Cryopreserved Human Otic Neuronal Spheroids Self-assemble for Functional Connectivity Analysis and Long-term...

Spiral ganglion neurons (SGNs) in the inner ear are indispensable for auditory function, and their irreversible damage causes permanent sensorineural hearing loss. Although current human pluripotent stem cell (hPSC)-derived otic lineages offer a valuable resource for SGN regeneration, they face challenges in terms of reproducibility and functional maturation. Here, a robust protocol is established to generate human otic neuronal spheroids (hONS) from cryopreserved hPSC-derived pre-placodal ectoderm (PPE) cells. Post-thaw PPE cells retained high purity and differentiation efficiency comparable to fresh PPE cells. These self-assembled hONS differentiated into functionally mature SGN-like neurons, showing specific maker expression, electrophysiological activity, AMPA receptor-mediated glutamate response, and extensive neurite extension. In tripartite cocultures incorporating murine cochlear explants and human cortical organoids, hONS formed bidirectional functional synaptic connections, validated through live-cell imaging, optogenetic stimulation, and synaptic immunostaining. Notably, hONS exhibited heightened sensitivity to ototoxic insults. Short-term cisplatin exposure induced dose-dependent alterations in cellular and calcium dynamics, whereas prolonged exposure impaired glutamatergic neural functionality and triggered progressive neuronal death. Co-treatment with sodium thiosulfate attenuated cisplatin-induced damage. The hONS model also demonstrated concentration-dependent toxicity to neomycin. Collectively, this hONS model provides a reliable platform for investigating SGN regeneration and conducting preclinical evaluation of ototoxic drug.