The effect of mechanical strain on fetal rat lung cell proliferation: Comparison of two-and three-dimensional culture systems

Summary Normal growth of the fetal lung is dependent on fetal breathing movements. We have previously reported that an intermittent strain, which simulates normal fetal breathing movements, stimulates DNA synthesis and cell division of mixed fetal rat lung cells maintained in organotypic cultures. To examine which cell type is responding to mechanical strain and to investigate whether the effects of strain on cell proliferation and mechanotransduction are affected by tissue architecture, we isolated fetal lung cells and subjected them to intermittent strain either as two-dimensional monolayer cultures or as three-dimensional organotypic cultures. Strain enhanced DNA synthesis of mixed cells, epithelial cells, and fibroblasts when cultured in a three-dimensional configuration. In contrast, no stimulatory effect on cell proliferation was observed depending on the culture conditions. These results suggest that mechanical strain stimulates the proliferation of both epithelial cells and fibroblasts and that the response of fetal lung cells to mechanical strainin vitro depends on cellular architecture.     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Differentiation of human airway epithelia is dependent on erbB2

A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, we treated an in vitro model of well-differentiated human airway epithelia with trastuzumab or heregulin-alpha, an erbB ligand. In addition, coculturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-alpha treatment increased epithelial height and decreased the number of metaplastic and nonciliated columnar cells, whereas it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factor-alpha, heparin-binding epidermal-like growth factor, epiregulin, heregulin-alpha, and amphiregulin, all of which are erbB ligands. Cocultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-alpha treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that may direct various pathways of epithelial differentiation.     

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.     

Detection of endothelin-like immunoreactivity in epithelium and fibroblasts of the human umbilical cord

We studied tissue sections of freshly obtained full-term and premature human umbilical cords using polyclonal antibody to endothelin and immunocytochemistry. Endothelin immunoreactivity was detected in the cytoplasm of epithelial cells and primitive fibroblasts, but not in the endothelial cells of both full-term and premature umbilical cords. Immunoelectron microscopy using indirect immunogold staining technique localized endothelin immunoreactivity to the cytoplasm of the epithelial cells and fibroblasts but not confined to any particular structures. No endothelin immunoreactivity was detected in the nucleus or on the cell membrane. Pre-absorption tests with synthetic endothelin-1, -2, and -3 independently established that the immunoreactivity represented endothelin-1 and -2, but not -3. The presence of endothelin-1 and -2-like immunoreactive materials in epithelial cells and fibroblasts of human umbilical cord suggests a role of endothelin in parturition.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.     

A comparison of primary oesophageal squamous epithelial cells with HET‐1A in organotypic culture

Background Information. Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5‐year survival of less than 15%. Recent evidence suggests that stromal—epithelial interactions are fundamental in carcinogenesis. The advent of co‐culture techniques permits the investigation of cross‐talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET‐1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. Results. When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET‐1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET‐1A cells were highly proliferative and did not express the epithelial markers E‐cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N‐cadherin. Conclusion. Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET‐1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.     

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3^- or Cl^-. Bestrophin genes represent a newly identified group of calcium-activated Cl^- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.     

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.     

