ENHANCING MORPHOLOGICAL, MOLECULAR, AND FUNCTIONAL MATURATION IN IPSC-DERIVED GLUTAMATERGIC NEURONS USING AN ALTERNATIVE TRANSCRIPTION FACTOR

The study of psychiatric diseases using iPSC-derived neurons is of major importance, as it enables translational research in human-relevant cellular models. Most current studies rely on the overexpression of Neurogenin-2 (NEUROG2) to generate cortical glutamatergic neurons; however, several limitations remain, including molecular heterogeneity and structural and functional immaturity. In addition, the majority of these studies are conducted in two-dimensional (2D) cultures, despite the brain being a three-dimensional (3D) structure, where spatial organisation plays a crucial role in network complexity and dendritic spine development. In this study, we show that an alternative transcription factor, TF-X, improves neuronal maturation and suitability for disease modelling in 2D and 3D neuronal structures. Human NEUROG2- and TF-X-derived neurons are imaged using two-photon laser scanning microscopy to assess cell morphology. Transcriptomic differences are assessed via bulk RNA-Sequencing (for 2D cultures) and single-nucleus RNA-Sequencing (for 3D cultures). A microelectrode array (MEA) is used to gain further insight into the electrical properties at a network level. Compared to NEUROG2-derived neurons, TF-X-derived neurons exhibit a more complex morphology as well as an increased network activity in pilot experiments. The ongoing transcriptomic analysis indicates improved maturity and increased expression of cortical markers. These findings indicate that TF-X enables the generation of neurons with improved morphological, molecular, and functional properties, providing a robust platform for studying psychiatric diseases and for advancing human-relevant disease modelling and therapeutic development.