The well-characterized human teratocarcinoma line Ntera2 (NT2) can be differentiated into mature neurons. We have significantly shortened the time-consuming process for generating postmitotic neurons to approximately 4 weeks by introducing a differentiation protocol for free-floating cell aggregates and a subsequent purification step. Here, we characterize the neurochemical phenotypes of the neurons derived from this cell aggregate method. During differentiation, the NT2 cells lose immunoreactivity for vimentin and nestin filaments, which are characteristic for the immature state of neuronal precursors. Instead, they acquire typical neuronal markers such as beta-tubulin type III, microtubule-associated protein 2, and phosphorylated tau, but no astrocyte markers such as glial fibrillary acidic protein. They grow neural processes that express punctate immunoreactivity for synapsin and synaptotagmin suggesting the formation of presynaptic structures. Despite their common clonal origin, neurons cultured for 2-4 weeks in vitro comprise a heterogeneous population expressing several neurotransmitter phenotypes. Approximately 40% of the neurons display glutamatergic markers. A minority of neurons is immunoreactive for serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase. We have found no evidence for a dopaminergic phenotype. Subgroups of NT2 neurons respond to the application of nitric oxide donors with the synthesis of cGMP. A major subset shows immunoreactivity to the cholinergic markers choline acetyl-transferase, vesicular acetylcholine transporter, and the non-phosphorylated form of neurofilament H, all indicative of motor neurons. The NT2 system may thus be well suited for research related to motor neuron diseases.

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Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. Approximately a third of the differentiated neurons expressed cell markers related to cholinergic neurotransmission. To examine whether this human cell model system can be directed toward a motoneuronal fate, postmitotic neurons were co-cultured with mouse myotubes. Outgrowing neuronal processes established close contact with the myotubes and formed neuromuscular junction-like structures that bound a-bungarotoxin. To determine how grafted precursor cells and neurons respond to embryonic nerve tissue, NT2 cells at different stages of neural development were injected into chick embryo neural tube and brain. Grafted NT2 neurons populated both parts of the nervous system, sometimes migrating away from the site of injection. The neural tube appeared to be more permissive for neurite extensions than the brain. Moreover, extending neurites of spinal grafts were approaching the ventral roots, thus resembling motoneuronal projections. Developmental Dynamics 239:496-504,