体外培养的牛耳成纤维细胞的细胞周期研究

通过细胞体外培养,对牛耳成纤维细胞的周期分布进行了研究。不同代数的细胞在生长至70-85％汇合和血清饥饿72h两种状态的细胞周期分布无显著差异。对于体外培养的第8代细胞,生长至50-60％、70-85％和完全汇合时,细胞周期分布存在显著差异;而培养时间对血清饥饿培养和正常培养至充分汇合时的细胞周期分布无显著影响。结果表明,体外培养代数及培养方法不会明显影响细胞周期的分布,但同代细胞因处于不同生长阶段细胞周期分布会存在显著差异。     

Culture and characterization of dental follicle cells from rat molars

Summary Because the dental follicle is necessary for the eruption of teeth of limited eruption, it was the objective of this study to determine if the cells of the follicle could be cultured in vitro. To achieve this, dental follicles and associated enamel organs were dissected from the first and second mandibular molars of 6–7-day-old rats (secretory stage of amelogenesis), and then cultured in a medium that promotes fibroblast growth — the predominant cell type of the dental follicle. The cultured cells grew to confluency and were kept through 3 passages before experimentation. The cultured cells were fibroblastic in shape, elongate with processes, and transmission electron microscopy revealed that they contained an abundant rough endoplasmic reticulum, but did not form desmosomes. Immunofluorescent staining for anti-vimentin showed that all the cells stained and electron-microscopic immunogold labeling indicated that the antibody was associated with intermediate filaments. As revealed by SDS-polyacrylamide gel electrophoresis and Western blotting, the cultured cells synthesized and secreted the extracellular matrix molecules fibronectin and procollagens. Subsequent immunofluorescence staining of permeabilized and non-permeabilized cells confirmed the presence of fibronectin and type I collagen both intra- and extracellularly. Thus, based on all the above characteristics, the cultured cells appeared to be fibroblasts derived from the dental follicle, although a few of the fibroblasts may be derived from undifferentiated mesenchymal cells interposed between the alveolar bone and follicle. Experiments now can be conducted to determine how these cultured cells respond directly to growth factors that alter the rates of tooth eruption.     

Decrease in the density of DNA topological turns during in vitro aging of Syrian hamster cells

Primary cultures of Syrian hamster embryonal fibroblasts were grown until the culture flask bottom was completely covered, then passed into a fresh medium with the dilution 1:2 and grown again until complete sealing of the bottom glass surface was achieved. The period required to attain cellular confluency and the density of topological turns (‘titratable superhelical turns’) in nuclear chromosomal superhelical DNA loops were measured in the course of cultivation. Embryonal cells of Syrian hamsters were found to undergo up to twelve culture doublings (passages) in a medium containing 10% bovine serum. The period required to reach confluency increased from 1–2 days for early passage cells, to 4–8 days for late passage cells, which were often able to reach confluency only after change of growth medium. The density of topological turns in nuclear DNA loops was found to decrease, from a range of −0.076 to −0.083 turns per 10 base pairs (bp) in cells of 1–3 passages, to −0.062 turns per 10 bp in cells of 11–12 passages. This decrease in density of DNA-topological turns may play a part in switching the activity of the genes responsible for cellular proliferation and differentiation in the course of aging of primary cell culture.     

Erythropoietin production in long-term cultures of human renal carcinoma cells : The role of cell population density

The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density.     

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures

Background aimsFirst-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.MethodsWe set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.ResultsGrowth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.ConclusionsOur findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.     

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.     

The bimodal growth response of Swiss 3T3 cells to the B subunit of cholera toxin is independent of the density of its receptor, ganglioside GM1

The B subunit of cholera toxin, a protein which binds specifically to cell surface ganglioside GM1, has been shown to have a bimodal effect on DNA synthesis in Swiss 3T3 fibroblasts. The B subunit induced cellular proliferation of confluent and quiescent cells while it inhibited the growth of the same cells when they were sparse and rapidly dividing. The amount of cell surface GM1 increased when the cells reached confluency. To examine the hypothesis that the variation in levels of GM1 was responsible for the bimodal effect, we increased GM1 levels in rapidly dividing cells by insertion of exogenous GM1 or by treatment of the cells with neuraminidase to convert polysialogangliosides to GM1. Even after the level of GM1 was increased to levels similar to those found in confluent cells, the B subunit still inhibited, rather than stimulated, their growth. Therefore, this result indicates that the bimodal response to the B subunit is not solely a function of the concentration of cell surface GM1; rather it is the growth stage that determines the fate of the signal transduced by the interaction of the B subunit and ganglioside GM1.     

