells (which include the putative stem cells) are efficient scavengers of [3H]TdR, and this might be related to the level of thymidine kinase activity within the cells, and/or to changes in the availability of endogenous thymidine (break-down products) which compete with exogenous [3H]TdR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of the intestinal epithelium in B10.A mice to infection with Trichinella spiralis

Previous studies on intestinal trichinosis have dealt mainly with areas other than the intestinal epithelium. Since the epithelium is now known to be the parasite's habitat, its response to infection is important. Infection with Trichinella spiralis in immunologically slow-responding B10.A mice was associated with crypt hyperplasia and villus atrophy. With similar infection levels in both primary and challenge infections, there was no difference in the maximal degree of atrophy or hyperplasia between the 2 groups. However, challenged mice underwent these mucosal changes in about half the time. Expulsion of worms always occurred during regeneration of the intestinal epithelium suggesting that the host's defense mechanism of altering the kinetics of the epithelium was not the prime factor causing expulsion. Pulse labelling of enterocytes with [3H] thymidine showed that there was no significant increase in the relative size of the proliferation zone. This indicates that the crypt cell output was not altered by this parasite. Atrophy of the villus was analysed with respect to its 3-dimensional shape. There was a decrease in both height and width of the villus but not thickness. Thus, there is a real decrease in the size of the enterocyte population per villus. Histochemical staining of the enterocyte brush border by an alkaline phosphatase method showed that (1) hyperplastic crypts have an enlarged maturation zone and (2) the villus epithelium is composed entirely of mature cells. The distribution of the nematode population was compared to these changes in the intestine. Trichinella spiralis showed a marked anteriad (distal to proximal) migration prior to expulsion. Thus, utilizing a novel approach to study intestinal trichinosis, the response of the mucosal epithelium has been characterized.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Influence of irradiation or thymidine (TdR) on the pattern of 3H-TdR incorporation at each cell position in the crypts of the small intestine of the mouse

Using autoradiographic methods it was noted that S phase cells at the bottom of the crypts in the small intestine were the most efficient scavengers of exogenous injected thymidine. The efficiency of the incorporation of 3H-TdR (salvage pathway of DNA synthesis) by cells at the crypt base (stem cell zone) was twice as high as for the S phase cells at the top of the crypt (maturing proliferative cells). There were no such position-dependent differences in incorporation of 3H-UdR (de novo pathway of DNA synthesis). Radiation (0.75-5.0 Gy 137Cs gamma-rays) inhibited the incorporation of 3H-TdR very rapidly and this was also cell-position dependent. The cells at the bottom of the crypt were the most affected. The injection of cold thymidine before 3H-TdR changed the pattern of the incorporation of 3H-TdR along the side of the crypt in a very similar way to radiation, and the grain number was decreased predominantly in the cells at lower positions. The possibility of the existence of a regional gradient of endogenous thymidine (reutilization from intestinal sources), and the influence of irradiation on the gradient of thymidine incorporation resulting from direct and abscopal effects of whole body exposure, are discussed.     

CHANGES IN INTESTINAL CELL KINETICS IN THE SMALL INTESTINE OF LACTATING MICE

The enlargement of the small intestine of mice during lactation is due, at least in part, to hyperplasia in the mucosal crypts and villi. The number of cells per crypt increases by 130% and the cell production rate by 63% after 15 days of lactation. These parameters were measured from crypt squashes and sections using both double-label and PLM techniques. Neither the numbers of crypts and villi in the small intestine nor the turnover time of post-mitotic cells on the villi changed. A number of factors appear to act in concert during lactation to trigger this increase in epithelial cell number in the small intestine. The experiments reported suggest a role for the increased quantity of food consumed by the lactating animal, for changing hormonal levels, and for the increased demands placed on the body by milk production.     

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.     

Cell-specific effects of insulin receptor substrate-1 deficiency on normal and IGF-I-mediated colon growth

Insulin-like growth factor I (IGF-I) potently stimulates intestinal growth. Insulin receptor substrate-1 (IRS-1) mediates proliferative and antiapoptotic actions of IGF-I in cell lines, but its in vivo relevance in intestine is not defined. This study tested the hypothesis that there is cell type-specific dependence on IRS-1 as a mediator of IGF-I action. Length, mass, crypt cell proliferation, and apoptosis were measured in small intestine and colon of IRS-1-null mice and wild-type (WT) littermates and in colon of IRS-1-null or WT mice expressing IGF-I transgenes. Expression of IGF-I receptor and signaling intermediates was examined in intestine of WT and IRS-1-null mice, cultured intestinal epithelial cells, and myofibroblasts. Absolute IRS-1 deficiency reduced mucosal mass in jejunum and colon, but effects were more pronounced in colon. Muscularis mass was decreased in both segments. In IGF-I transgenics, IRS-1 deficiency significantly attenuated IGF-I-stimulated growth of colonic mucosa and abolished antiapoptotic but not mitogenic effects of IGF-I transgene on crypt cells. IGF-I-induced muscularis growth was unaffected by IRS-1 deficiency. In intestinal epithelial cells, IRS-1 was expressed at higher levels than IRS-2 and was preferentially activated by IGF-I. In contrast, IGF-I activated both IRS-1 and IRS-2 in intestinal myofibroblasts and IRS-2 activation was upregulated in IRS-1-null myofibroblasts. We conclude that the intestinal epithelium but not the muscularis requires IRS-1 for normal trophic actions of IGF-I and that IRS-1 is required for antiapoptotic but not mitogenic effects of IGF-I in the intestinal crypts in vivo.     

THE EFFECT OF INTESTINAL RESECTION ON THIRY-VELLA FISTULAE OF JEJUNAL AND ILEAL ORIGIN IN THE RAT: EVIDENCE FOR A SYSTEMIC CONTROL MECHANISM OF CELL RENEWAL

Local and systemic control mechanisms have been postulated to explain the maintenance of steady state cell renewal in intestinal epithelium. Permanent alterations of cell renewal resulting in a new steady state imply alterations in control. Intestinal resection appears to cause such alterations resulting in hyper-plasia of the residual intestine. To test the hypothesis of a systemic control, the effect of 60% mid-intestinal resection on Thiry-Vella fistulae of both jejunal and ileal origin was observed in rats. Results showed that hypoplasia occurred in fistulae without resection of the remaining intestine in continuity. Cell counts of crypt and villus columns and tritiated thymidine uptake in isolated whole crypts were reduced. Scanning electron microscopy showed marked hypoplastic alterations in villi. However, when 60% of the intestine in continuity was resected, hyperplasia occurred not only in the residual intestine but in the fistulae of both jejunal and ileal origin. Cell counts of villus and crypt columns were increased along with increased tritiated thymidine uptake per crypt. Neutral cc-glucosidase and non-specific esterase activities did not change as a result of resection but the activities of both enzymes were greater in ileal fistulae than in ileum in situ. Observations on the different resection response of the jejunal versus ileal fistulae lead to a distinction between inherent and induced differences within the small intestine. This study suggests a systemic control of cell renewal. A possible mechanism involving intestinal vascular physiology is discussed.