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.