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Electrophysiological characterization of mature neurons derived from mouse embryonic stem cells by Sox-1 Lineage selection and directed differentiation

R.J. Lang1, J. Haynes2, J. Kelly3, J. Johnson3, E. Mulholland1, L. Baker3, C. Pouton2, 1Physiology, Monash University, Clayton, VIC, Australia, 2Victorian College of Pharmacy, Monash University, Parkville, VIC, Australia, 3Stem Cell Sciences Ltd, Collingwood, VIC, Australia

Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. Directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6-7 days post plating (D6-7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials arising from the activation of tetrodotoxin (TTX)-sensitive Na+ channels. Neurons approached their maximal cell capacitance after D6-7PP, however ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca2+-free, 2.5 mM Mg2+ physiological saline. Synaptic currents of rapid decay time constants (<20 ms) were reduced in amplitude with membrane depolarization and preferentially blocked by the nonNMDA glutamatergic receptor antagonists, CNQX or NBQX. Ca2+ levels within ES cell-derived neurons increased in response to glutamate receptor agonists L-glutamate, AMPA, N-methyl-D-aspartate (NMDA) and kainic acid (KA) and to acetylcholine, ATP and dopamine. NBQX displaced the concentration-Ca2+ response curve to AMPA but not to glutamate or KA. NMDA evoked a cationic membrane current which reversed at -11 mV and displayed a Mg2+-dependent outward rectification (block) at negative potentials. Glycine and GABA evoked Cl--selective currents which reversed at -70 and 78 mV, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic inputs and respond to various neurotransmitters in a manner similar to primary central neurons.