Tissue Culture Studies in Bovine Leukosis

By examining cover slips stained at regular intervals the development of primary lymph node tissue cultures from 2 cases of juvenile bovine leukosis and 2 “normal” foetuses was studied. Secondly, comparisons were made between cell lines prepared from 23 bovines — “normal” animals and foetuses, cases of adult leukosis, juvenile leukosis and skin leukosis — with respect to susceptibility to infection with interferon sensitive viruses (VSV and PRV) and/or growth rate in the presence of sera from “normal” and leukotic animals. Nuclear budding, nuclear fragmentation, lymphocyte adsorption and giant cell formation were observed — though to a much greater extent in cultures prepared from the leukosis animals — in all 4 cases studied. No indications of different susceptibility for the test virus infections appeared between cell lines prepared from “normal” and leukotic animals. The growth rate of the cell lines seemed similar irrespective of the kind of serum used.     

Enzyme activity during the growth and aging of human cells in vitro

Acid phosphatase, alkaline phosphatase, and lactic dehydrogenase activities have been compared in normal human diploid cell strains and in SV₄₀-transformed heteroploid cell lines derived from them. A higher level of acid phosphatase activity was observed in diploid cultures derived from adult lung than in cultures derived from fetal lung of similar passage levels. The alkaline phosphatase activity of normal diploid fibroblasts was significantly higher than that of SV₄₀-transformed cell lines derived from them. Generally, the lactic dehydrogenase activities of all these cell cultures were similar. Human diploid cells in culture “age,” in the sense that their ability to proliferate decreases with time during serial subcultivation. Evaluation of the activities of these three enzymes during the “aging” process showed that, although alkaline phosphatase and lactic dehydrogenase activities were similar in “young” and “senescent” cells, acid phosphatase showed a small but significant increase in the senescent cells.     

Differentiation of lens in cultures of neural retinal cells of chick embryos

When dissociated cells of neural retinae of 8-day-old chick embryos were cultured, monolayer sheets of epithelial cells were obtained. These cells proliferated actively. After about 30 days of culture, both lentoid bodies and pigment cells were differentiated in all plates. In the second and the third generation cultures, both differentiations were also observed. Lentoid bodies showed positive immunofluorescence for fluorescein-isothiocyanate-conjugated antiserum against δ-crystallin. Molecular constituents of lentoid bodies were very similar to those of lenses developing in situ, as revealed by immunodiffusion tests. Several lines of evidence for the “neural retinal” origin of lentoid bodies, as opposed to their being derived from lens cells inadvertently included in the original culture inocula are given. Some implications of the present results for the problem of “determination” are discussed.     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

A radiographic approach to childhood illness in precolumbian inhabitants of southern Peru

A total of 108 individuals from six different cultures found in the Department of Ica, Peru, were studied for the presence of Harris's Lines. Such lines or “bone scars” are formed due to cessation of bone growth due to episodes of starvation or illness and possibly other causes. These individuals covered a span of time of nearly 2,600 years. The individuals from mountain cultures had fewer lines and possibly a healthier childhood than those from coastal cultures. The modern population surveyed in this series still show a pattern of Harris's Lines similar to people from the Inca culture of 450 years ago.     

The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture

This paper describes the physiological and pharmacological parameters of the response of mature muscle fibers that develop from myoblasts in vitro to iontophoretically applied acetylcholine (ACh) and the distribution of ACh sensitivity over fibers innervated in vitro by spinal cord cells and uninnervated (control) fibers. Peaks of sensitivity were detected near nerve terminals on functionally innervated fibers, but the “extrasynaptic” chemosensitivity remained high. The distribution of chemosensitivity over uninnervated fibers is not uniform: peaks or “hot spots” were detected over most fibers. Autoradiography of cultures exposed to ¹²⁵I-α-bungarotoxin is consistent with the uneven distribution detected by iontophoresis. Sensitivity peaks were usually located in the immediate vicinity of obvious muscle nuclei and conversely the membrane near most nuclei was more sensitive than that over other regions along the same cell. The relation between innervation and distribution of ACh sensitivity is discussed.     

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.     

