In vitro generation of spiral ganglion neurons from embryonic stem cells

Spiral ganglion neurons (SGNs) are crucial for transmitting auditory signals from the inner ear to the brainstem, playing a pivotal role in the peripheral hearing process. However, SGNs are usually damaged by a variety of insults, which causes permanent hearing loss. Generating SGNs from stem cells represents a promising strategy for advancing cell-replacement therapies to treat sensorineural hearing loss. SGNs comprise two subtypes of neurons (types 1 and 2); however, it remains a challenge to regenerate SGN subtypes. This study aimed to investigate the generation and characterization of SGN subtype neurons induced from embryonic stem cells (ESCs) in vitro. ESCs were cultured and treated with retinoic acid, followed by neuronal induction. The differentiated cells showed protein expressions of multiple neuronal markers, suggesting the generation of neuron-like cells. Protein expressions of vGlut-1 and GATA-3 indicate the generation of glutamatergic otic neuron-like cells. ESC-derived neuron-like cells cultured for 6 days showed co-expressions of calretinin, calbindin, and POU4F1 antibodies, suggesting an early stage of SGN subtype induction. However, 14-day in vitro induction generated cells showing two distinct SGN subtypes: a group of cells expressed calretinin (subtype 1a/2 precursor), and the other group expressed calbindin and POU4F1 (subtype 1b/c). These results suggest that in vitro generation of SGN subtypes from ESCs is culture time dependent.