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.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

A comparison of the specificity of phosphatidylcholine synthesis by human fetal lung maintained in either organ or organotypic culture.

Human fetal lung (14-18 weeks gestation) was maintained in either organ or organotypic culture. By 4 days in organ culture or 14 days in organotypic culture, epithelial cells within both culture systems exhibited well-developed apical microvilli and possessed numerous intracellular lamellar bodies characteristic of surfactant phospholipid stores. However, analysis of the pattern of synthesis of individual molecular species of phosphatidylcholine by [14C]choline incorporation and reversed-phase h.p.l.c. showed that this apparent maturation was not paralleled by an increased synthesis of the dipalmitoyl species in either culture system. By contrast, the fractional synthesis of dipalmitoyl phosphatidylcholine, expressed as a percentage of total [14C]choline incorporation, decreased with time in both organ and organotypic culture. Moreover, these fractions were not significantly different from those measured in parallel monolayer cultures of mixed human fetal lung cells that displayed mainly fibroblast morphology. These results suggest that the synthesis pattern of phosphatidylcholine species by lung cells in culture is determined principally by their incubation conditions and not by their state of apparent maturation.     

Activities of enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II pneumocytes

Although differentiated fetal and adult type II pneumocytes are ultrastructurally similar, it is not known whether there are metabolic differences between them. We measured the activities of selected enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II cells, in late gestation fetal rat lung explants and in intact lung from rat fetuses of comparable gestational age. The activity of 1-acylglycerophosphocholine acyltransferase was significantly greater in adult type II cells than in fetal type II cells, fetal explants or intact fetal lung. The activity of CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase was similar in fetal and adult type II cells, but significantly lower in explants and intact fetal lung. There was a significant positive correlation between the percentage of alveolar epithelial cells in the cultures and tissue studied and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity. This suggests that the previously reported correlation between phosphatidylglycerol synthesis and the percentage of alveolar epithelial cells in various lung culture systems may be related to the activity of this enzyme. Phosphatidylglycerol synthesis and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity may be metabolic markers of type II cells, whereas the acyltransferase activity may be an indicator of type II cell maturation.     

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.     

Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture

Type II alveolar epithelial cells were isolated from fetal rat lung by differential adherence in monolayer culture. The preparation had a high degree of purity, as assessed by phase contrast microscopy and immunocytochemistry. Purity, based on reactivity with specific anti-adult lung serum (SAALS), which recognizes only type II cells, was 91% for cells isolated from 19-day fetal lungs and 79% for cells isolated from 21-day fetal lungs. The lower purity of type II cells in cultures derived from 1-day postnatal rat lungs (51% cells reactive with SAALS) is probably due to a lower tendency of the type II cells from neonatal rats to adhere to culture dishes than of type II cells from fetal rats. Type II cells isolated from 21-day fetal lungs contained a higher percentage phosphatidylglycerol and incorporated [Me-3H]choline faster into phosphatidylcholine (PC) than type II cells isolated from 19-day fetal lungs. Moreover, in cell preparations derived from lungs at fetal day 21, a higher percentage of epithelial cells contained lamellar bodies than in preparations derived from lungs at fetal day 19. The observation of these differences in the stage of maturation indicates that these differences, which are typical features of the original material, are not obliterated by differentiation during the culture. Type II cells isolated according to the present procedure were capable of synthesizing PC with a high percentage of the disaturated species. This method for the isolation of fetal type II cells may be a useful tool in studies concerning surfactant synthesis and its regulation in the fetal lung.     

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.     

Accelerated fetal lung maturation by estrogen is associated with an epithelial-fibroblast interaction

Summary The role of epithelial-mesenchymal interactions in the stimulation of lung development by estrogen is now investigated using organ cultures of lung from male and female fetal rats taken from Days 17 to 21 of gestation. Estradiol at 1 μg/ml was found to reduce cell proliferation in explants taken during a rapid growth phase (Day 18) and to stimulate surfactant synthesis in both males and females only in Day 20 explants when cell division is much slower. At this time more epithelial cells from estrogen-treated explants contained lamellar bodies, which were also secreted to fill the air sacs. These cultures also showed a significant increase in the frequency of cell-to-cell contacts between epithelial cells and fibroblasts. Uptake of tritiated estradiol by explants increased from Day 18 onward, and by autoradiography, labeling was located predominantly over fibroblasts. Using pure cultures of fetal and adult cells, uptake of labeled estradiol was significantly higher in fibroblasts than in corresponding epithelial cells, and estradiol did not directly enhance palmitate incorporation into epithelial cells. The results suggest that the earlier maturation and increased surfactant synthesis in female fetal lung is related at least in part to enhanced binding of estrogen by the fibroblast with subsequent transfer of a maturation factor to the fetal epithelium. This research project was supported by grants from the Medical Research Council of Canada and the Council for Tobacco Research, U.S.A., Inc.     

