Effect of hydrocortisone on the maturation of human foetal kidney explants in serum-free organ culture

The effect of hydrocortisone on the in vitro maturation of human foetal kidney was investigated. Following legal therapeutic abortions, explants of renal cortex from foetuses aged 13-18 weeks were cultured for 5 days in serum-free Leibovitz's L-15 medium at 37 degrees C in a mixture of 95% air - 5% CO2, without hormone (controls) or with hydrocortisone at concentrations of 12.5, 25, or 50 ng/mL, which are the levels representative of different gestational periods. During the studied period of culture, the overall architecture of the renal structures was preserved without any evident signs of nephrogenesis induced by hydrocortisone. DNA synthesis was measured by incorporation of [3H]thymidine and was stimulated on day 5 by 80% with the addition of hydrocortisone at 12.5 ng/mL, and by 131% with 50 ng/mL. In autoradiograms, the sites of [3H]thymidine incorporation were the same after hydrocortisone addition, but the number of labelled nuclei was higher in 5-day explants supplemented with hydrocortisone at 50 ng/mL. The activities of some brush border enzymes (leucylnaphthylamidase, maltase, and alkaline phosphatase) were not influenced by hydrocortisone when compared with controls. Trehalase activity was decreased on day 5 with 12.5 and 50 ng/mL. A concentration of 12.5 ng/mL diminished gamma-glutamyltransferase activity by 29% on day 5. The incorporation of [3H]leucine into proteins was not influenced by any concentration of the glucocorticoid hormone. This study indicates that hydrocortisone directly influences cell proliferation and certain brush border enzymic activities in human developing kidney maintained in organ culture.     

Normal human endometrial cells in culture: Characterization and immortalization of epithelial and stromal cells by SV 40 large T antigen

Summary— Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derived.     

Enhanced bone marrow stromal cell adhesion and growth on segmented poly(ether ester)s based on poly(ethylene oxide) and poly(butylene terephthalate)

In previous studies in rats and goats, hydrophilic compositions of the PEOT/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro on these hydrophilic copolymers. In this paper two ways of surface modifying these materials to improve in vitro bone marrow stromal cell attachment and growth are discussed. Two different approaches are described: (1) blending of hydroxyapatite (HA) followed by CO(2) gas plasma etching; (2) surface modification using CO(2) gas plasma treatments. It was observed that not only HA but also the CO(2) plasma treatment by itself has a positive effect on bone marrow stromal cell attachment and growth. Gas plasma treatment appeared to be the most successful approach, resulting in a large increase in the amount of bone marrow stromal cells present on the surface (determined by a DNA assay). The amount of DNA present on the plasma-treated copolymer 1000/70/30 PEOT/PBT, based on poly(ethylene oxide, M(w) = 1000, 70 m% soft segment), was comparable to the amount present on PDLLA and significantly higher than the amount present on PCL after 7 days of cell culturing. The fact that after gas plasma treatment bone marrow stromal cells do attach to PEOT/PBT copolymers, enables in vitro bone marrow stromal cell culturing, making bone tissue engineering applications of these materials possible.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Expression and action of hepatocyte growth factor in bovine endometrial stromal and epithelial cells in vitro.

Hepatocyte growth factor (HGF) is a pleiotropic growth factor that acts on various epithelial cells. The objectives of this study were to determine whether HGF altered the proliferation and prostaglandin (PG) secretion of bovine endometrial stromal and epithelial cells in vitro. We also observed HGF and HGF receptor (c-met) mRNA expression in cultured bovine endometrial stromal and epithelial cells by RT-PCR. Stromal and epithelial cells obtained from cows in early stage of the estrous cycle (days 2-5) were cultured in DMEM/Ham's F-12 supplemented with 10% calf serum. The cells were exposed to HGF (0-10 ng/ml) for 2, 4, or 6 days. HGF significantly increased the total DNA in epithelial (P < 0.05), but not stromal cells. In another experiment, when the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA containing HGF 0-100 ng/ml and the cells were cultured for 24 hr. The HGF stimulated PGF2alpha secretion in epithelial, but not stromal cells. RT-PCR revealed that mRNA of HGF is expressed only in stromal cells, and that c-met mRNA is expressed in both stromal and epithelial cells. These results suggest that HGF plays roles in the proliferation and the regulation of secretory function of bovine endometrial epithelial cells in a paracrine fashion.     

