In vitro differentiation of a mouse neuroblastoma

Proceedings of the National Academy of Sciences, 1969

Clonal lines of neurons were obtained in culture from a mouse neuroblastoma. The neuroblastoma cells were adapted to culture growth by the animal-culture alternate passage technique and cloned after single-cell plating. The clonal lines retained the ability to form tumors when injected back into mice. A striking morphological change was observed in the cells adapted to culture growth; they appeared as mature neurons, while the cells of the tumor appeared as immature neuroblasts. Acetylcholinesterase and the enzymes for the synthesis of neurotransmitters, cholineacetylase and tyrosine hydroxylase were assayed in the tumor and compared with brain levels; tyrosine hydroxylase was found to be particularly high, as described previously in human neuroblastomas. The three enzymes were found in the clonal cultures at levels comparable to those found in the tumors. Similarly, there were no remarkable differences between the three clones examined.

Cancer research, 1970

When neuroblastoma cells are obtained from bone marrow aspirates or pleural fluid and placed into tissue culture, a characteristic growth pattern is seen. Cells are found attached to the culture vessel walls and in suspension. By serial decanting of cells in the tissue culture media, cultures can be obtained which are actively dividing and remain in suspension without agitation. The growth rate of these cells decreases to barely detectable levels within 2 months after initiation of the cultures. Cell growth can be recovered by addition of mouse salivary gland extract to the cultures. A similar stimulation is found when ascitic fluid from a patient with neuroblastoma is added to the cultures. The cells are capable of degrading norepinephrine-7-3 H to metabolites similar to those found with solid neuroblastomas, and the

The murine embryonal carcinoma cell line P19S1801Al devel- ops into neuronlike cells after treatment with retinoic acid (Ed- wards and McBurney, 1983). We have analyzed the expression of cell surface carbohydrate antigens and intracellular cyto- skeletal antigens in differentiating OlAl cells in order to iden- tify the cell types present in the cultures and to characterize the differentiation process. Undifferentiated OlAl cells express the SEA-1 antigen, GD, ganglioside, and the D1.l ganglioside antigen, carbohydrate markers that are found on early embry- onic cells and neuroepithelial germinal cells in viva. The cells also bind tetanus toxin, cholera toxin, and monoclonal antibody A2B5, probes that bind to gangliosides found on the surfaces of neurons and immature astrocytes in viva and in vitro. They con- tain vimentin-type intermediate filament antigens but have no detectable neurofilament or glial filament protein antigens. Af- ter aggregation of the cells in medium containing ...

Developmental Biology, 1981

The embryonal carcinoma line C17-Si clone 1003 is multipotential in vivo. When the cells are grown in vitro in serum-containing medium most of them remain undifferentiated, while a few differentiate into a unique morphologic type of epithelioid cell. If 1603 cells are passaged into a defined medium containing insulin, transferrin, selenium, and fibronectin they grow for six to eight generations at the same rate as in serum-containing medium. During this time, all the cells of the culture differentiate into a limited number of phenotypes with neuroepithelial and neuronal cells predominating. Differentiation could be obtained in the defined medium at relatively low cell densities. Exogenous fibronectin is required for cell attachment to the substratum, and when absent the cells form aggregates in which differentiation still occurs. Low amounts of serum added to the defined medium allow multiplication and maintenance of cells of undifferentiated phenotype and prevent differentiation into neuronal cells.

The Journal of neuroscience : the official journal of the Society for Neuroscience, 1988

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.

Differentiation, 1977

Neuroblastoma cells, grown in monolayer, transform, emit cytoplasmic processes, and acquire morphological and functional properties resembling those of mature neurons, whereas in suspension culture they remain in the undifferentiated anaplastic form. The appearance of intermediate (10 nm) filamentous structures in neuroblastoma cells is generally considered to indicate a state of cellular differentiation, one of a progressive sequence of maturing phases which lead the cell to the final differentiated state.We have examined by electron microscope murine C 1300 neuroblastoma cloned cells, grown in suspension or in monolayer cultures in the presence or absence of BrdU as an inducing agent and have compared the expression of intermediate filaments. These filaments were present in five clones of cells grown in suspension still in undifferentiated anaplastic form. One clone in particular showed a massive expression of filaments, particularly visible in the perinuclear region. One hundred per cent of the cells observed presented filaments whose number apparently increased when cells were grown in the presence of BrdU in suspension or in monolayer. One clone never showed intermediate filaments under any circumstances. The original line from which clones were derived showed poor expression of filaments which were visible only in cells grown in monolayer. These results suggest that the expression of intermediate filaments in neuroblastoma cells should be viewed as the result of a positive genetic control of phenotype expression rather than the result of a progressive sequence of differentiating events.

