Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate culture

Abstract

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.