Clonal growth characteristics of adult human prostatic epithelial cells

Summary The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined. In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement. It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 μg/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm² dish. Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result. The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25%. When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates. The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

Effect of certain growth factors on proliferation in serum-free collagen gel culture of vaginal epithelial cells from prepuberal mice exposed neonatally to diethylstilbestrol

Neonatal treatment with diethylstilbestrol (DES) induces ovary-independent vaginal epithelial changes in mice. The response of vaginal epithelial cells from intact prepuberal BALB/cCrgl mice treated neonatally with 2 micrograms of DES for 5 days to growth-stimulatory and -inhibitory factors was studied using a serum-free collagen gel culture system that sustains the growth of normal vaginal epithelial cells. Cells from control and DES-exposed mice at 21 days of age showed about a 5-fold increase in number during 10 days in a serum-free medium supplemented with transferrin, bovine serum albumin fraction V, insulin, and epidermal growth factor. Epidermal growth factor and insulin stimulated dose-related proliferation of vaginal epithelial cells from both control and DES-exposed mice; however, cells from DES-exposed mice showed a reduced growth response to epidermal growth factor and an increased growth response to insulin, compared with control cells. Insulin-like growth factor I (1-100 ng/ml) tested in the absence of insulin failed to stimulate cell growth. Transforming growth factor-beta (0.05-5 ng/ml) consistently inhibited cell growth in a dose-dependent manner.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Basic fibroblast growth factor and insulin-like growth factor I are strong mitogens for cultured mouse keratinocytes

Basic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six-to-nine fold increase in 3H-thymidine (3H-dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum-free and growth factor-free medium with a Ca++-concentration below 0.1 mM. The half-maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin-like growth factor I/somatomedin C (IGF-I) and insulin act as mitogens. IGF-I shows half-maximal stimulation with 2-3 ng/ml, insulin with 100-500 ng/ml. Basic FGF, IGF-I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half-maximal stimulation at a concentration between 1.5-2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF-I receptor.     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

Growth of normal oral keratinocytes and squamous cell carcinoma cells in a novel protein-free defined medium

Summary A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.     

Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Summary Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64–80, 1984). This serum-free system was used to investigate the activity of cells. bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca²⁺ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major α-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-β) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes. Editor’s Statement This report contributes significantly to our knowledge of keratinocyte cell biology in two ways. First, a serum-free medium has been developed that can now be used by many investigators to define growth versus differentiation factors for these cells. This is important since several impure or relatively crude preparations of factors are known to influence these cells. Second, the finding that TGF-Beta is an inhibitor of keratinocyte growth opens new avenues to investigate the biochemical events leading to differentiation. David A. Sirbasku     

Keratinocyte growth-promoting activity from human placenta

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is non-dialyzable and is destroyed at 100 degrees C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.     

Establishment and characteristics of Gottingen minipig skin in organ culture and monolayer cell culture: relevance to drug safety testing

Skin from Gottingen minipigs was used as a source of tissue for organ and cell culture and compared to human skin for growth conditions and sensitivity to irritants. Optimal organ culture conditions were determined, based on the preservation of the histological structure. These included serum-free, growth factor-free conditions with a calcium concentration of 1.5mM. Formulations in which the calcium concentration were low (0.075-0.15mM) failed to support tissue viability (even in the presence of dialyzed serum). Epidermal keratinocytes were grown from tissue explants and as single cells from enzyme-disrupted tissue. Optimal keratinocyte growth was achieved using a serum-free, growth factor-supplemented culture medium with a calcium concentration of 0.15mM. Fibroblasts were optimally grown from explant cultures using a medium with 1.5mM calcium and 10% fetal bovine serum. The conditions that were optimal for maintenance of intact pig skin, as well as for the isolated cells, are the same conditions that have been shown previously to be optimal for intact human skin and skin cells. In additional studies, pig skin keratinocytes and fibroblasts were exposed to a panel of contact irritants and contact sensitizers. Using growth inhibition as the response, the median effective dose values with each agent were very similar to the values previously determined for human epidermal keratinocytes and human dermal fibroblasts. Taken together, these data suggest that the skin from the Gottingen minipig can be used as a surrogate for human skin in ex vivo skin safety studies.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Purification of an equine apotransferrin variant (thyromedin) essential for thyroid hormone dependent growth of GH1 rat pituitary tumor cells in chemically defined culture

