The hiPSC-derived neural networks on dish are expected to improve the predictability of CNS adverse effects at the non-clinical stage in drug development. In human brain, EAATs are responsible for removal of an excess amount of L-glutamate (L-Glu) and prevent neuronal damage caused by excitotoxicity. In this study, we studied the role of EAATs in the hiPSC-derived neural networks.

Commercially-available hiPSC-derived neurons were seeded at 3.0×10⁵ cells/cm². At 63 DIV, the synchronized burst firing activity, which was indicative of network formation, were confirmed by microelectrode array system. Synapsin1 puncta were adjacent to PSD95 puncta, supporting synaptic maturation in these neural networks. When 100 µM L-Glu was applied exogenously at 63 DIV, L-Glu concentration in the medium ([L-Glu]_o) became almost zero in 1 hr and cell viability was not affected. In the presence of TFB-TBOA (TFB), a non-specific EAATs inhibitor, [L-Glu]_o was not changed at all and the cell viability was severely decreased. When AP5, an NMDAR antagonist, was co-applied with TFB, the cells were significantly rescued. These results indicate that EAATs are functional in the hiPSC-derived neural networks and protect neurons from excitotoxicity. It is also suggested that hiPSC-derived neural networks can be applied for the development EAAT-related drugs.