The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity

The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.     

Differentiation of the pulmonary surfactant system. Disaturated phosphatidylcholine accumulation in fetal rat lung in vivo and in vitro

Fetal rat lung was placed in organ culture at 15 days gestation (22 days total gestation period), before biochemical and morphological development of the pulmonary surfactant system. At the fifth day of culture numerous Type II cells containing lamellar bodies were present as determined by electron micrography. Phospholipid accumulation in the cultures increased abruptly beginning at 6 days in culture. The phospholipid which accumulated between the sixth and twelfth culture days was composed of 21--27% disaturated phosphatidylcholines. Both the percent of disaturated phosphatidylcholines in the phospholipid fraction and the qualitative pattern of accumulation as a function of time were similar to observations for fetal rat lung developing in vivo. The data presented provide evidence for development of the pulmonary surfactant system in organ culture in vitro.     

Metabolism and function of platelet-activating factor in fetal rabbit lung development

Previously, platelet-activating factor (PAF, PAF-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) had been identified in association with a lamellar-body-enriched fraction of human amniotic fluid obtained from women in labor. In consideration of the fact that fetal lung is the source of lamellar bodies, we have investigated the capacity of the developing lung to synthesize PAF. The specific activity of the PAF biosynthetic enzyme, 1-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase, increased from 116 pmol/min per mg protein in day 21 fetal rabbit lung to 332 pmol/min per mg protein by day 31. Although this enzymatic activity in fetal kidney also increased, it never reached the level found in lung. In contrast, the actyltransferase activity decreased by 80% in fetal liver between days 21 and 31. The acetyltransferase activity in lung was primarily localized in the microsomal fraction (105 000 X g pellet); however a significant proportion of the activity was found in the 18 000 X g pellet. The specific activity of acetyltransferase in adult alveolar type II rat pneumonocytes was significantly higher than that of adult rat lung or rat alveolar macrophages, suggesting that type II cells make a significant contribution to the actyltransferase activity of lung tissue. PAF acetylhydrolase remained relatively constant throughout the gestation in all tissues. The concentration of PAF in the fetal lung increased by 3-fold from 12 to 35 fmol/mg protein, between day 21 and day 31 of development. The concentrations of the PAF precursors, 2-lyso-PAF (1-alkyl-2-lyso-sn-glycero-3-phosphocholine) and the 2-acyl derivative, were several orders of magnitude higher than the PAF concentration. The pulmonary glycogen content decreased from 163 at day 21 to 35 micrograms/mg protein at day 31 of gestation. We suggest that the increase in PAF concentration may participate in the regulation of glycogen breakdown in fetal lung as it does in perfused rat liver (Shukla, S.D., Buxton, D.B., Olson, M.S. and Hanahan, D.J. (1983) J. Biol. Chem. 258, 10212-10214). The formation of PAF in the developing lung and its secretion, in association with lamellar bodies, into amniotic fluid is discussed in relation to parturition.     

ErbB4 regulates fetal surfactant phospholipid synthesis in primary fetal rat type II cells

Insufficient fetal surfactant production leads to respiratory distress syndrome among preterm infants. Neuregulin signals the onset of fetal surfactant phospholipid synthesis through formation of erbB receptor dimers. We hypothesized that erbB4 downregulation in fetal type II epithelial cells will downregulate not only fetal surfactant phospholipid synthesis, but also affect proliferation and erbB receptor localization. We tested these hypotheses using small interfering RNA (siRNA) directed against the erbB4 gene to silence erbB4 receptor function in cultures of primary day 19 fetal rat lung type II cells. ErbB4 siRNA treatment inhibited erbB4 receptor protein expression, fibroblast-conditioned medium induced erbB4 phosphorylation, and fetal surfactant phospholipid synthesis. Cell proliferation, measured as thymidine incorporation, was also inhibited by erbB4 siRNA treatment. Downregulation of erbB4 receptor protein changed erbB1 localization at baseline and after stimulation, as determined by confocal microscopy and subcellular fractionation. We conclude that erbB4 is an important receptor in the control of fetal lung type II cell maturation.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

