EFFECTS OF DEPOLARIZATION PATTERNS ON THE NEURONAL DEVELOPMENT AND MATURATION OF MOUSE AND HUMAN STEM CELLS

During development, terminal neuronal phenotypes are determined by the interplay of intrinsic properties and extrinsic factors such as bioelectric patterns manifested by early network oscillations and giant depolarization potentials. Consequently, modulating embryonic electrical activity can drive neuronal differentiation.
In this study, we optogenetically stimulated channelrhodopsin-2 expressing mouse neuroectodermal stem cells (NE-4C) and human iPSC-derived neuronal precursors (NPCs) for 48h. During this time, 2 msec flashes evoking action potentials were applied in a rhythmic [(i) 5s-long 20 Hz oscillation repeated every minute, (ii) 5s-long random Poisson distribution repeated every minute] and non-rhythmic [(iii) continuous Poisson distribution] manner. Dark-kept cultures were used as controls. Neuronal maturation was tracked by qRT-PCR, bulk RNA-seq, calcium imaging, and whole-cell patch-clamp techniques.
Our results demonstrate that the oscillatory pattern was the most potent driver of maturation, by significantly increasing the proportion of neurons exhibiting robust action potentials in both mouse and human cultures. Additionally, in NE-4C cells, the prevalence of inward-rectifying K-currents (KIR) and the expression of vGluT1 and vGaT1 transporters also increased. In human NPCs, we observed faster calcium wave kinetics and significant transcriptomic enrichment of genes associated with cytoskeleton remodeling. On the other hand, the continuous Poissonian pattern was ineffective in mouse cells, but significantly reduced the ratio of active cells and perturbed the gene expression in human NPC-derived neurons.
These findings suggest that the temporal structure and duration of depolarization differently regulate the maturation of mouse and human neurons, highlighting the importance of species-specific bioelectric environments during embryonic development.