# Cryopreserved Human Otic Neuronal Spheroids Self‐assemble for Functional Connectivity Analysis and Long‐term Ototoxicity Evaluation

## TL;DR

This study creates human otic neuronal spheroids from frozen stem cells that can form functional connections and test drug toxicity for hearing loss.

## Contribution

A reproducible method to generate functional human otic neuronal spheroids from cryopreserved cells for ototoxicity testing.

## Key findings

- hONS differentiated into mature SGN-like neurons with functional properties and synaptic connections.
- hONS showed high sensitivity to ototoxic drugs like cisplatin and neomycin.
- Cocultures with cochlear explants and cortical organoids validated bidirectional synaptic connectivity.

## Abstract

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.

This study establishes a robust and reproducible protocol for generating human otic neuronal spheroids (hONS) from cryopreserved hPSC‐derived pre‐placodal ectoderm cells. These hONS further differentiate into functional SGN‐like neurons, which extend neurite projections toward both murine hair cells and human cortical organoids, thereby forming functional synaptic connections in tripartite coculture systems. Moreover, these hONS demonstrate heightened sensitivity to ototoxic drugs.