Development of fetal rat intestine in organ and monolayer culture

Maturation and differentiation of intestinal epithelial cells was demonstrated in segments of fetal rat small intestine, maintained for more than a month in suspension organ culture, by ultrastructural, biochemical, and immunological criteria. Over a 5-7 d period, fragments of fetal intestine evolved into globular structures covered with a single columnar epithelium ultrastructurally similar to suckling villus cells. Loose mesenchymal cells, cellular debris, and collagen were present inside the structures. After 6 d in culture, goblet cells, not present in the fetal intestine at day 18, were numerous and well developed. Intestinal endocrine cells were also observed. Immunofluorescence studies employing monoclonal antibodies specific for villus and crypt cells in vivo, and various enzyme assays, have demonstrated a level of differentiation and maturation of the cultured epithelial cells similar but not identical to that of suckling intestinal mucosa in vivo. Crypts and crypt cell markers were not observed in the the cultures. Addition of glucocorticoids to the culture medium resulted in the induction of sucrase-isomaltase but failed to promote most of the functional changes characteristic of the intestinal epithelium at weaning in vivo. Epithelial cells were identified in explants derived from the organ cultures by their specific expression of intestinal cytokeratin. Differentiation-specific markers, present in the epithelial cells in primary cultures, were lost upon selection and subculturing of pure epithelial cell populations. These results suggest a requirement for mesenchymal and/or extracellular matrix components in the maintenance of the differentiated state of the epithelial cells. The fetal intestinal organ cultures described here present significant advantages over traditional organ and monolayer culture techniques for the study of the cellular and molecular interactions involved in the development and differentiation of the intestinal epithelium.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.     

Effects of interleukin 3, interleukin 4, and fibroblasts on cultures of human lung mast cells in bronchoalveolar lavage fluids

The effects of T cell factors, including interleukin (IL)-3 and IL-4, and fibroblasts on the growth and differentiation of human lung mast cells (MCs) obtained by bronchoalveolar lavage (BAL) were examined. The number of MCs identified by alcian blue-safranin staining was twice that of the control culture without conditioned medium (CM) when BAL cells were cultured for 2 weeks in RPMI 1640 containing 10% fetal calf serum and partially purified CM derived from PHA-stimulated lymphocytes. In the presence of both recombinant (r) IL-3 and rIL-4, the number of MCs was twice as high as the control without increase in the per-cell histamine content after 2 weeks' culture. In umbilical cord blood cultures, IL-3 plus IL-4 augmented basophilic cells about 20-fold more than the control when cultured for 2 weeks. In some cases, the percentage of safranin-positive MCs was about 2-5 fold greater, with 2-7 fold higher histamine content, when cultured for 10 days with CM and fibroblasts derived from human embryonic lung. However, in all BAL experiments, there was no increase in the total number of MCs after culture compared with the initial number of MCs, unlike the umbilical cord blood cultures. These results suggest that T cell factors, including IL-3 and IL-4, and fibroblasts may influence the phenotype and the survival of lung mast cells in BAL, whereas there was no evidence for the presence of MC precursors in BAL fluids.     

A three-dimensional model of differentiation of immortalized human bronchial epithelial cells

A therapeutic approach being investigated for a variety of pathologies is tissue regeneration using a patient's own cells. Such studies have been hampered due to the difficulty in growing epithelial cells for prolonged periods in culture. Replicative senescence due to short telomeres and p16 induced by culture stress work together to inhibit cell growth. Forced expression of telomerase (hTERT) can prevent replicative senescence, and expression of the cell cycle protein cdk4 can sequester p16, thereby immortalizing epithelial cells in culture. In the present study, we used this method to immortalize human bronchial epithelial cells (HBECs) to determine whether immortalized HBECs retain the ability to differentiate normally. HBECs were plated atop contracted collagen gels containing lung fibroblasts. This three-dimensional (3D) tissue model was cultured initially submerged, then raised to the air/liquid interface for up to 28 days. Normal differentiation was assessed by the presence of ciliated cells, goblet (mucin-producing) cells, and basal epithelial cells. Scanning electron microscopic observations revealed both ciliated and non-ciliated cells in these 3D tissues. Histological examination revealed the presence of mucin-producing cells, and immunohistochemistry using antibodies against p63 and keratin 14 showed the presence of basal cells. These results demonstrate that immortalized HBECs retain the capacity to differentiate into each of three cell types: basal, mucin-producing, and columnar ciliated epithelial cells. Such cells will be useful cellular reagents for research in aging, cancer progression, as well as normal bronchial epithelial differentiation and will help progress the use of engineered cells to enhance tissue regeneration.     