The effect of population density on phenylalanine hydroxylase activity in rat-hepatoma cells in culture

The expression of phenylalanine hydroxylase activity in rat-hepatoma cells in culture (line H4-II-E-C3) is a function of culture density: under normal growth conditions in the presence or in the absence of exogenously added hydrocortisone, the levels of this enzyme are low in subconfluent cell populations, but increase steeply as cultures attain confluency. This phenomenon (i) is not an artifact of the subcultivation process and (ii) is not produced by some alteration in the growth medium effected by high-density cultures. The levels of phenylalanine hydroxylase in high-density cultures of H4-II-E-C3 cells in the presence of serum plus added hydrocortisone are at least 80% of those seen in adult-rat liver. It is concluded that this density-associated phenomenon is the result of an intrinsic property of H4-II-E-C3 cells and possibly constitutes a form of epigenetic control governing the sensitivity of these cells to stimulation by serum or by serum plus hydrocortisone.     

Glucocorticoid stimulation of metabolism and glycerol-3-phosphate dehydrogenase activity in cultured heart cells

The direct effects of the glucocorticoids hydrocortisone and corticosterone on myocardial metabolism were studied in cultured heart cells by assessing several parameters previously unreported. Hormone and growth factor concentrations were carefully controlled by using a serum-free medium, which also allowed maintenance of cells in the absence of glucocorticoids. Heart cell beating rate, glucose uptake rate, and CO2 evolution from radioactively labeled glucose were increased by the addition of 0.03 microM corticosterone to the medium of cells maintained in culture for 11 days. There were no further changes in these parameters as steroid concentration was increased to 14.43 microM. The activity of NAD-linked sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) was increased by both corticosteroids and was dose dependent between 0.06 and 1.44 microM corticosterone. The difference between glycerol-3-phosphate dehydrogenase activity in cells maintained with hydrocortisone as compared to cells maintained without hydrocortisone increased with days in culture. The protein and DNA contents of dishes maintained with corticosteroid were depressed, demonstrating an inhibitory effect on cellular replication. Glucocorticoids have numerous direct effects on cardiac cell metabolism, and the nature of these effects suggests that secondary responses of the cell to chronic exposure are significant.     

Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia-prostaglandin E2

Cell strains and cell lines rat mammary (Rama) 350-353 have been isolated from the slowly adherent stromal fraction of enzymatically digested rat mammary glands. Primary cultures of this fraction yield fat cels on extended culture. Their proportion can be increased with horse serum or growth hormone in the medium, and this increase is associated with a 100-fold or more increase in the release of radioimmunoassayable prostaglandins of the E type (PGE). The stromal cell strains and lines that are capable of yielding fat cells also secrete elevated levels (greater than 100 ng/mg/24 hr) of PGE; the fast-sticking epithelial fraction in primary cultures and the epithelial cell lines derived from it secrete 10-100 times less. Chromatography and radioisotopic labeling of the culture media from Rama 352 cells identify the PG as PGE2. PGE2 with insulin and hydrocortisone maximally stimulates [3H]DNA synthesis of epithelial cell lines and primary cultures from normal and tumorous glands by 2-4-fold at concentrations (10-20 ng/ml) well below those released by the preadipocytic stromal cells (20-100 ng/ml). Medium exposed to most cultured cells stimulates [3H]DNA synthesis of one epithelial cell line, Rama 25, by 2-4-fold. Prevention of the synthesis of PGE2 in Rama 352 cultures with indomethacin or flurbiprofen abolishes the mitogenic activity present in the culture medium, and the PG receptor antagonist polyphloretin phosphate inhibits completely the mitogenic activity for Rama 25 cells. Myoepithelial-like cell lines normally secrete moderate levels of PGE (10-100 ng/mg/24 hr) but the mitogenic activity for Rama 25 cells released from one such line, Rama 29, is not abolished by preventing the synthesis of PG's nor by PG-receptor antagonists.     

