Growth induction by serum or polyoma virus inhibits the aggregation of trypsinised suspensions of BHK21 tissue culture fibroblasts

BHK21 hamster tissue culture fibroblasts, when brought into suspension with trypsin, aggregate spontaneously in serum-free medium. The amount of aggregation appears to depend on the proportion of quiescent (growth inhibited) cells which were present in the culture. It is greatest in cells from cultures whose growth is inhibited, either by high cell density or by low serum concentration. When serum is added to low serum cultures, growth is induced and S phase and mitosis follow at 14 and 20 h respectively. It has now been found that aggregation ceases less than 2 h after the addition of serum. Growth can also be induced in such cultures by infection with high multiplicities of polyoma virus. Infected cells enter S phase about 28 h after infection. It has been found that aggregation ceases between 12 and 20 h after infection. Thus in both these cases loss of aggregation precedes S phase by about 12 h. It is concluded that the absence of aggregation in suspensions derived from polyoma virus-transformed lines of BHK21 cells is a consequence of their resistance to density-dependent inhibition of growth. Failure to undergo spontaneous aggregation appears to be an indicator of an early surface change associated with the induction of growth.     

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.     

DOES COLCHICINE AFFECT AGGREGATION OF NORMAL AND TRANSFORMED BHK CELLS DIFFERENTLY?

The effect of colchicine and vinblastine on cell aggregation was studied, using BHK cells and their transformed derivatives (pyBHK cells). When cells were dissociated with EDTA and the assay was made in a Ca²⁺-containing medium, the aggregation of transformed cells was prevented by colchicine and vinblastine, whereas the aggregation of normal cells was unaffected. When a Ca²⁺-free medium was used for aggregation, neither type of cell was influenced by these drugs. BHK and pyBHK cells, dissociated by trypsin in the presence of Ca²⁺, can aggregate only in the Ca²⁺-containing medium and the aggregation of both cell types was equally prevented by colchicine and vinblastine. Based on these results, it was concluded that colchicine and vinblastine inhibited the Ca²⁺-dependent mechanism of cell adhesion, but not the Ca²⁺-independent one which occurs in the Ca²⁺-free aggregation medium.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

Somatic and germ cell interactions during histogenetic aggregation of mouse fetal testes.

In the present study we examined the capacity of somatic and germ cells dissociated from fetal mouse testes at various stages to reform seminiferous cords in culture. We found that after 12 h in culture, seminiferous cords became segregated from stromal cells. Although Sertoli cells were incorporated into seminiferous cords at all stages studied, the germ cells dramatically changed their histogenetic behavior with age. Most germ cells which had been dissociated at 12.5 days postcoitum (dpc) were incorporated into the seminiferous cords, whereas at 14.5 dpc or later the majority remained among the stromal cells or as clusters on the surface of the aggregates. We considered three possible causes for this change in behavior of germ cells: (i) Failure to deposit some extracellular matrix components in the aggregates. (ii) Decrease in adhesiveness of prospermatogonia to either extracellular matrix components or Sertoli cells. (iii) A change in adhesiveness of Sertoli cells to germ cells with age. We found that laminin and fibronectin were similarly deposited in aggregates at 12.5 and 15.5 dpc. When prospermatogonia at 15.5 dpc labeled with colloidal gold were reaggregated with somatic cells at 12.5 dpc, 50% were incorporated into seminiferous cords. Moreover, [3H]thymidine-labeled Sertoli cells at 15.5 dpc formed heterochronic seminiferous cords with Sertoli cells at 12.5 dpc. These results suggest that mouse Sertoli cells change their surface property which is essential for binding to germ cells when they enter the mitotic resting stage (T-prospermatogonia).     