Pituitary tumor cells require thyroid hormones for growth in vivo [Sorrentino, J. M., Kirkland, W. L., & Sirbasku, D. A. (1976) J. Natl. Cancer Inst. 56, 1155-1158]. In vitro, GH1 rat pituitary tumor cells were studied in a serum-free defined medium (PCM-10) formulated with Ham's F12 and Dulbecco's modified Eagle's media (1:1, v/v) supplemented with 2.2 g/L sodium bicarbonate, 15 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulfonic acid (pH 7.2), 10 micrograms/mL human transferrin, 50 microM ethanolamine, 10 micrograms/mL insulin, 10 ng/mL selenous acid, 0.1 nM 3,5,3'-triiodothyronine (T3) and 500 micrograms/mL bovine serum albumin and in the same medium without T3 (PCM-0). The cells only grew in PCM-10 when low concentrations of horse serum were added. Attempts to replace the serum factor requirement with known growth factors and adhesion proteins were unsuccessful. The Mr 65,000-72,000 serum factor regulating T3-induced growth (thyromedin) was purified to homogeneity and identified as equine transferrin R and/or D by amino acid sequencing. The ED50 in PCM-10 was 17-40 micrograms/mL (260-620 nM) while in PCM-0 half-maximum growth was not achieved at 200 micrograms/mL. Concentrations of 75 micrograms/mL in PCM-10 caused 80% of serum-stimulated growth rate. Removal of iron from thyromedin, and assay in iron salts reduced PCM-10, increased the specific activity 110-270-fold to ED50 150 ng/mL (2.3 nM); at 1.0 micrograms/mL, growth in PCM-10 was 16-fold greater than in PCM-0. Iron saturation of thyromedin caused total loss of biological activity. We conclude that the horse transferrin variant isolated in this report is active as apotransferrin.     

Interleukin-6 promotes human epidermal keratinocyte proliferation and keratin cytoskeleton reorganization in culture

We have studied the effects of interleukin-6 (IL-6) on human epidermal keratinocytes by using serum-free culture conditions that allow the serial transfer, differentiation, and formation of well-organized multilayered epithelia. IL-6 at 2.5 ng/ml or higher concentrations promoted keratinocyte proliferation, with an ED₅₀ of about 15 ng/ml and a maximum effect at 50 ng/ml. IL-6 was 10-fold less potent than epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) and supported keratinocyte growth for up to eight cumulative cell generations. IL-6-treated keratinocytes formed highly stratified colonies with a narrower proliferative/migratory rim than those keratinocytes stimulated with EGF or TGF-α; confluent epithelial sheets treated with IL-6 also underwent an increase in the number of cell layers. We also examined the effect of IL-6 on the keratin cytoskeleton. Immunostaining with anti-K16 monoclonal antibodies showed that the keratin network was aggregated and reorganized around cell nucleus and that this was not attributable to changes in keratin levels. This is the first report concerning the induction of the reorganization of keratin intermediate filaments by IL-6 in human epidermal keratinocytes.This work was supported in part by CONACyT grant nos. 1314P-N9507 and G28272-N.     

Propagation of differentiating normal human tracheobronchial epithelial cells in serum-free medium

Serial-passage cultures of normal human tracheobronchial (TB) epithelial cells that exhibit functional differentiation have been established in serum-free medium supplemented with bovine pituitary extract (25 micrograms/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), EGF (5 ng/ml), 10(-6)M each of ethanolamine and phosphoethanolamine, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 80 hours. Further, the passaged cultures retained differentiated morphology as evidenced by secretion of glycoproteins, binding of concanavalin A lectin, and presence of alcian blue and periodic acid Schiff-positive material in their cytoplasm. Ultrastructural observations further supported the functional epithelial nature of the cultures. Most cells exhibited characteristic microvilli on cell surfaces and showed junctional complexes between them. The cytoplasm contained a large number of perinuclear secretory vesicles, a characteristic feature of the differentiated cells. These cultures provide an excellent model to study factors that regulate synthesis and secretion of glycoproteins in normal human TB cells.