A mucus-secreting human colonic epithelial cell line responsive to cholinergic stimulation

The human colonic epithelial cell line Cl.16E grows in culture as a polarized monolayer which differentiates at confluency into typical goblet cells secreting their mucin content into the culture medium. Polyclonal antibodies raised against these mucins were used in an ELISA to measure the amount of mucins secreted by the Cl.16E cells. Carbachol caused a transient and significant increase in mucus secretion with a maximal stimulation occurring at 30 min. A dose-dependent effect was found with a maximal stimulation with 10-3M carbachol. This effect was inhibited by atropine. These results indicate that the effects of carbachol are mediated by muscarinic receptors present on mucus-secreting epithelial cells.     

Trefoil peptide expression and goblet cell number in rat intestine: effects of KGF and fasting-refeeding

The trefoil factor family peptides TFF1, TFF2, and TFF3 are important for gut mucosal protection and restitution. Keratinocyte growth factor (KGF) stimulates proliferation and differentiation of epithelial cells with potent effects on goblet cells. To investigate interactions between food intake and KGF, rats were fed ad libitum (control), fasted for 72 h, or fasted for 72 h and then refed for 72 h with or without KGF (3 mg. kg(-1). day(-1)). With fasting, goblet cell number in duodenum increased, TFF3 mRNA in duodenum and jejunum decreased, and TFF3 protein did not change or increased. KGF during fasting stimulated colonic growth, normalized TFF3 mRNA in duodenum and jejunum, and broadly upregulated gut goblet cell number and TFF3 protein expression. With fasting-refeeding, KGF increased small bowel and colonic mucosal growth, goblet cell number, and TFF3 protein but had variable effects on TFF3 mRNA. KGF induced TFF2 mRNA and protein in duodenum and jejunum with both nutritional regimens. We conclude that nutrient availability modifies rat intestinal goblet cell number, TFF3 mRNA, and the gut-trophic effects of KGF in a region-specific manner. KGF enhances TFF2 expression in proximal small bowel and increases goblet cell number and TFF3 protein content throughout the intestine independent of food intake.     

Similar protective effects of BQ-123 and erythropoietin on survival of neural cells and generation of neurons upon hypoxic injury

Our recent study [Danielyan et al., 2005. Eur. J. Cell Biol. 84, 567-579] showed an additive protective effect of endothelin (ET) receptor A (ETA-R) blockade and erythropoietin (EPO) on the survival and rejuvenation of rat astroglial cells exposed to hypoxia. Whether the effects observed with rodent astroglial cells can be reproduced in human astrocytes and whether these effects of ETA-R blockade and EPO on astrocytes are associated with neuronal survival remained open. Therefore, in the present study, the effects of the ETA-R antagonist BQ-123 and EPO on the maintenance of the neuronal population and survival of the human fetal astroglial cell line (SV-FHAS) under normoxic and hypoxic conditions (NC and HC, respectively) were investigated. Rat brain primary cultures exposed to BQ-123 and/or EPO revealed an increase in the number of beta-III tubulin-positive neurons under NC. The hypoxia-caused loss of neurons was abolished by administration of BQ-123 or EPO. Simultaneous application of EPO and BQ-123 led to an additive protective effect on the generation of neurons under NC only. By contrast, BQ-788, the selective ETB-R antagonist, diminished the neuronal population both in NC and HC. Both under NC and HC the number of non-differentiated nestin+/GFAP- neural cells increased upon application of EPO or BQ-123. SV-FHAS responded to BQ-123 or EPO by a decrease in LDH activity in the culture medium under NC (35%) and HC (26% LDH decrease). Concomitant effects of EPO and BQ-123 were illustrated in an additional increase in the survival of human astrocytes (33% under NC and 17% under HC). These data hint at a neuroprotective therapeutic potency of ETA-R blockade, which either alone or in combination with EPO may improve the survival of astroglial and neuronal cells upon hypoxic injury.