Myogenesis in chick embryo somites in vitro

Development ◽  
1969 ◽  
Vol 21 (2) ◽  
pp. 341-346
Author(s):  
M. L. Ellison ◽  
E. J. Ambrose ◽  
G. C. Easty
Keyword(s):  
Chick Embryo ◽  
Environmental Factors ◽  
Conditioned Medium ◽  
Cell Density ◽  
Phenotypic Expression ◽  
Culture Conditions ◽  
Tissue Mass ◽  
Latent Form

Evidence from a number of studies (e.g. Holtzer, 1964; Coon & Cahn, 1966; Konigsberg, 1963) has suggested that the differentiation of some types of cells in vitro may be influenced by environmental factors. Culture conditions can permit or prevent the phenotypic expression of characteristics already determined in the cells. The length of time involved in the stabilization of differentiation appears to vary widely according to the conditions of culture. Coon (1966), using chondrocytes, and Simpson & Cox (1967), using lizard tail regenerate, among other examples, have demonstated that determination could be maintained over many mitotic divisions in a latent form, before being expressed eventually under the stabilizing conditions. A range of environmental factors has been shown to be effective during stabilization, including protein constituents of the medium (Coon, 1966; Cahn & Cahn, 1966), conditioned medium (Konigsberg, 1963), cell density (Abbott & Holtzer, 1966; Umansky, 1966) and tissue mass (Grobstein, 1964; Wessells & Cohen, 1967).

Effect of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in monolayer culture

1992 ◽  
Vol 133 (2) ◽  
pp. 291-NP ◽  
Author(s):  
C. Ohlsson ◽  
A. Nilsson ◽  
O. G. P. Isaksson ◽  
A. Lindahl
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Density ◽  
Monolayer Culture ◽  
Culture Conditions ◽  
Factor I ◽  
Sulphate Uptake ◽  
Igf I ◽  
Matrix Production

ABSTRACT The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture. Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture. Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 μg/l (60 ± 11% stimulation over control) and for IGF-I at 10 μg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake. In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm2. GH had no significant effect at a low (< 100 000 cells/cm2) or a high (>400 000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm2, but was reduced at higher cell densities (over 250 000 cells/cm2). Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice. The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions. Journal of Endocrinology (1992) 133, 291–300

The relationship between intracellular calcium levels and limb bud chondrogenesis in vitro

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 73-81
Author(s):  
J.A. Bee ◽  
R. Jeffries
Keyword(s):  
Phenotypic Expression ◽  
Culture Conditions ◽  
Limb Bud ◽  
Concentration Effects ◽  
Cartilage Differentiation ◽  
Standard Culture ◽  
The Relationship ◽  
Plateau Level

Under standard culture conditions, chondrogenic expression by stage-21 embryonic chick limb bud mesenchyme is dependent upon high cell plating densities. Alternatively, when cultured in suspension aggregating limb bud cells differentiate exclusively as cartilage. We have previously demonstrated that the aggregation of prechondrogenic limb bud cells is specifically mediated by a Ca2+ -dependent mechanism. In the present paper, we examine the involvement of calcium cations in chondrogenic expression in vitro. During cartilage differentiation, we demonstrate that limb bud cells elevate their intracellular Ca2+ levels to achieve a conserved plateau level. This increase in intracellular Ca2+ levels does not occur in sparse cell cultures, which also fail to demonstrate cartilage differentiation. Although elevation of extracellular Ca2+ concentration effects precocious chondrogenesis, ultimately this is substantially lower than in control cultures. In contrast, elevation of intracellular Ca2+ levels by the addition of 0á1 μm-A23187 readily stimulates precocious and extensive cartilage differentiation. 0á1μm-A23187 initially elevates intracellular Ca2+ levels to that required for cartilage differentiation but this then continues to increase concomitant with a reduction in cartilage nodule size. 10μm-retinoic acid completely inhibits chondrogenesis in vitro and elevates intracellular Ca2+ to particularly high levels. Our data indicate the central role of controlled intracellular Ca2+ levels to normal chondrogenic expression. Deviation from this level by cells that either fail to achieve or that exceed it inhibits subsequent cartilage development, and can cause a loss of phenotypic expression by differentiated cartilage.

scholarly journals Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production.

