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

Area of Science:

• Neuroscience
• Regenerative Medicine
• Otic Biology

Background:

• Spiral ganglion neurons (SGNs) are vital for hearing; their damage causes permanent sensorineural hearing loss.
• Existing human pluripotent stem cell (hPSC)-derived otic lineages for SGN regeneration face reproducibility and maturation challenges.

Purpose of the Study:

• To establish a robust protocol for generating human otic neuronal spheroids (hONS) from cryopreserved hPSC-derived pre-placodal ectoderm (PPE) cells.
• To validate the functional maturation and synaptic integration of hONS.
• To utilize the hONS model for assessing ototoxicity.

Main Methods:

• Generation of hONS from post-thaw PPE cells.
• Assessment of SGN-like neuron differentiation, including marker expression and electrophysiology.
• Tripartite co-culture system with murine cochlear explants and human cortical organoids for synaptic connection validation.
• Ototoxicity assays using cisplatin and neomycin.

Main Results:

• Post-thaw PPE cells yielded reproducible hONS with high differentiation efficiency.
• hONS differentiated into mature SGN-like neurons exhibiting functional characteristics and extensive neurite outgrowth.
• hONS formed functional synaptic connections with cochlear explants and cortical organoids.
• hONS demonstrated sensitivity to ototoxic agents like cisplatin and neomycin, with protective effects observed for sodium thiosulfate.

Conclusions:

• The developed protocol provides a reliable platform for generating functional SGN-like neurons.
• The hONS model serves as a valuable tool for investigating SGN regeneration strategies.
• This model enables preclinical evaluation of ototoxic drug effects and potential protective agents.