Inhibitory effects of dextran sulfates on aggregation of embryonic quail liver cells in culture

The effects of cAMP and DB cAMP on the aggregation of dissociated embryonic quail liver cells were examined in rotation-mediated cell culture. Both cAMP and DB cAMP had concentration-dependent inhibitory effects on the aggregation of cells. At a concentration of 0.6 mg/ml of cAMP, aggregates formed after 24 h and 48 h of rotation culture had half the mean diameter of those obtained in respective control cultures. DB cAMP had stronger inhibitory effects than cAMP at the same concentrations. When aggregates formed after 24 h in media containing cAMP at various concentrations were transferred to normal medium and cultured for a further 24 h, they recovered their cohesiveness to form larger aggregates. By contrast, aggregates cultured for 24 h with DB cAMP lost almost completely their aggregability in further cultivation in normal medium.     

Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells

In the retinal pigment epithelium (RPE) of fish, melanosomes (pigment granules) migrate long distances through the cell body into apical projections in the light, and aggregate back into the cell body in the dark. RPE cells can be isolated from the eye, dissociated, and cultured as single cells in vitro. Treatment of isolated RPE cells with cAMP or the phosphatase inhibitor, okadaic acid (OA), stimulates melanosome aggregation, while cAMP or OA washout in the presence of dopamine triggers dispersion. Previous studies have shown that actin filaments are both necessary and sufficient for aggregation and dispersion of melanosomes within apical projections of isolated RPE. The role of myosin II in melanosome motility was investigated using the myosin II inhibitor, blebbistatin, and a specific rho kinase (ROCK) inhibitor, H-1152. Blebbistatin and H-1152 partially blocked melanosome aggregation triggered by cAMP in dissociated, isolated RPE cells and isolated sheets of RPE. In contrast, neither drug affected melanosome dispersion. In cells exposed to either blebbistatin or H-1152, then triggered to aggregate using OA, melanosome aggregation was completely inhibited. These results demonstrate that (1) melanosome aggregation and dispersion occur through different, actin-dependent mechanisms; (2) myosin II and ROCK activity are required for full melanosome aggregation, but not dispersion; (3) partial aggregation that occurred despite myosin II or ROCK inhibition suggests a second component of aggregation that is dependent on cAMP signaling, but independent of ROCK and myosin II.     

Monocyte requirement for mitogen-induced aggregation of human peripheral mononuclear leukocytes in vitro

Formation of distinct multicellular aggregates is one of the phenomena associated with activation of quiescent human mononuclear leukocytes in vitro. Aggregate formation involves active cell motility and enhances cell-cell interactions required for an optimal proliferative response of T-cells stimulated with agents like phytohemagglutinin. We have developed an assay to quantitate the rate at which motile cells form aggregates on a flat surface. This assay follows the time rate of deviation of cells in undisturbed culture away from an initial random distribution using an "aggregation index." We used this assay to establish minimal culturing conditions required to observe an aggregation response for a partially purified mononuclear leukocyte population. We also studied the ability to aggregate of various subpopulations enriched for T- and B-lymphocytes and monocytes and found evidence for a monocyte requirement for lymphocyte aggregation. In a second assay, we followed the rate of entry of esterase positive monocytes into aggregates and compared this to the rate of entry of mononuclear cells in toto. We found that monocytes are preferentially associated with non-esterase positive cells within one hour of PHA stimulation. The results support the conclusion that monocytes play a central role in directing the motility of human T-lymphocytes leading to their aggregation response in tissue culture.     

Alkalinity-induced aggregation in Chlorella vulgaris I. Changes in cell volume and cell-wall structure

In Chlorella vulgaris cell aggregation, the clustering of singlecells into groups is induced by an alkaline pH (9.5). The processof alkalinity-induced aggregation may be divided into two stages:the first stage (0–24 hr after exposure to the alkalinepH) is characterized by enhanced precipitation of cells fromthe medium, as well as by a seven fold increase in cell volume.The second stage (24–120 hr) is associated with a furtherincrease in the extent of cell precipitation in the culture,which seems to result from the aggregation of clusters of enlargedcells. Electron micrographs reveal the existence, at this phase,of a number of autospores in the cells within a modified multi-layeredmother cell wall. The pectin content of cells at this stageis twice that of control cells grown at pH 6.3. In addition,the relative content of the different pectin fractions is modifiedas a result of the exposure to alkalinity. It is suggested thatthe aggregates result from the repeated failure of the cellsto detach from their original mother cell walls, thus formingclusters which represent several generations of cells. ¹Present address: Division of Food Storage, ARO Beit Dagan,P.O.B. 6, Israel. (Received September 3, 1979; )     

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.     