Culture of differentiated and undifferentiated type II cells from fetal rat lung

We have developed a relatively simple and reproducible method for the isolation and culture of both differentiated and undifferentiated type II cells from fetal rat lung. The technique involves an initial period of explant culture in serum and hormone free medium, followed by enzymatic dissociation of the explants, differential adhesion to remove fibroblasts, incubation of the cell pellet to promote aggregation of the type II cells and monolayer culture of the type II cells. The type II cells form clusters which are surrounded by scattered fibroblasts. When the technique was performed with three differential adhesion steps, cultures contained 86.0 +/- 1.4% type II cells. To obtain a higher degree of purity and greater yield, two differential adhesions followed by gentle trypsinization of the cultures which selectively removes the isolated fibroblasts was performed. This resulted in cultures with 89.4 +/- 1.7% type II cells. The differentiated fetal type II cell cultures were prepared from 19-day fetal rat lungs which were initially maintained in explant culture for 48 h. These differentiated cells demonstrated the characteristic morphologic features of type II cells including lamellar bodies and microvilli. Undifferentiated fetal cells were prepared in a similar manner from 18-day fetal rat lung maintained in explant culture for 24 h. These cells did not contain intracellular osmiophilic granules; the appearance of these granules could, however, be induced by hormones. For this reason they are considered to be pre-type II cells. The viability of the cultured cells was 97%. Both the differentiated and undifferentiated fetal type II cells specifically bound the Maclura pomifera lectin, a type II cell surface marker. The phospholipid profile of the fetal cells was similar to that of adult rat type II cells; the differentiated fetal cells, however, synthesized less phosphatidylcholine than the adult cells did, but more than the undifferentiated fetal cells. The differentiated fetal cells secreted phosphatidylcholine at a basal rate of 0.6% +/- 0.1% during a 90-min incubation. There was dose-dependent stimulation of phosphatidylcholine secretion after exposure to terbutaline. Maximum stimulation (76%) was observed at a concentration of 10 microM. This culture system provides a valuable model for studies of the maturation of the undifferentiated fetal type II cell and surfactant metabolism and secretion in the differentiated fetal type II cell.     

Time-dependent effects of maternal continuous propranolol on fetal lung development in rats

Previous studies have demonstrated a role for the beta-adrenergic system in the maturation of the fetal alveolar epithelium. Chronic blockade of beta-adrenergic binding sites has been shown to adversely effect physiologic and biochemical indices of fetal lung maturation. In the present study timed-pregnant female Sprague-Dawley rats were treated with a continuous 0.5 mg/hr dose of propranolol HCl, or saline, via an osmotic pump. The treatment periods were days 18-21, or 20-23 of gestation. Fetal body weights were obtained, and the morphology of the fetal lungs studied by light and electron microscopy. Cytoplasmic volume densities of lamellar inclusion bodies and glycogen within developing type II alveolar epithelial cells were also determined. In addition, total phospholipids (as phosphorus) and glycogen content were determined biochemically. The fetuses from females treated from day 20-23 demonstrated no differences between saline-treated and propranolol-treated groups, in either fetal weight or the morphologic appearance of the developing lung. In contrast, the fetuses from mothers treated from day 18-21 with propranolol were significantly smaller, and their lungs appeared less mature than saline-treated counterparts. The glycogen content of developing type II alveolar epithelial cells was significantly more abundant (as judged by stereologic and biochemical analyses) in the propranolol-treated fetuses. In addition, total phospholipids were decreased in the propranolol-treated 21-day fetuses. The results of the present study suggest that the development of the alveolar epithelium is sensitive to continuous beta-adrenergic blockade by propranolol during a critical time late in gestation.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

Effects of Anoectochilus formosanus Hayata extract and glucocorticoid on lung maturation in preterm rats.

We investigated the effects of maternal administration of Anoectochilus formosanus extract and dexamethasone on lung maturation in preterm rats. A. formosanus group mothers were tube-fed A. formosanus extract (300 mg/kg body wt./day) for 7 days from days 12-18 of gestation. Dexamethasone group mothers were injected intraperitoneally with dexamethasone (0.2 mg/kg body wt.) in saline on day 18 of gestation. Control group mothers were similarly injected with saline alone. On day 19 of gestation, fetuses were delivered by cesarean section. A. formosanus treatment significantly increased the fetal lung/body weight ratio, as compared to dexamethasone treatment. Saturated phosphatidylcholine levels in fetal lung tissue and growth hormone levels in maternal serum were significantly increased in the A. formosanus- and dexamethasone-treated groups as compared to controls. The histological appearance of preterm rat lungs revealed extensive branching of intermediate airways, denser mesenchyme, and more epithelial tubules in the dexamethasone and A. formosanus groups as compared with the control group. These results suggest that antenatal A. formosanus treatment may play a role in accelerating fetal rat lung maturation.     

Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures

Summary The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3–8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3–8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.     

Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.