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.     

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Summary Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.Supported by a research grant from the Medical Research Council of Canada (J.H.)     

Aquaporin-9 is expressed in a mucus-secreting goblet cell subset in the small intestine

We analyzed the expression of aquaporins (AQPs) in the small intestine to elucidate their functions, and found that AQP9, which had not previously been detected there, is present in duodenum, jejunum, and ileum. AQP9 is expressed in colon as well, but not in stomach. Also, its expression in these intestinal sections is limited to the basolateral membranes of a goblet cell subset. Our finding that AQP9 is present specifically in goblet cells as mucus-secreting cells suggests its involvement in the synthesis and/or secretion of a certain kind of mucus which may protect the intestinal surface and smooth the flow of intestinal contents.     

Targeted ablation of secretin-producing cells in transgenic mice reveals a common differentiation pathway with multiple enteroendocrine cell lineages in the small intestine.

The four cell types of gut epithelium, enteroendocrine cells, enterocytes, Paneth cells and goblet cells, arise from a common totipotent stem cell located in the mid portion of the intestinal gland. The secretin-producing (S) cell is one of at least ten cell types belonging to the diffuse neuroendocrine system of the gut. We have examined the developmental relationship between secretin cells and other enteroendocrine cell types by conditional ablation of secretin cells in transgenic mice expressing herpes simplex virus 1 thymidine kinase (HSVTK). Ganciclovir-treated mice showed markedly increased numbers of apoptotic cells at the crypt-villus junction. Unexpectedly, ganciclovir treatment induced nearly complete ablation of enteroendocrine cells expressing cholecystokinin and peptide YY/glucagon (L cells) as well as secretin cells, suggesting a close developmental relationship between these three cell types. In addition, ganciclovir reduced the number of enteroendocrine cells producing gastric inhibitory polypeptide, substance-P, somatostatin and serotonin. During recovery from ganciclovir treatment, the enteroendocrine cells repopulated the intestine in normal numbers, suggesting that a common early endocrine progenitor was spared. Expression of BETA2, a basic helix-loop-helix protein essential for differentiation of secretin and cholecystokinin cells was examined in the proximal small intestine. BETA2 expression was seen in all enteroendocrine cells and not seen in nonendocrine cells. These results suggest that most small intestinal endocrine cells are developmentally related and that a close developmental relationship exists between secretin-producing S cells and cholecystokinin-producing and L type enteroendocrine cells. In addition, our work shows the existence of a multipotent endocrine-committed cell type and locates this hybrid multipotent cell type to a region of the intestine populated by relatively immature cells.     

GLP-1 and GIP are colocalized in a subset of endocrine cells in the small intestine

BACKGROUND: The incretin hormones GIP and GLP-1 are thought to be produced in separate endocrine cells located in the proximal and distal ends of the mammalian small intestine, respectively. METHODS AND RESULTS: Using double immunohistochemistry and in situ hybridization, we found that GLP-1 was colocalized with either GIP or PYY in endocrine cells of the porcine, rat, and human small intestines, whereas GIP and PYY were rarely colocalized. Thus, of all the cells staining positively for either GLP-1, GIP, or both, 55-75% were GLP-1 and GIP double-stained in the mid-small intestine. Concentrations of extractable GIP and PYY were highest in the midjejunum [154 (95-167) and 141 (67-158) pmol/g, median and range, respectively], whereas GLP-1 concentrations were highest in the ileum [92 (80-207) pmol/l], but GLP-1, GIP, and PYY immunoreactive cells were found throughout the porcine small intestine. CONCLUSIONS: Our results provide a morphological basis to suggest simultaneous, rather than sequential, secretion of these hormones by postprandial luminal stimulation.     

Individual subtypes of enteroendocrine cells in the mouse small intestine exhibit unique patterns of inositol 1,4,5-trisphosphate receptor expression.

Enteroendocrine cells are a complex population of intestinal epithelial cells whose hormones play critical roles in regulating gastrointestinal and whole-animal physiology. There are many subpopulations of enteroendocrine cells based on the major hormone(s) produced by individual cells. Intracellular calcium plays a critical role in regulating hormone release. Inositol 1,4,5-trisphophate (IP3) receptors regulate calcium mobilization from endoplasmic reticulum-derived calcium stores in many endocrine and excitatory cells and are expressed in the intestine. However, the specific subtypes of enteroendocrine cells that express these receptors have not been reported. Immunohistochemical (IHC) studies revealed that enteroendocrine cells did not express detectable levels of type 2 IP3 receptors, whereas nearly all enteroendocrine cells that produced chromogranin A and/or serotonin expressed type 1 and type 3 IP3 receptors. Conversely, enteroendocrine cells that produced glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, cholecystokinin, or somatostatin did not express detectable levels of any IP3 receptors. Subsets of enteroendocrine cells that produced substance P or secretin expressed type 1 (33% or 18%, respectively) and type 3 (10% or 62%, respectively) IP3 receptors. Thus, different subtypes of enteroendocrine cells, as well as individual cells that express a particular hormone, exhibit remarkable heterogeneity in the molecular machineries that regulate hormone release in vivo. These results suggest that therapeutic agents can be developed that could potentially inhibit or promote secretion of hormones from specific subtypes of enteroendocrine cells.     

Detection and characterization of hemopoietic stem cells in the adult human small intestine

The concept of lymphoid differentiation in the human gastrointestinal tract is controversial but is the focus of this study, which examined adult human small intestinal tissue for the presence of CD34(+)CD45(+) hemopoietic stem cells (HSCs) and lymphoid progenitors. Flow cytometry demonstrated that over 5% of leukocytes (CD45(+) cells) isolated from human gut were HSCs coexpressing CD34, a significantly higher incidence than in matched peripheral blood or control bone marrow. HSCs were detected in cell preparations from both the epithelium and lamina propria of all samples tested and localized to the intestinal villous and crypt regions using immunofluorescence. A high proportion of gut HSCs expressed the activation marker CD45RA, and few expressed c-kit, indicating ongoing differentiation. The vast majority of intestinal HSCs coexpressed the T cell Ag, CD7 (92% in the epithelium, 80% in the lamina propria) whereas <10% coexpressed the myeloid Ag CD33, suggesting that gut HSCs are a relatively mature population committed to the lymphoid lineage. Interestingly, almost 50% of epithelial layer HSCs coexpressed CD56, the NK cell Ag, compared with only 10% of the lamina propria HSC population, suggesting that the epithelium may be a preferential site of NKR(+) lymphoid differentiation. In contrast, bone marrow HSCs displayed low coexpression of CD56 and CD7 but high coexpression of CD33. The phenotype of intestinal HSCs, which differs significantly from circulating or bone marrow HSCs, is consistent with a role in local lymphoid development.     

Putrescine uptake and release by a normal rat small intestine crypt cell line, IEC-6

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earle's balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.     

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.