Transcript-protein discrepancy of glutamatergic receptor subunits in human iPSC-derived neurons: Implications for neurotoxicity testing

The development of robust human in vitro models is crucial for advancing neurotoxicology and reducing animal testing. Human-induced pluripotent stem cell (hiPSC)-derived neuronal models hold great promise, but still show limitations in recapitulating certain neurodevelopmental processes. Currently, rodent primary cultures remain the gold standard for studying complex processes such as synaptogenesis. A key mechanism in glutamatergic synapse maturation is the GluN2B/GluN2A switch, which promotes the recruitment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, increasing the structural and functional complexity of the synaptic spines. This study characterizes the development of the glutamatergic machinery in hiPSC-derived neurons, focusing on the expression and maturation of N-methyl-D-aspartate (NMDA) and AMPA receptors. The increase of neuronal markers and the reduction of progenitor markers confirmed the differentiation efficiency. However, discrepancies emerged between transcriptional and protein profiles of key receptor subunits. GluN2A mRNA levels increased over time, while protein levels remained similar to those of neural progenitor cells (NPCs). Conversely, the GluN3A transcript increased at 30 and 60 days in vitro (DIV), while protein abundance decreased. Similar transcript-protein mismatches were observed for some AMPA receptor subunits. These results suggest that this model does not reach full glutamatergic maturity within the tested timeframe. Therefore, optimizing differentiation conditions (such as extending culture duration or adding maturation cues) may be necessary to better reproduce receptor dynamics. Finally, this study highlights the need to integrate protein-level analyses with transcriptional data to improve the reliability of hiPSC-derived neuronal models for neurotoxicity and NMDA receptor-mediated excitotoxicity studies.