Differentiation of human teratocarcinoma cell line into motor neurons

The aim of this project was to develop cell culture techniques for the directed differentiation of the human teratocarcinoma cell line (Ntera-2) into postmitotic motoneurons. Immunocytochemical and electrophysiological methods were used to characterize the cellular properties of the differentiated neurons in vitro. To investigate integration, differentiation and maturation of the NT2 cells in an in vivo system, they have been transplanted into the nervous system of chick embryos. Upon treatment with RA, and a proliferation step in free-floating cell aggregates, NT2 precursor cells can be induced to differentiate into postmitotic neurons (NT2N) within one month. NT2N cells showed neuronal morphology and immunoreactivity to a number of neuronal markers including cytoskeletal proteins such as β- III-tubulin, microtubule-associated protein 2, and phosphorylated tau. Moreover, neural processes displayed a punctate immunoreactivity for synapsin and synaptotagmin. Using the outside-out patch-clamp configuration, the kinetics of currents elicited by a rapid application of the amino acid neurotransmitters glutamate and GABA were measured. Spontaneous postsynaptic currents could be detected in glia free cell cultures, indicating the formation of functional synapses. Differentiated NT2 neurons when cultured in vitro, are a heterogeneous population displaying glutamatergic markers, serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase immunoreactivity. Subpopulations 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 (ChAT), vesicular acetylcholine transporter and the nonphosphorylated form of neurofilament H, all indicative of motor neurons. In co-cultures of NT2 neurons with mouse myotubes, morphological structures resembling neuromuscular junctions were found that stain for ChAT, synapsin and α-bungarotoxin. Human NT2 neurons have been successfully transplanted by many groups into the nervous tissue of adult experimental animals and a limited number of human neurological patients. For the first time, a study of integration and differentiation of various NT2 cells in the cells in the environment of the chick embryo was conducted. I have injected NT2 precursor cells (NT2), NT2 pretreated with retinoic acid (NT2+RA), NT2 pretreated with retinoic acid and Sonic hedgehog (NT2+RA+Shh), and NT2 neurons (NT2N) into the spinal cord and brain. Monitoring of integration, differentiation and outgrowth ratio between transplanted cell types was done after 3-5 days of incubation by morphological analysis and staining for β-III-tubulin. The spinal cord appears to facilitated neurite extension more than the brain. In addition, extending neurites of spinal grafts were often approaching the ventral roots, thus resembling motoneuronal projections. In summary, NT2 neurons and pretreated NT2 cells transplanted into chick embryos, may serve as a unique model for the in vivo study of human neuronal cells development.