Spiral Ganglion Neurons (SGNs) are crucial for transmitting auditory signals from the cochlea to the brain, with their damage contributing significantly to sensory hearing loss. Human pluripotent stem cell-derived otic neural progenitors (ONPs) provide a promising approach for regenerating SGNs. However, the diversity, electrophysiological maturation, and subtype specification of SGNs derived from human embryonic stem cells (hESCs) remain poorly understood. Moreover, despite advances in differentiating SGNs from hESCs leveraging the molecular mechanisms governing otic tissue development, protocols to generate specific subtypes, such as Type I and Type II SGNs, remain limited due to an incomplete understanding of the molecular cues required for their differentiation. To address this knowledge gap, this research investigates the differentiation and maturation of SGNs derived from hESC-ONPs and focuses on their molecular and electrophysiological properties. Additionally, the role of BMP signalling in directing subtype specification was assessed using BMP inhibitors (LDN) and agonists. Differentiated neurons were collected on days 7, 14, and 21 to assess morphological changes, examine gene and protein expression using immunofluorescence and qPCR, and evaluate electrophysiological properties via whole-cell patch-clamp recordings. Results revealed that ONPs differentiated into SGNs with distinct subtype markers: ~50-60% expressed Type I markers, and ~15-30% expressed Type II markers. Electrophysiological recordings showed diverse firing patterns and potassium current profiles, including inactivating currents resembling Type II SGNs and non-inactivating currents characteristic of Type I SGNs. Morphologically, neurons exhibited extended neurites and complex branching indicative of maturation. Moreover, immunostaining and qPCR results indicate that LDN treatment enhanced Type II marker expression. However, electrophysiology revealed the modulatory effect influencing Type I and Type II SGNs' ion channel activity. In conclusion, hESC-derived ONPs differentiate into functional SGNs with molecular and electrophysiological heterogeneity. BMP signaling modulation, particularly inhibition, promotes Type II SGN differentiation, providing insights for enhancing SGN maturation and subtype specificity in vitro.
