Basal lamina formation by epithelial cell lines correlates with laminin A chain synthesis and secretion.

Laminin is a major component of the basal lamina upon which all epithelial cells reside in vivo. The synthesis of basal lamina components and their subsequent assembly into a morphologically distinct basal lamina is a differentiated function of epithelial cells in vivo. Ultrastructural studies in our laboratory show that some epithelial cell lines (P-MDCK) form a basal lamina when cultured on membrane-permeable substrate (Millipore Millicells or type I collagen gels). Under the same conditions other epithelial cell lines (MDCK-AA7, M-mTAL-1P, and T84) do not form a basal lamina. When metabolically labeled with [35S]methionine, laminin A and B chains can be immunoprecipitated from the culture medium and culture lysates of P-MDCK cells. In contrast, labeled laminin chains cannot be immunoprecipitated from the culture medium of MDCK-AA7, M-mTAL-1P, and T84 cells. Immunoprecipitates of MDCK-AA7, M-mTAL-1P, and T84 culture lysates demonstrate the presence of one or both B chains but not A chains. These results suggest that laminin B chain synthesis is constitutive in MDCK-AA7, M-mTAL-1P, and T84 cells and that B chains, in the absence of A chains, are not secreted. Furthermore, laminin secretion and basal lamina formation are not required to maintain structural and functional polarity in these cell lines.     

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

Summary Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.     

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.     

Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells

Summary Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro. One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE). A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture. Here we report that by growing the HTE cells at 32° instead of 37°C we can totally prevent lysis of the collagen gel. Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media. We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture. The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals. This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells. This work was supported in part by grants HL-19717 and HL-36854 from the National Institutes of Health, Bethesda, MD.     

Human breast epithelium in vitro: The re-expression of structural and functional cellular differentiation in long-term culture

Summary Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/ cell contact or topological organisation of cells prior to embedding within the collagen matrix.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line

The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR). Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media. The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates. Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components. The laminin-HSPG complex was deposited and retained in the ECM. This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels. At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium. The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures. Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG. This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.     

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.     

Regulation of differentiation and keratin protein expression by vitamin A in primary cultures of hamster tracheal epithelial cells.

Hamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A-deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum-free, vitamin A-supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8, 18, and 19). In contrast, vitamin A-deficient HTE cells, which were squamous-like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5, 6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A-deficient hamster tracheal organ cultures. Thus it appears that HTe cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchogenic tumors.     

Fibroblasts contribute to the deposition of laminin 5 in the extracellular matrix

Laminin 5 (alpha3beta3gamma2) is specifically present in the basal lamina underneath epithelia with secretory or protective functions, where it is essential for anchoring basal epithelial cells to the underlying extracellular matrix. Laminin 5 is produced by epithelial cells as a 480-kDa precursor that is converted into forms of 440 and 400 kDa. To analyse the processing of laminin 5, we have used monolayer and co-cultures of epithelial cells and fibroblasts. The processing of the 180-kDa laminin alpha3 chain to 165 kDa in the cell culture medium, and to both 165 and 145 kDa polypeptides in the cell layer, are not modified by the presence of fibroblasts. In contrast, cleavage of the laminin gamma2 chain, occurring in the cell culture medium and in the cell layer, is enhanced by the presence of fibroblasts. Further analysis by immunofluorescence staining and laser-scanning microscopy reveals that deposited laminin 5 is present in a fibroblast-associated filamentous meshwork. Only laminin 5 containing a fully processed gamma2 chain is present in this fibroblast-associated fraction. These studies show that, although laminin 5 is a product of epithelial cells, fibroblasts contribute to its integration into the extracellular matrix architecture.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

A biphasic chamber system for maintaining polarity of differentiation of culture respiratory tract epithelial cells

Summary A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whicutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo. Editor's statement Supported in part by grants from NCI (CA42097) and American Cancer Society (BC-465) to R. W., and grants from The Council for Tobacco Research-USA, and Cystic Fibrosis Foundation to K.B.A.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

Differentiated cultures of primary hamster tracheal airway epithelial cells

Summary Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The, secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI as judged by the appearance of β tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.     

Extracellular Matrix Components Regulating Glandular Differentiation and the Formation of Basal Lamina of a Human Pancreatic Cancer Cell Line in Vitro

The interaction between the extracellular matrix and human tumor-cell clones S2-013 and S2-020, derived from a pancreatic cancer cell line (SUIT-2), was examined in vitro, using various cell differentiation-promoting matrices in two- and three-dimensional cultures. S2-013 cells (well-differentiated tubular adenocarcinoma in xenografts in nude mice) cultured in Matrigel formed glandular structures. Ultrastructural observation revealed a morphological polarity of cells and a distinct basal lamina. On the other hand, S2-020 cells (poorly differentiated tubular adenocarcinoma in xenografts) cultured in Matrigel formed neither glandular structures nor a basal lamina, but only cell aggregates. The morphology of these two sublines cultured in Matrigel expressed the histological degree of differentiation which they presented in nude mice. In contrast, in type I collagen gel, S2-013 cells formed glandular structures without a basal lamina, and in soft agar, they were able to form neither glandular structures nor a basal lamina. S2-020 cells cultured in type I collagen gel or soft agar formed the same simple cell aggregates as in Matrigel. Matrices used in a three-dimensional culture influenced the degree of differentiation in S2-013 cells but had no effect on the morphological differentiation in S2-020 cells. To detect the factors which induce basal lamina formation, S2-013 cells were cultured on a microporous membrane coated with extracellular matrix components such as laminin, type IV collagen, and fibronectin. S2-013 cells formed a basal lamina only on the laminin. These cell lines may be useful in investigating the mechanisms regulating the formation of glandular structures and basal lamina.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.