Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Characterization of rat colonic epithelial cell populations

Colonic epithelial cells from male Sprague-Dawley rats were isolated by incubating everted colon with hyaluronidase suspended in Puck's saline F with an average cell yield of 120 x 10(6). These cells were fractionated by discontinuous Ficoll gradient and by short-term cell culture techniques. Centrifugation of isolated cells on discontinuous Ficoll gradient (15-35%) yielded populations differing in their proliferative activity. Additionally, a short-term cell culture technique was standardized to fractionate these cells according to their proliferating activity as judged by their DNA synthesis and thymidine kinase activity. Viability of these cells were judged by trypan blue exclusion, capacity to oxidize glucose and incorporation of precursors into protein DNA, RNA and glycoproteins. These fractionated cells were examined and identified by cytological studies. Cells showing proliferative activity sedimented at heavier regions of the Ficoll gradient, and the majority of these cells attached to the surface under conditions of short-term culture. Columnar mature absorptive cells and mucus-secreting goblet cells that showed very little proliferative activity sedimented at lighter zones of the Ficoll gradient and a major portion of these cells failed to attach by the cell culture method.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

Prostate epithelial stem cell culture

The prostate gland is the site of the second most common cancer in men in the UK, with 9,280 deaths recorded in 2000. Another common disease of the prostate is benign prostatic hyperplasia and both conditions are believed to arise as a result of changes in the balance between cell proliferation and differentiation. There are three types of prostatic epithelial cell, proliferative basal, secretory luminal, and neuroendocrine. All three are believed to be derived from a common stem cell through differentiation along different pathways but the mechanisms behind these processes is poorly understood. In particular, there has until recently been very little information about prostate stem cell growth and differentiation. This review will discuss ways of distinguishing these prostate cell types using markers, such as keratins. Methods available for the culture of prostate epithelial cells and for the characterisation of stem cells both in monolayer and three-dimensional models are examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.     

Characterization of the rabbit gastric epithelial lineage progenitors in short-term culture

Little is known about the mechanisms that establish and maintain the proliferation and differentiation programs of the gastric epithelium. This is largely due to the complexity of the gastric epithelial units and the presence of the different epithelial lineage progenitors among heterogeneous populations of various mature cell types. This study is undertaken to establish an in vitro system highly enriched for gastric epithelial lineage progenitors. By using adult male rabbits, a simple method of isolating gastric epithelial cell fractions enriched in lineage progenitors was applied. Cultured cells labeled with bromodeoxyuridine were characterized by using lectin and immunohistochemical studies at light- and electronmicroscopical levels. Analysis of primary cultures derived from the progenitor cell region of the epithelial units revealed that this system can support the proliferation and some of the differentiation programs of the progenitor cells. Cultured cells can be maintained for up to 5 days, while retaining most of the morphological features, molecular markers, and dynamic behavior of gastric epithelial progenitors. Differential cell counts at 1-day culture revealed that, while the undifferentiated progenitors formed about 30% of all attached cells, pre-pit, pit, and preparietal cells represented about 30%, 10%, and 2%, respectively. By 3 days, the increase in the percentage of pit and preparietal cells up to 25% and 9%, respectively, reflected their production in vitro. In conclusion, we have established a culture system enriched for gastric epithelial lineage progenitors that would hopefully allow the identification of factors and mechanisms involved in controlling their proliferative activity and differentiation pathways.     

Kupffer cells modulate natural killer cell activity in vitro by producing prostaglandins

The effect of Kupffer cells on natural killer (NK) cell-mediated cytotoxicity was examined. Kupffer cells prepared from rat liver suppressed NK activity against K562 cells and other tumor cell lines through a soluble factor secreted into the culture supernatant. When human peripheral blood mononuclear cells were incubated with the Kupffer cell-culture supernatant, a significant reduction of the cytotoxic activity was observed in the 6-hr chromium-release assay. This activity was dose dependent and was evident at various effector/target cell ratios. Lipopolysaccharide stimulated generation of the suppressive factor released from Kupffer cells in a dose-dependent manner. Suppression of the NK activity was observed when the Kupffer cell-culture supernatant was present in the assay system, whereas pretreatment of effector/target cells with the supernatant had minimal inhibitory effects. Autologous monocytes in human peripheral mononuclear cells were not related to this suppression. The suppressive factor in the fraction had a molecular weight below 10,000. Indomethacin, an inhibitor of prostaglandin synthesis, ameliorated the suppressive effects. These results suggest that Kupffer cells may modulate NK activity by producing PGs (E1, E2, and F2 alpha).     

