Evaluating Neuron Differentiation and Maturation

Human iPSC-derived iNSCs were seeded at 6 × 10⁴ cells in a 24-well microelectrode array (MEA) plate (Axion Biosystems, Atlanta, GA, USA) containing 16 electrodes for the assay of neuron differentiation and maturation. All procedures were conducted by following the user’s manual of Axion Biosystems. MEAs system can capture the extracellular field potentials of electrically active cells by recording their neuronal activity [21 (link)]. To examine the spontaneous electrical activity of differentiated neuron, we collected the activity data from neuronal networks for 8 min by MEA every two or three days starting from day 14 after neuronal differentiation (3–4 replicates per experiment). Spike and burst rates represented the overall activity of the neuronal network, with more spikes and bursts corresponding to a higher electric activity. Network events include synchronous network spikes and bursts in which more than 35% electrodes captured the activity simultaneously. For electrical stimulation, after differentiation for 8 weeks the neurons received current injection at 50 μA/1000 μs/1000 mV every 5 s for five consecutive stimulations.