The role of cell-cell contact in "contact" inhibition of cell division: a review and new evidence

The term “contact inhibition of cell division” was borrowed from “contact inhibition of cell movement.” We prefer the term “postconfluence inhibition of cell division” as being more operational and less mechanistically biased; it is operationally defined as a pronounced depression of the mitotic rate in a postconfluent culture which displays a stationary density despite periodic nutrient renewal, the inhibition being locally reversibly by removal of the adjacent cells. The mechanism of postconfluence inhibition is of considerable interest because of the inverse correlation between postconfluence inhibition and the tumorigenicity of a number of cell lines. Several hypotheses, involving direct cell-to-cell contacts or locally restricted diffusion gradiens, could explain postconfluence inhibition. With the goal of discriminating among these hypotheses, time-lapse films were taken of carefully regulated, perfused cultures of 3T3 mouse cells, in which the transition from rapid growth to the stationary phase was recorded. Measurements of cell-to-cell contact, local cell density, and generation times were made on an individual cell level and analyzed with the aid of a computer. We observed that all-around cell-cell contact or a high local cell density present throughout G1 often did not produce immediate inhibition of cell division. We conclude that either (i) simple visible cell-cell contacts or a high local cell density are not the direct cause of postconfluence inhibition of cell division, or (ii) their effects often do not inhibit cell division until after a delay of about one cell generation time. Such a delay may be partly responsible for the 50% overshoot past the stationary density that we observed in 3T3 cultures.     

Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2-deoxy-D-glucose is very low. Upon raising the pH to 7.4 and adding serum (“turn-on”) the uptake of 2-deoxy-D-glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. The uptake of 2-deoxy-D-glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum (“turn-off”). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been “turnedoff” during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if “turned-on” again within 2.5 hours. If the cultures have been “turned-off” for 5.5 hours before restoring the “turn-on,” there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results indicate that chick embryo cells do not become committed to the initiation of DNA synthesis until shortly before, or at the time of the onset of the S period. Up to 96% of the cells in post-confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to ³H-thymidine. Seventy-eight percent of the cells in serum-deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G₁ period rather than by shifting cells into a qualitatively distinct G₀ period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Differentiation of actin-containing filaments during chick skeletal myogenesis.

Actin-containing filaments in cultures of differentiating chick skeletal muscle were examined by indirect immunofluorescence and transmission electron microscopy (TEM). As early as 20 h in culture, a large proportion of the pre-fusion population appeared as elongated, bipolar cells which contained actin filaments parallel to the longitudinal axis of the cell. During fusion, most of the mononucleated cells were bipolar and contained actin filament bundles which appeared to extend the entire length of the cell body and lie in close proximity to the plasma membrane. Striations were observed within actin filament bundles only after fusion had been completed. The small number of non-myogenic cells present in the cultures were not observed to display a bipolar morphology, orientation of actin fibers parallel to the longitudinal axis of the cell, or striations in their actin filament bundles.     

Enhanced rates of fluid pinocytosis during exponential growth and monolayer regeneration by cultured arterial endothelial cells

Rates of fluid pinocytosis by bovine aortic endothelial cells were measured during various manipulations of growth status in vitro. Sparsely seeded cultures grew exponentially until a confluent monolayer was formed, at which time growth slowed. This change in growth rate coincided with a decline in the rate of pinocytosis to about one-third that in the growing cultures. During the subsequent attainment of maximal cell density in the confluent monolayer, the pinocytic rate remained constant. There was close correlation between ³H-thymidine labelling indices, as measured by autoradiography, and the rates of pinocytosis. Mechanical “wounding” of the confluent monolayer resulted in cell migration and proliferation. Twenty-four hours after “wounding,” rates of pinocytosis per mg. cell protein were significantly enhanced. When regeneration of the monolayer was blocked by cytochalasin B, pinocytosis remained at the same rate as in the uninjured, confluent monolayer. These experiments support, and extend to endothelium, earlier observations that in growing cells pinocytosis proceeds at a higher rate than in non-growing, quiescent cells. Furthermore, they raise the possibility that the transendothelial transport of macromolecules such as lipoproteins by receptor-in-dependent fluid pinocytosis in vivo may be altered by the growth status of the endothelium.     

Sphingomyelinase activities in neuronal cell cultures.

Human and rat brains have been previously demonstrated to contain three sphinomyelinases, one lysosomal with a pH optimum of 5.0, one with a pH optimum of 7.4 and a dependence on magnesium and another with a pH optimum of 7.0 and no divalent cation requirement. Using brain cell cultures and clonal cell lines of both neuronal and glial origin the activities of the pH 5.0 and pH 7.4 (magnesium-dependent) sphingomyelinase were examined. Sphingomyelinase activity measured at pH 5.0 was found in all the cell lines tested including G26, C6, N18 (differentiated and undifferentiated), mouse “L” cells, human skin fibroblasts, fetal mouse brain surface cultures and fetal mouse brain reaggregate cultures. However, pH 7.4 (magnesium-dependent) sphingomyelinase activity was found only in the N18 cell lines and the reaggregate cultures suggesting the probable localization of this activity in neurons. Although the pH 7.4 sphingomyelinase activity was found in the fetal mouse brain used for the surface cultures this activity was rapidly lost. This enzyme may play an important role in neuronal development and maturation.     

Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds

In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26. Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ-cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.     