Ammonium uptake in carrot cell structures is influenced by pH-dependent cell aggregation

Semicontinuously grown wild carrot ( Daucus carota L.) cells were used in an investigation of the effect of culture medium pH on ammonium uptake in suspension cultures as a first step in exploring the relationship between pH and anthocyanin biosynthesis. In contrast to published data showing decreasing uptake rates with decreasing culture medium pH, ammonium-limited, semicontinuous carrot cell cultures showed a 25% greater ammonium uptake rate at pH 4.5 than at pH 5.5. When cells that had been grown semicontinuously in medium with a pH of 4.5 or 5.5 were grown in batch cultures at pH 4.5, 5.5 or 6.5 the ammonium uptake rates were those of the semicontinuous cultures, indicating that the pH of the batch culture medium had no effect on ammonium uptake rates over 7 days. The cell culture was composed of very small aggregates when it was grown semicontinuously in medium at pH 4.5, but was composed of large aggregates when it was grown semicontinuously in medium at pH 5.5. The aggregation/disaggregation of the cells was pH dependent, as changing the pH of the semicontinuous culture medium altered the extent of the aggregation. We conclude that the change in culture medium pH caused the cells to aggregate or disaggregate which in turn decreased or increased the rate of ammonium uptake from the medium.     

Cultivation and Differentiation of Dissociated Cells of Chick Embryo Encephalon

Dissociated cells from cerebral hemispheres of chick embryo at stages 17–18, 12–13 and 9–10, were cultivated for seven days. The cells were cultivated either completely covered with the nutrient medium in an atmosphere containing 5 percent CO₂ or they were covered by only a thin film of nutrient medium in contact with air.
For the embryos at stages 17–18 or 12–13, under both culture conditions neurons differentiated after 3 or 4 days in culture, while for the embryos at stage 9–10, no neuronal differentiation occurred under either condition. The cells remained morphologically undifferentiated and formed aggregates of about 50 cells. Some fibroblasts were found to grow on the collagen matrix.
It is concluded that dissociated cells from embryos at stage 9–10 are incapable of auto differentiation under the present culture conditions. The possible causes of this inability to differentiate are discussed.     

Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.     

Hypoblast cells of chick pre-streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro . Primary and secondary hypoblast cells from embryos at various pre-streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum-supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum-free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.     

Different aggregation properties of sea urchin embryonic cells at different developmental stages. I. Stage specificity of aggregation factors solubilized by butanol

Surface proteins solubilized with butanol from purified plasma membranes of sea urchin embryos at different developmental stages were tested for their aggregation promoting activity on dissociated cells. Cells used for the assays were obtained either from blastulae or from embryos at the 16 cell stage. Results show that a strong enhancement of cell aggregation was produced only when extracted proteins and dissociated cells were obtained from embryos at the same developmental stage.     

Temperature-dependent aggregation of pH-sensitive phosphatidyl ethanolamine-oleic acid-cholesterol liposomes as measured by fluorescent spectroscopy.

pH-sensitive liposomes made of phosphatidyl ethanolamine-oleic acid-cholesterol (4:2:4 molar ratio) at neutral pH values aggregate at approximately 40 degrees C. The aggregation is accompanied by liposome destabilization and by the release of intraliposomal fluorescent marker (calcein). Both aggregation and calcein leakage start at the temperature corresponding to the lipid phase transition into hexagonal phase. In the system studied the phase transition temperature interval is within 45 to 55 degrees C as estimated with the use of the fluorescent probe 1,6-diphenylhexatriene. The presence of cell cultivation medium RPMI 1640 decreases liposome aggregation temperature. The addition of 10% serum to the system decreases the temperature at which the aggregation proceeds still further. The conclusion that serum-free media should be used for cell experiments involving pH-sensitive liposomes is made.     

