Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

An in vitro system for the study of tracheal epithelial cells

Enzymatically dissociated hamster tracheal epithelial (HTE) cells were cultured on collagen coated Millicell filters. Within 3-5 days after being placed in culture large numbers of ciliated and mucus cells began to appear. By 1 week the HTE cells closely resembled those seen in vivo, i.e. columnar morphology and organelle polarity. After 4 weeks in vitro the HTE cultures were still able to maintain the polarity and overall columnar morphology found in in vivo tissue. There was, in addition, no apparent degradation of the collagen substrate. Auto-radiographic data indicated that there was an initial period of high DNA synthesis during the first 3 days in vitro. This was followed by a second phase in which, by day 6, the amount of DNA synthesis was greatly reduced. Analysis of the numbers of ciliated cells relative to non-ciliated cells demonstrated that between days 5 and 8 there was an increase in the percentage of ciliated cells, suggesting that cellular differentiation (i.e. ciliogenesis) follows cellular proliferation. The results of this study show that when HTE cells are grown on collagen-coated Millicell filters there is a significant improvement in cell growth and morphology yielding cells that are very similar to those present under in vivo conditions. Moreover, since there is no degradation of the collagen substrate, HTE cultures may be suitable for long-term studies of respiratory tract epithelia.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Immunocytochemical localization of laminin in hamster tracheal epithelial cell cultures

EM examination of 28 day cultures of enzymatically dissociated hamster tracheal epithelial (HTE) cells grown on collagen coated millipore filters reveals that fragments of basal lamina may be present at the basal plasmalemma. Since the basal lamina consists of several major components including type IV collagen, heparan sulfate proteoglycans, entactin/nidogen, and laminin, questions naturally arise concerning the presence of such a structure in this cell culture system. When immunocytochemical procedures utilizing anti-laminin antibody and PAP techniques are carried out with paraffin sections of HTE culture at 1,2,3, and 4 weeks in vitro, LM analysis reveals that a thin, dense line of reaction product is present between the basal surface of the HTE cells and the underlying collagen substrate. Immunoblotting evaluation carried out with supernatants of 7d HTE cell homogenates and HTE cell conditioned media also indicate that laminin is being produced by the tracheal cells. Thus, the presence of basal lamina-like fragments, the immunocytochemical localization of laminin, and immunoblot identification of laminin in hamster tracheal epithelial cell cultures, suggest that, although basal lamina components may be produced by HTE cells, at the time points tested, they are not yet being organized into a complete basal lamina.     

An in vitro airway wall model of remodeling

Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1). mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2). inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma.     

Secretion of lactoferrin and lysozyme by cultures of human airway epithelium

Lactoferrin and lysozyme are important antimicrobial compounds of airway surface liquid, derived predominantly from serous cells of submucosal glands but also from surface epithelium. Here we compared release of these compounds from the following human cell cultures: primary cultures of tracheal epithelium (HTE), Calu-3 cells (a lung adenocarcinoma cell line frequently used as a model of serous gland cells), 16HBE14o- cells (an SV40 transformed line from airway surface epithelium), T84 cells (a colon carcinoma cell line), and human foreskin fibroblasts (HFF). For lysozyme, baseline secretory rates were in the order Calu-3 > 16HBE14o- > HTE T84 > HFF = 0; for lactoferrin, the only cell type showing measurable release was HTE; for mucus, HTE > Calu-3 > 16HBE14o- T84 > HFF = 0. A wide variety of neurohumoral agents and inflammatory stimuli was without effect on lactoferrin and lysozyme release from HTE or Calu-3 cells, although forskolin did stimulate secretion of water and lysozyme from Calu-3 cells. However, the concentration of lysozyme in the forskolin-induced secretions was much less than in airway gland secretions. Thus our data cast doubt on the utility of Calu-3 cells as a model of airway serous gland cells but do suggest that HTE could prove highly suitable for studies of mucin synthesis and release.     

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.     

Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3–4-month-old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell-free medium under conditions in which all of a polyanionic protein [³H]-labeled mucin, was precipitated without carrier. [¹⁴C] galactosamine-labeled AB precipitate was β-eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio-Gel P₂ column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse--labeled for 1.0 hr with [³H] leucine or [¹⁴C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

Physiological 3D tissue model of the airway wall and mucosa

This protocol describes the setup, maintenance and characteristics of a tissue-engineered model of the human bronchial mucosa that can be used for basic physiology and pathophysiology studies. The model includes a well-differentiated epithelium with functional cilia, mucus secretion and subepithelial fibroblasts within type I collagen. The tissue is created within porous polymeric wells to prevent gel contraction and allow culture at the air-liquid interface. It requires at least 2 wk to be established and can be maintained thereafter for over 4 wk, with tissue differentiation moving towards a more physiologically relevant phenotype with increasing time in culture. Over time, the extracellular matrix also remodels, depositing proteins such as types III and IV collagen and fibronectin. Because it recapitulates many key anatomical and functional features of the airway wall, this model is well suited for a wide range of studies, including those on airway remodeling, transepithelial transport and inflammatory cell interactions with the mucosa. The entire protocol takes 4-6 wk, including cell expansion, depending on the extent of ciliogenesis desired.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Secretion of rat tracheal epithelial cells induces mesenchymal stem cells to differentiate into epithelial cells

Bone marrow MSCs (mesenchymal stem cells) can differentiate into various tissue cells, including epithelial cells. This presents interesting possibilities for cellular therapy, but the differentiation efficiency of MSCs is very low. We have explored specific inducing factors to improve the epithelial differentiation efficiency of MSCs. Under inducing conditions, MSCs differentiated into epithelial cells and expressed several airway epithelial markers using RTE (rat tracheal epithelial) cell secretions. Rat cytokine antibody array was used to detect cytokines of the RTE secretion components, in which 32 kinds of protein were found. Seven proteins [TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), TGFβ1 (transforming growth factor β1), MMP-2 (metalloproteinases-2), OPN (osteopontin) and activin A in RTE secretions] were assayed using ELISA kits. The four growth factors (VEGF, BDNF, TGFβ1 and activin A) were involved in regulating stem cell growth and differentiation. We speculated that these four play a vital role in the differentiation of MSCs into epithelial cells by triggering appropriate signalling pathways. To induce epithelial differentiation, MSCs were cultured using VEGF, BDNF, TGFβ1 and activin A. Differentiated MSCs were characterized both morphologically and functionally by their capacity to express specific markers for epithelial cells. The data demonstrated that MSCs can differentiate into epithelial cells induced by these growth factors.     

Growth and differentiation of primary tracheal epithelial cells in culture: regulation by extracellular calcium

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum-free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40-60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (greater than 0.3 mM). High calcium medium also increased cell-cell interactions, formation of desmosomes, and multi-cell layering. The relative content of mucous cells, which were identified by a mucin-specific monoclonal antibody and the presence of mucus-secreting granules at the ultrastructural level, was greater in the high-calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H-glucosamine into mucous glycoprotein fractions, was also increased more than 5-fold in media containing high calcium content (greater than 0.6 mM). In contrast, MTE cells cultured in low calcium medium (less than 0.15 mM) were squamous-like with prominent tonofilaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.     

IL13 activates autophagy to regulate secretion in airway epithelial cells

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.     

Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures

We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.