The effect of ascorbate on embryonic chick sternal chondrocytes cultured in agarose

Primary cultures of embryonic chick sternal chondrocytes were embedded in a three-dimensional matrix of 1% solid agarose which was overlaid with nutrient media. The chondrocytes divided and formed nests of spherically shaped cells which were surrounded by an extensive extracellular matrix containing high molecular weight proteoglycans. Using light and electron microscopy, condensation of proteoglycan was observed pericellularly, often forming septa between cells of a nest, and as part of the outer boundary of the cell nest. No cross-striated collagen fibers were observed in the extracellular matrix although proteoglycan appeared to decorate a network of fine strands. Upon the addition of ascorbate to the nutrient media high molecular weight proteoglycans were synthesized, but there was a marked decrease in the synthesis of proteoglycans after a 10 day exposure to ascorbate. Morphologically, the decrease in proteoglycan synthesis was manifested in the discontinuous arrangement of the pericellular matrix as well as the diffuse form of the cell-nest boundary. Both of these structures were clearly defined in control cultures and were enriched in proteoglycan as demonstrated by ruthenium red staining. This study demonstrates that embryonic chondrocytes remain differentiated when cultured in solid agarose for a period of up to 15 days. They continue to synthesize their tissue specific macromolecules and are phenotypically stable when exposed to ascorbate for extended periods of time.     

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02?mg/g DW in comparison with the nontransformed roots (up to 0.687?mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.     

Peptides of normal and variant cells of tobacco

Polypeptides solubilized from established normal and variant cell lines of Nicotiana tabacum L cv “Wisconsin 38” have been analyzed by one-dimensional and two-dimensional gel electrophoresis. There was little variability observed in the polypeptide profile in an established cell line; polypeptides present in different clonal lines of cells, all derived from an initial established cell culture, were very similar, if not identical. However, a large fraction of the observed polypeptides present in cytokinin-habituated cell lines (up to 3.8% of the total polypeptides analyzed by two-dimensional gel electrophoresis) were different from those found in the cytokinin-requiring cells from which they were selected. The habituated nature of the selected cell lines was demonstrated to be epigenetic; tissue cultures that were reisolated from plantlets regenerated from habituated cell lines did require cytokinin. Further observations demonstrate: (1) that epigenetic events that alter a cellular phenotype change the expression of a relatively large number of polypeptides, (2) that a single epigenetic phenotype may be the result of any one of a number of possible patterns of gene expression, and (3) that epigenetic events are not random events.     

THE NEAR-SPINELESS TRACHELOMONAS GRANDIS (EUGLENOPHYCEAE) SUPERFICIALLY APPEARS SPINY BY ATTRACTING BACTERIA TO ITS SURFACE1,2

Trachelomonas grandis Singh has a mucilaginous, highly porous mineralized lorica (envelope) generally without ornamentation except occasionally for a few short, tapered, anterior or posterior spines. However, as our first cultures of this species aged, rod-shaped structures appeared on the loricas. That these surface projections were bacteria was determined by scanning and transmission electron microscopy. The bacteria, 2-6 μm long with rounded apices, were oriented perpendicular to the exterior lorica surface and were attached on one end by apically produced tie-down fibers. The bacteria also secreted fibers over their entire surface, forming a network between them that collapsed during specimen preparation for scanning electron microscopy. The density of the surface bacteria increased with time until the lorica took on a “spiny” appearance superficially similar to lorica extensions of algal origin. In mature algal specimens, an estimated 1200-1800 bacteria per lorica occurred as a monolayer, the maximum number related to the surface area of the lorica available for bacterial colonization. The bacteria, always motionless while attached, showed putative evidence of budding. Fission formed short chains of up to three cells on the lorica. Our cultures maintained this association for 8 years (1972-1979). However, cultures ordered for further study in the past year have failed to develop loricas with more than just a few bacterial cells, and most have none.     

Properties of cell lines derived from altered-cell foci in baby mouse skin cultures

Long-term cell lines were readily developed from a proportion of either baby mouse skin (BMS) cultures passing through colchicine-induced crisis or altered-cell foci selected from BMS cultures exposed to light and/or oxygen followed by colchicine. The developing cell lines behaved as though they passed through a continuing, profound genetic reshuffling process, which was usually lethal but which in some cases eventually yielded a gene set that favored long-term survival. Some cell lines have passed 120 cell doublings, and none has shown a second crisis or signs of senescence. As soon after isolation as measurement was possible (19–50 days) the cell lines were predominantly or entirely tetraploid or subtetraploid. Although BMS cells and all of the cell lines were density inhibited, the BMS, C14, C21 and C23 cells overgrew (formed colonies on) monolayers of the same cells in most combinations. The cell lines retained a variety of neoplastic morphological characters, although their morphology was more normal than in the original focus. No cell line, however, showed anchorage-independent growth or formed tumors in syngeneic hosts. The cell lines may all, therefore, be regarded as preneoplastic.