1981 ◽  
Vol 90 (2) ◽  
pp. 332-338 ◽  
Author(s):  
R P Mecham ◽  
G Lange ◽  
J Madaras ◽  
B Starcher
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Elastic Fiber ◽  
Calf Serum ◽  
Phenotypic Expression ◽  
Fetal Tissue ◽  
Culture Conditions ◽  
Population Doubling

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.

scholarly journals Role of Plant Cell Conditioned Medium in the Phenotypic Expression of Nitrogenase Activity of Rhizobium trifolii Strain T1

1980 ◽  
Vol 33 (5) ◽  
pp. 613 ◽  
Author(s):  
Minocher Reporter ◽  
Mary L Skotnicki ◽  
Barry G Rolfe
Keyword(s):  
Oxidative Phosphorylation ◽  
Conditioned Medium ◽  
Plant Cell ◽  
Nitrogenase Activity ◽  
Phenotypic Expression ◽  
Atp Synthesis ◽  
Atp Production ◽  
Cell Conditioned Medium

The influence of substances from a conditioned medium of cultured plant cells on nitrogenase activity, respiration and ATP synthesis was investigated in R. tri/olii strain Tl. Nitrogenase activity in strain Tl was dependent on the addition of the plant cell conditioned medium. Studies showed that the initial effects of the plant substances on rhizobial cells was to increase their respiration rate and ATP production. Mutants of strain Tl which were uncoupled in their oxidative phosphorylation, were also tested. However, the plant factors had no effect on respiration and ATP synthesis and also failed to elicit in vitro nitrogenase activity in these mutants. It is proposed that these plant factors act by increasing the efficiency of oxidative phosphorylation, making more ATP available, and thus stimulating nitrogenase activity of R. tri/olii cells.

The Effect of Environmental Factors on the Differentiation of the AUantoic Endoderm in Organ Culture

Development ◽  
1954 ◽  
Vol 2 (4) ◽  
pp. 348-352
Author(s):  
B. Fell
Keyword(s):  
Chick Embryo ◽  
Environmental Factors ◽  
Organ Culture ◽  
Large Intestine ◽  
Granular Layer ◽  
Goblet Cells ◽  
Columnar Epithelium ◽  
Embryonic Life ◽  
Hind Gut

The allantois of the chick appears at about the 3rd day of incubation as an evagination from the hind gut; it soon becomes distended with fluid, enlarges rapidly, and fuses with the chorion to form the chorio-allantoic membrane. Out of curiosity, the allantoic sac of a 4-day chick embryo was explanted in a watchglass culture to see how it would behave in vitro. Under these conditions the allantoic endoderm did not develop into a flattened, granular layer as in normal embryonic life, but instead differentiated into a tall, columnar epithelium with many actively secreting goblet cells, which closely resembled that of the large intestine. This casual observation was investigated further and the results of the study are described below. The allantoic sac was removed from 3½-4½-day embryos, before fusion with the chorion had taken place; the neck of the allantois, uniting the latter with the gut, was excluded from the explants.

Chondrogenesis in chick embryo somites in vitro

Development ◽  
1969 ◽  
Vol 21 (2) ◽  
pp. 331-340
Author(s):  
M. L. Ellison ◽  
E. J. Ambrose ◽  
G. C. Easty
Keyword(s):  
Spinal Cord ◽  
Chick Embryo ◽  
Embryonic Development ◽  
Liquid Medium ◽  
Culture Conditions ◽  
Chick Embryos ◽  
Cartilage Formation

During the embryonic development of vertebral cartilages, cells from the somite mesoderm differentiate into chondrocytes around the spinal cord and notochord. Grafting experiments in amphibian and chick embryos have indicated that the spinal cord and notochord have some influence on this differentiation of somite cells to cartilage (Holtzer & Detwiler, 1953; Watterson, Fowler & Fowler, 1954). Further analysis in vitro has established that, under specified culture conditions, cartilage formation from somites is, in fact, dependent on the presence of either spinal cord and/or notochord or their extracts (Grobstein & Parker, 1954; Grobstein & Holtzer, 1955; Strudel, 1962, 1963; Lash, 1963). For example, Lash (1963) showed that in his system on a liquid medium stage 16 (Hamburger & Hamilton, 1951, stages) somites alone formed no cartilage, but that cartilage did develop when notochord or spinal cord was cultured with the somites. This suggested that the spinal cord and notochord were ‘inducing’ somite mesoderm cells to form chondrocytes.

