A line of embryonal carcinoma cells, PCC7-S, established in vitro from a spontaneous testicular teratocarcinoma, has been studied. Upon removing the cells from a low density monolayer culture system and permitting the cells to form aggregates in suspension, we observed a change of several physical and biochemical parameters: (a) reduction in average cell volume, (b) blockage and accumulation of cells in G1, (c) rise in secreted protease activity, (d) rise in acetylcholinesterase and choline acetyltransferase activities, and (e) disappearance of embryonic antigen F9. Although PCC7 aggregates did not undergo substantial morphological changes while suspended, when aggregates 4 or more days old were allowed to attach to plastic tissue culture dishes, substantial neurite outgrowth occurred over the next 1-3 d. This process was markedly enhanced by the addition to the growth medium of carboxymethylcellulose and inhibitors of DNA synthesis. Transmission electron microscopy disclosed a neurite ultrastructure consistent with that of neuronal processes. A veratridine-stimulated, tetrodotoxin-blocked sodium influx of 100 nmol/min per mg protein was also observed in these differentiated surface cultures. This cell line is discussed in terms of its utility for the study of early events leading to a commitment to cellular differentiation, as well as for the investigation of terminal differentiation to cholinergic neurons.

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