Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Distributions of AT- and GC-base pairs along the length of chromosomes 1, 2, 3 and 16 in primary cultures of embryonic fibroblasts and of peripheral blood leucocytes were studied by autoradiography with: 1. ³H-thymidine and ³H-deoxycytidine; 2. ³H-deoxyadenosine and ³H-deoxyguanosine. It has been shown that the two types of cells differ in the DNA content and proportion of AT- and GC-nucleotide pairs in the centromeric heterochromatin of chromosome I: this region contains more DNA in fibroblasts than in leucocytes mainly due to AT-pairs. In both types of cells the telomeric region of the short arm of this chromosome contains more GC- than AT-pairs. Similar results were obtained for C-heterochromatin of chromosome 16: the frequencies of labelling of this region by ³H-deoxyadenosine and ³H-thymidine in fibroblast cultures were higher than in case of ³H-deoxycytidine and ³H-deoxygyanosine, and in leucocyte cultures these frequencies were almost equal. No differences in the distributions of base pairs along the length of chromosome 2 and 3 were established in the two types of cells. — The nature of the established phenomenon may be connected with under-replication or loss in another way of part of the genetic material in the process of development and differentiation of cell systems.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Replication of regions of chromosomes 1, 2, 3, 16, and group 4–5 was studied at the termination of the S period in primary cultures of embryonic fibroblasts (two embryos) and in cultures of peripheral blood (two women). Distinct differences were established in the pattern of late replication of the studied chromosomes in the cultures of the two types of cells. These differences consern first of all the centromeric and neighbouring regions of the chromosome. The content of late label in this region is 1.5–3 times higher in the cultures of fibroblasts than in the corresponding regions of leucocyte cultures. The difference is most pronounced in chromosomes 1, 3 and 16. It is suggested that the difference between cultures of these two types of cells in chromosome replication may be connected with the different genetic functioning of the centromeric and neighbouring regions in them. It is also possible that this difference is due to underreplication (or partial loss in an other way) of heterochromatin DNA of centromeric and neighbouring regions in leucocytes functioning for a long period without division.     

Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [¹⁴C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.     

The position of chromosomes at metaphase in human fibroblasts and their DNA synthesis behaviour

The peripheral location of H³-thymidine labelled chromosomes in spreads of colcemid blocked metaphases from human fibroblasts has been investigated in several karyotypes. Chromosomes show non-random distribution in relation to the periphery of the plate. Longer chromosomes are significantly more peripheral than smaller chromosomes. Those chromosomes which contain large amounts of late-synthesising DNA (Y, 18 and 13) are significantly more peripheral than early synthesising chromosomes of similar length (21–22, 19–20 and 14–15). These locations are compared with those from peripheral leucocytes obtained by other workers. When location and grain number of homologues of nos. 1, 2, 3, 16 and late-X are compared it was found that in late-S labelled cells, the more peripheral of each pair was significantly more heavily labelled; in early-S labelled cells the reverse was the case. Location and patterns of labelling between late-X homologues of XXXXY cells showed that peripheral location was related to more delay in DNA synthesis. These results are discussed in terms of peripheral and central interphase distribution of condensed chromatin. It is concluded that the apparent asynchrony of replication of homologues is principally the result of different degrees of condensation of the chromatin in the late-S phase at the time of synthesis, and these differences are related to the proximity to the nuclear membrane.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Replication pattern of the X and Y chromosomes in partially synchronized human lymphocyte cultures

The replication pattern of the X and Y chromosomes at the beginning of the synthetic phase was studied in human lymphocyte cultures partially synchronized by the addition of 5-fluoro-2-deoxyuridine (FUdR). The data were evaluated statistically by an analysis of the distribution of silver grain counts over the X and Y chromosomes. —In cells from normal females, one of the X chromosomes began replication later than any other chromosomes of the complement. The short arm of the late replicating X chromosome started replication earlier than the long arm. The telomeric region of the short arm was a preferential site of DNA synthesis at the beginning of replication. —In partially synchronized lymphocyte cultures from a patient with the XXY syndrome, the Y chromosome started replication together with the late replicating X chromosome. The Y chromosome most frequently replicated synchronously with the short arm of the X. The centromeric region of the Y chromosome initiated synthesis before the telomeric region and appeared to replicate synchronously with the telomeric region of the short arm of the X. These findings are discussed with reference to the pairing of the X and Y chromosomes at meiosis.Supported in part by the National Institute of Health Research Grant HD-01979 and National Foundation Birth Defects Research Grant CRCS-40. Dr. Knight was a predoctoral fellow under National Institute of Health Training Program HD-00049-09.     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [³H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [³⁵S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon – ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

Studies of multipolar mitoses in euploid tissue cultures

Cultures of kidney epithelium and fibroblasts of 39 specimens of Microtus agrestis were investigated. In all 77 cultures multipolar mitoses were found. They were studied in living state and after pulse labelling with ³H-thymidine. The ploidy of the multipolar mitoses and of their daughter nuclei was determined by measuring the relative Feulgen-DNA content and by counting the predominantly constitutive heterochromatic sex chromosomes. Constitutive heterochromatin was demonstrated by late replication, retarded separation of the chromatids in anaphase, heteropycnosis and by the Giemsa technique of Arrighi and Hsu (1971). The latter stained also the spindle apparatus of mitoses.—In living cells, transformation of multipolar mitoses into bipolar mitoses was observed. The chromosomes of multipolar mitoses are separated into complete genomes; the daughter nuclei can be haploid, diploid, triploid or tetraploid. The chromosomes of haploid and triploid metaphases were studied with the Giemsa banding technique. The banding pattern shows an exact monosomy and trisomy, respectively, for each chromosome. Haploid nuclei are likely to be viable only in multinucleate cells, whereas triploid cells behave like diploid cells during the S period and the mitosis.Dedicated to Prof. Dr. K. Goerttler on the occasion of his 75th birthday.Supported by the Bundesministerium für Bildung und Wissenschaft of the Federal Republic of Germany.     