Adhesion, growth and morphology of human mesothelial cells on extracellular matrix

1986 ◽  
Vol 85 (1) ◽  
pp. 133-147
Author(s):  
M.J. Niedbala ◽  
K. Crickard ◽  
R.J. Bernacki
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Cell Density ◽  
Doubling Time ◽  
Cell Monolayer ◽  
Phenotypic Expression ◽  
Mesothelial Cells ◽  
Cellular Migration ◽  
Final Cell Density

Human mesothelial cells (HMC) cover a variety of serosal surfaces and have been shown to rest upon an underlying subcellular basement membrane in vivo. Bovine corneal endothelial cells produce an extracellular matrix (ECM) in vitro that mimics HMC subcellular basement membrane and was found to modulate HMC adhesion, morphology and proliferation in vitro. Our results indicated that within minutes after plating, a high percentage (greater than 80%) of HMC firmly attached to ECM. Active cellular migration and subsequent proliferation were observed leading to the formation of a well-organized closely apposed cell monolayer. However, when cells were plated on plastic, the rate of cell attachment was much lower and the proliferative rate of HMC grown on plastic also was strikingly lower (exponential doubling time 4.3 days) than that of cells grown on ECM (exponential doubling time 2.4 days). Cells upon reaching confluency on plastic were markedly enlarged as compared to confluent cells grown on ECM. These observations corroborated differences in final cell density where it was noted that HMC cultured on ECM demonstrated a 10-fold greater final cell density as compared to cells grown on plastic. Results from these studies illustrate the fact that phenotypic expression as well as proliferative responsiveness of HMC can be modulated by adhesive interactions with preformed ECM.

scholarly journals The cultured rodent follicle as a model for investigations of gonadotrophin surge-attenuating factor (GnSAF) production

Reproduction ◽  
2004 ◽  
Vol 127 (6) ◽  
pp. 679-688 ◽  
Author(s):  
Paul A Fowler ◽  
Norah Spears
Keyword(s):  
Conditioned Medium ◽  
Granulosa Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Suitable Model ◽  
Molecular Weight Range ◽  
Follicle Culture ◽  
Stage Of Development

Gonadotrophin surge-attenuating factor (GnSAF) bioactivity (the suppression of GnRH-induced but not basal LH and FSH secretion from pituitary gonadotrophs) is produced by granulosa cells in vitro. Previous studies to investigate this bioactivity used dispersed granulosa cells which lack some cell types and the structural components of the follicle in vivo. The aim of this study, therefore, was to investigate whether intact rodent follicle culture was a suitable model for the study of the production of GnSAF bioactivity, allowing GnSAF to be investigated in a more physiologically realistic environment while still retaining culture conditions from which, as with granulosa cell cultures, extraneous factors can be excluded. Follicles from 16-day-old rats and 21-day-old mice were cultured for 3–6 days in the presence or absence of FSH and/or LH. The follicle-conditioned medium, and matching samples of unconditioned culture medium were added to our established rat pituitary monolayer GnSAF bioassay. Both mouse and rat intact follicles produced GnSAF bioactivity, reducing GnRH-induced LH secretion significantly. GnSAF output from the mouse follicles was highest during days 1–3 of culture, when follicles were at an early antral stage of development, and fell on days 4–6 as the follicles grew to the mid antral stage. While the stimulatory effects of FSH on rat follicle GnSAF secretion was dose-dependent, LH alone did not increase GnSAF production. An antibody against human GnSAF blocked GnSAF bioactivity produced by rat follicles, and recognised proteins within the expected pI and molecular weight range for GnSAF in two-dimensional gels of rat follicle-conditioned medium, showing a good homology between rodent and human GnSAF proteins. In conclusion, the release of GnSAF bioactivity is principally from small follicles stimulated by FSH. Therefore, intact rodent follicle culture systems offer an excellent model for the investigation of factors controlling GnSAF production under relatively physiological conditions.

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