Cellular Commitment for Post-Gastrular Increase in Alkaline Phosphatase Activity in Xenopus laevis Development

Xenopus embryos were dissociated into cells and cultured in Ca²⁺-free medium to study the relationship between the cell-to-cell interaction and macromolecular synthesis. Under the conditions, cells did not aggregate at all, and remained isolated even while they were dividing actively. Synthesis of DNA and protein as studied by the incorporation of (³H)thymidine and (³H)leucine proceeded as in the aggregating cells. Also, the activity to synthesize rRNA, 5S RNA, and heterogeneous RNA as determined by the incorporation of (³H)uridine was not impaired. Such an increase in the activity of alkaline phosphatase, as occurs in embryos after the gastrula stage, was found to be inhibited greatly when early-blastula cells were cultured in the non-aggregating conditions. However, we found here that the inhibition was not observed with cells isolated from late-blastulae. Therefore, it appears that the increase in the activity of alkaline phosphatase during post-gastrular stages is dependent on some cellular commitment which may be established by cell-to-cell contact during the blastula stage.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Aggregation of poliovirus and reovirus by dilution in water.

Poliovirus and reovirus were found to aggregate into clumps of up to several hundred particles when diluted 10-fold into distilled water from a stock preparation of minimal aggregation in 0.05 M phosphate buffer, pH 7.2, plus 22 to 30% sucrose. Reovirus was also found to aggregate when diluted into phosphate-buffered saline. The aggregation was concentration dependent and did not occur when either virus was diluted into water 100-fold or greater. The aggregation of poliovirus was reversible by further addition of saline and produced a dispersed preparation of virus. Reovirus aggregation was not reversible. Both viruses aggregated when diluted into buffers at pH 5 and 3, and poliovirus aggregated at pH 6, and this aggregation of both viruses was reversible when returned to pH 7. Aggregation did not occur at alkaline pH values. Aggregation at low pH could be caused aggregation of either virus at pH 7. Calcium ions, however, were found to aggregate both viruses at a concentration of 0.01 M.     

Intercellular adhesion in butyrate-treated HeLa S3 cultures : Flow cytometric analysis

The application of DNA flow cytometry (FCM) for analysis of sodium butyrate-induced intercellular adhesion in human carcinoma (HeLa S3) cell cultures is described. To prepare cell suspensions for FCM, the monolayers of cells were treated with medium containing 10% serum, 0.2% non-ionic detergent Triton X-100 and 1 μg/ml DNA fluorochrome 4,6′-diamidino-2-phenylindole (DAPI). Total numbers of single cells, and aggregates containing two, three, four or more cells, were determined from DNA histograms. In cultures treated with 5 mM butyrate for 16 h, more than 80% of the cells were aggregated. Intercellular adhesion began to appear 8 h after addition of butyrate, was maximal at 16–24 h and stable in the presence of butyrate, but disappeared 24 h after its removal. Treatment with EDTA (0.2%) dissociated only 50%, whereas trypsin (0.1%) separated all cell aggregates into single cells. Actinomycin D (actD) (0.5 μg/ml) prevented cell adhesion while blocking of cells in S phase with 250 μM 5-fluorouracil or 10 μM methotrexate did not interfere with aggregation. The number of cell aggregates estimated from DNA histograms of butyrate-treated HeLa S3 cultures was the same after staining with DAPI in the presence of Triton X-100 or after vital staining with Hoechst 33342. The DNA content was used as a marker to estimate the cellular composition of aggregates in mixed cultures of HeLa S3 cells and human fibroblasts (U cells). Intercellular adhesion in these cultures was seen only between HeLa S3 cells, indicating specificity of butyrate-induced cell aggregation. FCM provides fast automatic measurement of cell aggregate formation, estimates frequency of aggregates containing different cell numbers, shows participation of cells at different cycle phases in aggregates, and allows the detection of homotypic from heterotypic cell aggregates if the interacting cells have different DNA ploidy.