Lectin-mediated stimulation of DNA synthesis in cultures of embryonic neural retina cells

Plant lectins and other agents which are mitogenic for lymphocytes and fibroblasts were tested for their effects on DNA synthesis in primary monolayer cultures of neural retina cells from 10-day chick embryos. Concanavalin A (ConA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), and anti-retina cell antiserum significantly stimulated [³H]TdR incorporation; the maximum increase was reached 15 h after exposure of the cultures to these agents. Cells stimulated by ConA to synthesize DNA subsequently divided. The divalent succinyl derivative of ConA had a considerably lesser effect than the native tetramer, suggesting that cross-linking of cell surface components may be an important aspect of the changes that lead to the stimulation of DNA synthesis in these cells.Using [¹²⁵I]ConA, the average number of ConA-binding sites per 10-day retina cell was estimated to be 1.7 × 10⁶ (under the culture conditions employed); binding of the lectin to 25–50% of these sites was sufficient to elicit the maximal stimulation of DNA synthesis. Continuous association of the lectin with the cell surface for up to 8 h was essential for the maximal effect, since removal of the lectin from the cell surface (with α-methyl mannose) prior to this time reduced or prevented the stimulation of DNA synthesis.The stimulation by ConA of DNA synthesis in these cultures was dependent on the cell density and was reduced or absent at lower than optimal densities. Examination of this effect suggested that the frequency of intercellular contacts or specific cell associations play a role in the responsiveness of these cells to stimulation of DNA synthesis by ConA.     

Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy

The population decline of the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from an animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we established and characterized fibroblast cell cultures from the skin sample of a newborn common hippopotamus in this study. By combining the tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 34 h according to the growth curve. Karyotyping based on Giemsa staining showed that the cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. The amplified mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with that of the registered H. amphibius complete mitochondrial DNA, confirming the species of origin of the cells. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4, and vimentin. In conclusion, we generated the fibroblast cell cultures from a common hippopotamus and identified their characteristics using multiple techniques. We believe the cryopreserved cells could be useful genetic materials for future research.     

The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bg ^J/bg^J mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bg ^J/bg ^J beige mutants were grown in D-valine medium and glycosphingolipids labeled with [³H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [³H]palmitate into glycospingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes. (Mol Cell Biochem 118: 61–66, 1992)     

Plasmodium falciparum: stage-specific lactate production in synchronized cultures

Sequential treatment with gelatin followed by sorbitol-produced Plasmodium falciparum cultures that remained synchronous for ? 144 hr. The schizont stage was associated with more lactate production than the other asexual erythrocytic stages of the parasite (11–21 vs 4–7 neq/hr-10⁶ parasitized red blood cells [PRBC]). Lactate production in asynchronous cultures was relatively constant at 8–11 neq/hr-10⁶ PRBC and was directly proportional to parasitemia (P < 0.001).     

Methylcellulose effect on cell proliferation and glucose utilization in chemically defined medium in large stationary cultures

Three different established strains of mammalian cells were grown in chemically defined medium in large cultures. The degree of proliferation of cells of an established strain from human skin in large stationary cultures was significantly greater in the presence of methylcellulose (medium NCTC 135M) than in its absence (medium NCTC 135). The relatively fragile cells of a derivative of monkey kidney LLCMK2 strain were carried in large stationary cultures through 11 transfer generations during 152 days. The presence of methylcellulose was associated with higher cell population levels, proliferation rates, and cell viability. Cells of this strain utilized glucose at an extremely high rate; during two representative periods the rate averaged 1.2 mg/10⁶ cells/day in cultures on medium 135M and 1.9 mg in medium 135. In a 53-day experiment with mouse fibroblast 2071-L cells, the cells in suspension culture during the first 28 days went through the normal lag, logarithmic plateau, and initial decline phases in medium 135M, and then were transferred to large stationary cultures, where they proliferated for 7 days at uniformly high rates in both medium 135 and medium 135M. It appeared that cells of strain 2071-L in such stationary cultures had no need for Methocel as a protective agent. Glucose utilization rates while these cells were carried in large stationary cultures averaged 2–4 times the rates while they were in suspension cultures: about 0.8 and 0.2 mg/10⁶ cells/day, repectively.     

Lipid synthesis during reinitiation of growth from stationary phase cultures of Candida albicans

Lipid synthesis has been studied in the dimorphic fungus Candida albicans. ¹⁴C-acetate incorporation into lipid material was used to measure new lipid synthesis in two cultures in which either yeast or mycelial growth was initiated from stationary phase yeast cells. When resuspended in fresh medium at 37 °C, cells resume growth and change morphology while at 30 °C cells resume budding growth. When resuspended at the appropriate temperature, both yeast and germ tube cultures immediately incorporated ¹⁴C-acetate into lipid material. The labeled lipid was more or less evenly divided between neutral and phospholipid. Phosphatidyl choline was the major phospholipid fraction and along with phosphatidyl ethanolamine accounted for 60–65 % of the total phospholipid. Lipid synthesis during growth initiation of either morphology showed a similar pattern, with no significant differences observed in neutral or phospholipid or phospholipid components between yeast and mycelial forms.     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.     

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [³H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED₅₀ of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED₅₀ of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.     

Osteogenesis in cultures of limb mesenchymal cells

The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 10⁶ cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 10⁶ cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.