Induction of hemoglobin synthesis in K562 cells by carbon dioxide deficiency

Proliferation and differentiation are inversely related in many cell culture systems. The study of inducible systems is facilitated by optimal growth conditions in order that whatever differentiation is observed may be attributed to a specific effect of the inducer, rather than to a nonspecific effect of adverse growth conditions. To investigate the role of CO2 supply in an inducible system, the K562 human leukemia cell line inducible for hemoglobin synthesis was studied at 10%, 5% and 1.5% CO2 concentrations. The lower the CO2 concentration, the higher the percentage of benzidine-positive cells but the slower the growth rate. This increase in benzidine positivity reflected hemoglobin synthesis as indicated by incorporation of 3H-leucine into globin chains. If, in addition to reducing CO2 concentration, the complete medium was replaced by a bicarbonate-free medium, the percentage of benzidine-positive cells was further increased and growth further slowed. However, if endogenously produced CO2 was retained by sealing the culture vessel, these effects were mitigated. Since addition of ribosides blocked these effects, the mechanism for these effects appears to be inhibition of riboside biosynthesis due to the depletion of CO2 as a substrate. The implication of this work is that, for reproducibility in studies of inducible systems in which reduction of proliferation may itself increase the probability of differentiation, the CO2 tension, the bicarbonate concentration in the medium and the rate of egress of endogenously produced CO2 must be kept constant.     

Growth of wool follicles in culture

Summary A procedure for the culture of isolated wool follicles from Merino sheep is described. Follicles were microdissected from midside skin samples of 2-yr-old wethers and transferred, individually, to 24-well tissue culture plates. When maintained in supplemented Williams’ E medium containing 5 to 10% fetal bovine serum (FBS), insulin, hydrocortisone, and a trace element mixture, fiber growth rates of 40 to 80 μm/day were observed. Follicles maintained their morphologic integrity for up to 7 days, incorporated [methyl-³H]thymidine into DNA and [³⁵S]methionine into intermediate-filament keratins of the growing fiber. Insulin and hydrocortisone stimulated fiber growth at concentrations of 10 μg/ml and 50 ng/ml, respectively, but higher doses were inhibitory. The growth of fibers in response to hydrocortisone and the changes in follicle morphology was similar to those induced in skin after systemic administration of cortisol in vivo. A positive interaction between hydrocortisone and trace elements for follicle survival and hydrocortisone, insulin, and FBS for fiber growth was also found. The successful culture of Merino sheep follicles provides a model with which to study the direct influence of endocrine, nutritional and local factors on wool keratin synthesis independently of systemic shifts in the animals’ metabolism.     

Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts

Summary The effects of hydrocortisone and ascorbic acid on growth parameters were measured in human diploid skin fibroblasts from fetal and adult donors. In the presence of culture medium containing 10% fetal bovine serum, 0.3 μM hydrocortisone produced a 20% increase in the population growth rate and a 50 to 70% increase in the confluent density of fibroblasts from adult donors. Daily addition of 28 μM ascorbic acid also stimulated the population growth rate and cell density at confluency. The effects of hydrocortisone and ascorbic acid on the final cell density were additive. The action of hydrocortisone was restricted to cells in log-phase growth, whereas ascorbic acid affected cells in both the log and the postconfluent phases of the growth cycle. In fibroblasts from fetal donors, ascorbic acid was stimulative but hydrocortisone was not. The data suggest that whereas both compounds stimulate cell growth in an additive manner, they do so by different cellular mechanisms. This investigation was supported in part by USPHS Grants AM 02456, AM 05020 and AM 15312, and by the Kroc Foundation, No. UW 63-2986. Dr. Rowe is a fellow of the Helen Hay Whitney Foundation. Dr. Fujimoto is a recipient of a Research Career Development Award, AM 47142, from NIAMDD.     

Corticosteroid-dependent differentiation of human marrow preadipocytes in vitro

A unique population of human bone marrow-derived, adherent fibroblastlike cells differentiates to adipocyte morphology when grown in vitro in the presence of horse serum and hydrocortisone sodium hemisuccinate. Over the initial 8-weeks growth at 37 degrees C, 7% CO2, these cells accumulate Oil Red O-positive lipid and form colonies of over 100 cells, which persist in confluent cultures for over 30 weeks. Similar to cultures derived from mouse marrow, corticosteroid-induced adipocyte differentiation is associated with long-term granulopoiesis. Human marrow preadipocytes, as well as human, mouse and rat embryo fibroblast cell lines, failed to differentiate to adipocyte morphology in the presence of insulin. In contrast, the 3T3-L1 insulin-dependent preadipocyte cell line was not induced to differentiate in the presence of hydrocortisone. These studies demonstrate that human marrow preadipocytes are dependent upon corticosteroid for differentiation in vitro.     

Cell cycle status of stromal cells in long-term haematopoietic cultures

This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.     

Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis

Rat mesenchymal stem cells (rMSCs) represent a small portion of the cells in the stromal compartment of bone marrow and have the potential to differentiate into bone, cartilage, fat, and fibrous tissue. These mesenchymal progenitor cells were maintained as primary isolates and as subcultured cells in separate closed modular incubator chambers purged with either 95% air and 5% CO(2) (20% or control oxygen) or 5% oxygen, 5% CO(2), and 90% nitrogen (5% or low oxygen). At first passage, some cells from each oxygen condition were loaded into porous ceramic vehicles and implanted into syngeneic host animals in an in vivo assay for osteochondrogenesis. The remaining cells were continued in vitro in the same oxygen tension as for primary culture or were switched to the alternate condition. The first passage cells were examined for in vitro osteogenesis with assays involving the quantification of alkaline phosphatase activity and calcium and DNA content as well as by von Kossa staining to detect mineralization. Cultures maintained in low oxygen had a greater number of colonies as primary isolates and proliferated more rapidly throughout their time in vitro, as indicated by hemacytometer counts at the end of primary culture and increased DNA values for first passage cells. Moreover, rMSCs cultivated in 5% oxygen produced more bone than cells cultured in 20% oxygen when harvested and loaded into porous ceramic cubes and implanted into syngeneic host animals. Finally, markers for osteogenesis, including alkaline phosphatase activity, calcium content, and von Kossa staining, were elevated in cultures which had been in low oxygen throughout their cultivation time. Expression of these markers was usually increased above basal levels when cells were switched from control to low oxygen at first passage and decreased for cells switched from low to control oxygen. We conclude that rMSCs in culture function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension.     

CO2对转基因鱼腥藻生长的影响

为了进一步探索转基因鱼腥藻高密度培养的方法,在小型气升式反应器中研究了CO2对转基因鱼腥藻7120培养的影响。结果发现转基因鱼腥藻培养过程中通入5％ CO2能促进藻细胞生长,12d生物量提高7．44％,由于光照限制,不能大幅提高15d收获生物量,但生长周期能缩短近20％;而高浓度(10％)的CO2抑制转基因鱼腥藻的生长。CO2是通过调节pH值和影响碳源利用来影响藻细胞生长的,合适浓度的CO2有利于转基因鱼腥藻的培养。     

Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture

Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFbeta) into the basolateral chamber ( approximately 70% > apical) and tumor necrosis factor-alpha (TNFalpha) into the apical compartment ( approximately 30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24-48 h increased epithelial cell TER ( approximately 70% higher than control) and decreased TNFalpha release into both the apical and basolateral chambers ( approximately 30%-50%). In contrast, TGFbeta release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNFalpha release persisted for 5-7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNFalpha without effecting TGFbeta release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

Characterisation of serum-induced intracellular Ca2+ oscillations in primary bone marrow stromal cells

Intracellular Ca2+ signalling is pivotal to cell function and [Ca2+]i oscillations permit precise and prolonged modulation of an array of Ca2+-sensitive processes without the need for extended, global elevations in [Ca2+]i. We have studied [Ca2+]i signalling in primary rat marrow stromal cells exposed to foetal calf serum (FCS) constituents at concentrations up to those required to promote growth and differentiation in culture. Spontaneous [Ca2+]i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca2+ oscillations in 41 +/- 3% of cells. Incidence of FCS-induced oscillations was dose-dependent, saturating at 0.5%. These oscillations were arrested by disruption of Ca2+ stores with 100 nM-1 microM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP3-receptors by 50 microM 2-amino-ethoxydiphenyl borate (2-APB) and inhibition of phospholipase C with 5 microM U73122. The oscillations decreased in frequency and amplitude following inhibition of Ca2+ influx with EGTA or La3+ but were poorly sensitive to nifedipine (1-10 microM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca2+]i oscillations are heat stable, insensitive to disulphide bond reduction with 20 mM dithioerythritol and retained by a 30 kDa molecular weight filter. Serum is routinely present in culture medium at 10%-15% [v/v] and marrow stromal cells maintained under culture conditions exhibited sustained oscillations. This is the first demonstration of agonist-induced complex Ca2+ signals in marrow stromal cells. We conclude that Ca2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation.     

Proliferation of human embryonic corneal stromal cells in a serum-free medium

Substantial multiplication of stromal cells from human embryonic corneas has been obtained in a basal medium MCDB 104 supplemented with 25 ng EGF/ml, 10 micrograms insulin/ml, 20 micrograms transferrin/ml, 25 ng MSA/ml, 500 micrograms ovalbumin/ml, 50 micrograms LDL/ml, 50 micrograms HDL/ml and 10(-6) M hydrocortisone. Even though the growth rate appears to be similar to that in 10% serum, the cells cease proliferating at a lower density.