A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a 1 year culture period. Here we report the isolation, continuous culture, and initial characterization of a spontaneously arising feeder-independent PICM-19 subpopulation, PICM-19FF, that maintained replication rate and hepatocyte functions over an extended culture period. PICM-19FF cells grew to 90–98 % confluency after each passage at 2 week intervals, and the cells maintained a high cell density after 2 years and 48 passages in culture (average of 2.6 × 10⁶ cells/T25 flask or 1 × 10⁵ cells/cm²). Morphologically, the PICM-FF cells closely resembled the finite feeder-independent PICM-19 cultures previously reported, and, as before, no spontaneous formation of 3D multicellular ductules occurred in the cells’ monolayer. Their bipotent stem cell nature was therefore not evident. Over extensive passage, cytochrome P450 (EROD) activity was maintained, although urea production was reduced on a per mg protein basis at later passages. Two other attributes of fetal hepatocytes, γ-glutamyl transpeptidase activity and serum-protein secretion, were also shown to be maintained by the PICM-19FF cells. The PICM-19FF cells therefore appear to have indefinite growth potential as a feeder-independent cell line and this should enhance the experimental usefulness of the cell line, in general, and may also improve its application to toxicological/pharmacological assays and artificial liver devices.     

Viral probe into the events of cellular (in vitro) aging

Viral infection by vesicular stomatitis virus (VSV) as well as cellular protection against it were studied in cultured human diploid fibroblast cells (WI38 strain) of varying senescence (passage) level. Full protection against viral infection can be achieved by pretreatment of cells with an interferon inducer (complex of polycytidylate and polyinosinate) or by pretreatment with concentrates of interferon itself. The late passages (over 45) require higher concentration of both agents than the medium passages (25–45); a complete failure of protective mechanisms occurs only close to cellular death. Furthermore, effects of confluency and of duration of induction impulse were studied. WI38 cells are sensitive to VSV infection through their entire life span; however, during the last passage before their death by senescence, VSV replication is significantly decreased. It is concluded that even in relatively senescent cells (a) protection mechanisms which were not used previously can be activated, (b) synthesizing machinery necessary for efficient support of viral replication is sustained, and (c) release of lysosomal enzymes ending the viral replication does not begin sooner than with cells of earlier passage level.     

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.     

Carnitine uptake and fatty acid utilization by diploid cells aging in culture

Uptake of carnitine by cultured human fetal lung flbroblasts (WI-38 and IMR-90) and by smooth muscle cells from calf aorta and from human uterus was found to be temperature dependent and saturable. IMR-90 cells showed an apparent K_m of 6–8 μM and a V of 21–28 pmol/h/10⁶ cells for l-carnitine. Transport was abolished by N-ethylmaleimide and was inhibited variably by octanoyl-d-carnitine, d-carnitine, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Although WI-38 and IMR-90 cells accumulate lipids as they age in culture, they take up carnitine as rapidly as do smooth muscle cells of aorta and uterus that do not exhibit such accumulation. Comparison of the rates of carnitine uptake by IMR-90 fibroblasts during the logarithmic phase of growth shows no difference between “young” and “old” cultures. In contrast, when confluent or postconfluent monolayers were compared and uptake expressed as a function of cell number, cells grown from late passages took up carnitine more rapidly than did cells grown from early passages. However, when account was taken of cell size, and carnitine expressed as a function of cell volume, the differences in carnitine uptake between early and late passages were no longer apparent for the confluent or postconfluent monolayers examined. Moreover, late passage fibroblasts took up and oxidized radioactive palmitate at least as rapidly as did cells from early passages. Our results suggest that accumulation of lipid in aging fibroblasts is not due to decreased carnitine uptake or fatty acid oxidation.     

Growth,morphologic, and invasive characteristics of early and late passages of a human endometrial carcinoma cell line (RL95-2)

Summary Two in vitro passages of a human endometrial adenocarcinoma continuous cell line (RL95-2), an early (subcultured <30 times) and a late passage (subcultured >200 times) have provided an interesting model to study the growth, morphologic, and invasive properties of endometrial tumors. The early passage, which has been shown to be estrogen-receptor positive, has characteristics closely resembling a primary tumor, whereas the estrogen receptor negative late passage exhibits several features of the metastatic phenotype. Compared to the early passage cells, the late passage cells were less serum dependent, formed foci, demonstrated a faster rate of growth (due to their shorter doubling times), and attained higher saturation densities. The late passage cells also displayed an altered morphology which was accompanied by alterations in the distribution of F-actin. Even though early and late passages showed similar invasive potential in an in vitro invasion assay, the late passage cells, by virtue of their several transformed characteristics, maintain distinctive properties compared with their early passage counterparts.     

In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.     

