Enhanced electrophysiological activity and neurotoxicity screening of environmental chemicals using 3D neurons from human neural precursor cells purified with PSA-NCAM

The assessment of neurotoxicity for environmental chemicals is of utmost importance in ensuring public health and environmental safety. Multielectrode array (MEA) technology has emerged as a powerful tool for assessing disturbances in the electrophysiological activity. Although human embryonic stem cell (hESC)-derived neurons have been used in MEA for neurotoxicity screening, obtaining a substantial and sufficiently active population of neurons from hESCs remains challenging. In this study, we successfully differentiated neurons from a large population of human neuronal precursor cells (hNPC) purified using a polysialylated neural cell adhesion molecule (PSA-NCAM), referred to as hNPC^PSA-NCAM+. The functional characterization demonstrated that hNPC^PSA-NCAM+-derived neurons improve functionality by enhancing electrophysiological activity compared to total hNPC-derived neurons. Furthermore, three-dimensional (3D) neurons derived from hNPC^PSA-NCAM+ exhibited reduced maturation time and enhanced electrophysiological activity on MEA. We employed subdivided population analysis of active mean firing rate (MFR) based on electrophysiological intensity to characterize the electrophysiological properties of hNPC^PSA-NCAM+-3D neurons. Based on electrophysiological activity including MFR and burst parameters, we evaluated the sensitivity of hNPC^PSA-NCAM+-3D neurons on MEA to screen both inhibitory and excitatory neuroactive environmental chemicals. Intriguingly, electrophysiologically active hNPC^PSA-NCAM+-3D neurons demonstrated good sensitivity to evaluate neuroactive chemicals, particularly in discriminating excitatory chemicals. Our findings highlight the effectiveness of MEA approaches using hNPC^PSA-NCAM+-3D neurons in the assessment of neurotoxicity associated with environmental chemicals. Furthermore, we emphasize the importance of selecting appropriate signal intensity thresholds to enhance neurotoxicity prediction and screening of environmental chemicals.