Primary culture of gill epithelial cells from the sea bassDicentrarchus Labrax

Summary We have developed the first explant technique that allows the in vitro study of gill physiology and biochemistry in marine species. Gill fragments were cultured at 17° C, in atmospheric Pco₂, with nutrient medium (Leibovitz L15), pH 7.8, supplemented with 10% fetal bovine serum and adjusted to the osmolarity of fish plasma (350 mOsm/liter). Coating plates with collagen, gelatin, or polylysin did not improve our results. Decrease in osmotic pressure, removal of bovine serum, or its replacement by fish serum inhibited growth from the explants. Approximately 50% of the explants produced cell growth, and after 4 days of culture a monolayer of contiguous cells was formed. This technique is rapid and does not require the use of enzymes. The cells appeared flat and thin with an epithelioid shape. They looked polygonal with a maximum length of 10 to 50 μm. Evidence that they are unique gill cells is the presence of polymorphic surface crenelations (microplicae), prominent Golgi apparatus, tight junctions and desmosomes. Comparison with in vivo tissue showed them to be epithelial cells having differentiated in a homogeneous population of respiratorylike (pavement) cells. They are polarized with their apical surface facing the culture medium. The development of this culture system represents a new tool for cellular approaches to determine precisely the functions and transport mechanism of gill cells.     

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified ³⁵SO₄-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.     

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

Summary The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.     

A colonic mineralocorticoid receptor cell model expressing epithelial Na channels

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of β- and γ-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.     

Induction of colonic epithelial cell apoptosis by p47-dependent oxidants

Exogenous oxidants appear capable of initiating both proliferative and death signals, but the role of endogenous oxidants in either tumorigenesis or tumor suppression is unclear. We found that expression of the NAD(P)H oxidase adapter p47^phox was suppressed in human colon carcinoma specimens relative to adjacent normal colon. Overexpression of p47^phox increased apoptosis in colon cancer cell lines independent of p53 and mismatch-repair competency. p47^phox was found to interact with the c-Abl adapter Abl interactor-1 (ABI-1), and p47^phox coprecipitated with both ABI-1 and c-Abl. Ectopic expression of p47^phox in colon cancer cells increased oxidant production with phosphorylation and activation of nuclear c-Abl and consequent apoptosis. Colonic epithelial p47^phox may be specifically targeted to a c-Abl-containing complex that serves a physiologic tumor suppressing function.     

Distribution of prostaglandins in rabbit kidney

Three prostaglandins (PGE(2), PGF(2alpha) and PGA(2)) are present in rabbit kidney medulla. An acidic lipid extract (0.165g) obtained from 2kg of frozen rabbit kidney cortex was separated by silicic acid chromatography to yield eluates containing fatty acids, possible non-polar prostaglandin metabolites, PGA, PGE and PGF compounds. Ultraviolet spectra of the eluates before and after treatment with sodium hydroxide did not yield chromophores typical of any known prostaglandins or related metabolites. By using more sensitive bioassay procedures (contraction of rabbit duodenum) weak activity equivalent to 60mug of PGE(2) and 10mug of PGF(2alpha) was detected in the PGE and PGF eluates respectively. Extraction and bioassay of fresh kidney cortex revealed no prostaglandin-like activity. Attempts to biosynthesize prostaglandins in fresh homogenates of rabbit kidney cortex from endogenous precursors and from added arachidonic acid were unsuccessful. When freshly prepared homogenates of rabbit kidney cortex were incubated with added PGE(1) no evidence of enzymic breakdown was obtained. It is concluded that rabbit kidney prostaglandins are present predominantly in the medulla and there are no cortical mechanisms for their biosynthesis or inactivation under normal conditions.     

Primary culture and immortalization of human fallopian tube secretory epithelial cells

Primary human fallopian tube secretory epithelial cell (FTSEC) cultures are useful for studying normal fallopian tube epithelial biology, as well as for developing models of fallopian tube disease, such as cancer. Because of the limited ability of primary human FTSECs to proliferate in vitro, it is necessary to immortalize them in order to establish a cell line that is suitable for long-term culture and large-scale in vitro experimentation. This protocol describes the isolation of FTSECs from human fallopian tube tissue, conditions for primary FTSEC culture and techniques for establishing immortal FTSEC lines. The entire process, from primary cell isolation to establishment of an immortal cell line, may take up to 2 months. Once established, immortal FTSECs can typically be maintained for at least 30 passages.