Ex vivo characteristics of human amniotic membrane-derived stem cells

Cells were isolated from four human amniotic membranes, and their biological characteristics analyzed during ex vivo expansion. Morphologically homogenous populations of fibroblast-like cells were obtained from the second or third passage. Under the appropriate culture conditions, these human amniotic membrane-derived mesenchymal cells (HAM) were shown to differentiate into adipocytes, osteocytes, chondrocytes and neuronal cells, as visualized by Oil Red O, von Kossa, alcian blue, anti-Neu N, and anti-Gal C antibody staining, respectively. Immunophenotype analysis of HAM cells revealed the presence of antigens for SSEA-3, SSEA-4, collagen type-I, -II, -III, -IV, -XII, fibronectin, alpha-SMA, vimentin, desmin, cytokeratin18 (CK18), HCAM-1, fibroblast surface protein, and human leukocyte antigen (HLA) ABC. ICAM-1 protein was weakly detectable, and proteins of TRA-1-60, VCAM-1, von Willebrand factor, PECAM-1, and HLA DR were not detected. HAM cells reached senescence after 14.5+/-0.9 passages, over a period of 146.8+/-8.9 days, and underwent an average of 36.9 4.7 population doublings. RT-PCR analysis showed that all four HAM cell lines consistently expressed genes of Oct-4, Rex-1, SCF, NCAM, nestin, BMP-4, GATA-4, HNF-4alpha, vimentin, and CK18, regardless of the passage number. The genes of Brachyury, FGF-5, Pax-6, and BMP2 were never expressed. Strikingly, alpha-fetoprotein (alphaFP), HLA ABC, and HLA DR genes were expressed in an earlier passage but not expressed in later passages. Telomerase activity of two HAM lines was discernable upon the third passage. These observations strongly suggest that HAM might be immune-privileged and, thus, advantageous as therapeutic cells.     

Transdifferentiation of "lentoid" structures in cultures derived from pigmented epithelium was inhibited by collagen.

Cells from pigmented retina of 8- to 9-day-old chick embryos were cultured under two different conditions: on noncoated (NS) or collagen-coated (CS) substrates. Although cells on CS seemed to start dividing 2 to 3 days earlier than those on NS, their early growth rates were basically similar. Cells on CS stopped growing after attaining confluency and formed a monolayer, while cells on NS continued to grow after confluency and overlapped each other. In early growth phase, cells on both substrates became depigmented. Cells became repigmented earlier on CS than on NS. The average melanin content of cells in confluent cultures on CS was two to three times higher than that of cells on NS. By Day 30 “lentoid bodies” were formed only in cultures on NS. Immunoelectrophoretic tests showed the presence of all crystallins (α-, β-, and δ) in cultures on NS but not in cultures on CS. It is concluded that a collagen substrate inhibits “transdifferentiation” of pigmented retinal cells into lens during cell culture.     

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

Accelerated cell cycle progression in osteoblasts overexpressing the c-fos proto-oncogene: induction of cyclin A and enhanced CDK2 activity

Transgenic mice overexpressing the c-Fos oncoprotein develop osteosarcomas that are associated with deregulated expression of cell cycle genes. Here we have generated osteoblast cell lines expressing c-fos under the control of a tetracycline-regulatable promoter to investigate the role of c-Fos in osteoblast cell cycle control in vitro. Three stable subclones, AT9.2, AT9.3, and AT9.7, derived from MC3T3-E1 mouse osteoblasts, expressed high levels of exogenous c-fos mRNA and protein in the absence of tetracycline. Functional contribution of ectopic c-Fos to AP-1 complexes was confirmed by electromobility shift assays and transactivation of AP-1 reporter constructs. Induction of exogenous c-Fos in quiescent AT9.2 cells caused accelerated S-phase entry following serum stimulation, resulting in enhanced growth rate. Ectopic c-Fos resulted in increased expression of cyclins A and E protein levels, and premature activation of cyclin A-, cyclin E-, and cyclin-dependent kinase (CDK) 2-associated kinase activities, although cyclin D levels and CDK4 activity were not affected significantly in these cell lines. The enhanced CDK2 kinase activity was associated with a rapid, concomitant dissociation of p27 from CDK2-containing complexes. Deregulated cyclin A expression and CDK2 activity was also observed in primary mouse osteoblasts overexpressing c-Fos, but not in fibroblasts, and c-Fos transgenic tumor-derived osteosarcoma cells constitutively expressed high levels of cyclin A protein. These data suggest that overexpression of c-Fos in osteoblasts results in accelerated S phase entry as a result of deregulated cyclin A/E-CDK2 activity. This represents a novel role for c-Fos in osteoblast growth control and may provide c-Fos-overexpressing osteoblasts with a growth advantage during tumorigenesis.     

Accumulation of cyclin E is not a prerequisite for passage through the restriction point

The restriction point (R) is defined as the point in G(1) after which cells can complete a division cycle without growth factors and divides G(1) into two physiologically different intervals in cycling cells, G(1)-pm (a postmitotic interval with a constant length of 3 to 4 h) and G(1)-ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G(1) regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G(1) cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.