Materials (Basel, Switzerland), 2017

Cellular attachment plays a vital role in the differentiation of pheochromocytoma (PC12) cells. PC12 cells are noradrenergic clonal cells isolated from the adrenal medulla of Rattus norvegicus and studied extensively as they have the ability to differentiate into sympathetic neuron-like cells. The effect of several experimental parameters including (i) the concentration of nerve growth factor (NGF); (ii) substratum coatings, such as poly-L-lysine (PLL), fibronectin (Fn), and laminin (Lam); and (iii) double coatings composed of PLL/Lam and PLL/Fn on the differentiation process of PC12 cells were studied. Cell morphology was visualised using brightfield phase contrast microscopy, cellular metabolism and proliferation were quantified using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, and the neurite outgrowth and axonal generation of the PC12 cells were evaluated using wide field fluorescence microscopy. It was found that doubl...

Brain Research, 1993

Neural crest tumor cells which have been pharmacologically induced in culture to undergo neuronal 'differentiation' have been proposed as a model for normal neural crest cell differentiation. We have previously reported that murine neuroblastoma cells treated with the antineoplastic agent neocarzinostatin (NCS) adopt the light microscopic appearance of differentiated neurons. After undergoing morphologic change, the cells no longer divide. As part of an effort to compare the process of differentiation in these cells with what is known about normal neural crest cells, we have examined the cellular distribution and isoform complement of neural cell adhesion molecules (NCAMs) in native and NCS-treated neuroblastoma cells. Our studies show that NCS induces profound changes in NCAM distribution. Immunohistochemical staining indicates that, in contrast to native neuroblastoma cells, more than 80% of treated cells display surface NCAM by 4 days following treatment. Unlike the case for normal neurons, NCAM is uniformly distributed over the treated cell surface. Neuroblastoma cells treated with NCS are more avidly adherent to culture plates coated with NCAM than are control neuroblastoma cells, reflecting the homophilic binding characteristics of NCAM. Interestingly, Western blot analysis for NCAM demonstrates similar total cellular content of a single NCAM species in both control and treated neuroblastoma cells. Furthermore, this 120 kDa mol. wt. NCAM is an isoform of NCAM not found on normally differentiated cerebellar neurons. While the presence of NCAM on these treated murine neuroblastoma cells is evidence for 'differentiation' along neuronal lines, the isoform complement and cell surface distribution of NCAM in treated cells are not normal. The change in surface staining for NCAM without a change in the total amount of NCAM suggests a redistribution of NCAM within the cell. This is supported by our finding that, while untreated cells do not stain superficially for NCAM, permeabilization of these cells leads to marked staining, implying that, upon treatment, cytoplasmic NCAM is translocated to the membrane.

Developmental Brain Research, 1985

Morphological characteristics of undifferentiated and differentiated human neuroblastoma cells were studied. Monolayer cultures of a human neuroblastoma, IMR-32 clone, were grown in Eagle's minimum essential medium with fetal calf serum in tissue culture dishes with polystyrene film liners. After 48 h, cultures were treated with either mitomycin C and 5-bromodeoxyuridine or prostaglandin E 1 (PGE1) and dibutyryl adenosine 3',5'-cyclophosphate (cAMP). A third dish was untreated to study as an undifferentiated control. Three days later, all cultures were processed for acetylcholinesterase staining, scanning and transmission electron microscopy and high performance liquid chromatography. Treatment with mitomycin/5-bromodeoxyuridine and PGE1/cAMP inhibited growth as seen by the growth curves and caused morphological differentiation as seen by the extension of long neurites. The treated cells showed increased acetylcholinesterase staining compared to the controls. With the scanning electron microscope, the differentiated cells showed long neurites, processes with beaded varicosities and growth cones. By transmission electron microscopy, these cells contained a large number of neurosecretory granules in their cytoplasm and neurites. Specialized cell contacts were also observed between the treated cells. This is the first study demonstrating that both the treated and control cells of IMR-32 clone contain large quantities of serotonin and comparatively small amounts of norepinephrine and dopamine.

Medical and Pediatric Oncology, 2001

A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. Procedure. We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were cocultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2Јdeoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferasemediated dUTP-X fluorescein nick end labelling (TUNEL) assay. Results. Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. Conclusions. The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated. Med. Pediatr.