Neurons derived from embryonic stem (ES) cells resemble normal neurons in their vulnerability to excitotoxic death

We determined whether embryonic stem (ES) cells could provide a model system for examining neuronal death mediated by glutamate receptors. Although limited evidence indicates that normal neurons can be derived from mouse ES cells, there have been no studies examining pathophysiological responses in mouse ES cell systems. Mouse ES cells, induced down a neural lineage by retinoic acid (RA), were found to have enhanced long-term survival when plated onto a layer of cultured mouse cortical glial cells. In these conditions, the ES cells differentiated into neural cells that appeared normal morphologically and displayed normal features of immunoreactivity when tested for neuron-specific elements. Varying the culture medium generated cultures of mixed neuronal/glial cells or enriched in oligodendrocytes. These cultures were viable for at least four weeks. Real-time PCR analysis of N-methyl-D-aspartate (NMDA) receptor subunits revealed an appropriate age-in-vitro dependent pattern of expression. Neurons derived from ES cells were vulnerable to death induced by a 24-h exposure to the selective glutamate receptor agonists NMDA, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This vulnerability to agonist-induced death increased with age in vitro, and related closely to expression of receptor subunits, as it does in cultured primary neurons. Experiments with selective receptor antagonists showed that glutamate receptors mediated the NMDA- and kainate-induced death. Neuronal differentiated ES cells therefore exhibited an excitotoxic response resembling that displayed by central nervous system (CNS) neurons. Thus, ES cells, which are very amenable to genetic manipulation, provide a valid system for studying glutamate receptor-mediated toxicity at the molecular level.