Deciphering auditory neuroprogenitor stemness for large-scale neuronal screening

Sensorineural hearing loss can arise from damage to sensory hair cells, auditory neurons or their synaptic connections, with auditory neuropathy and cochlear synaptopathy highlighting the importance of neural integrity for hearing. The mammalian cochlea has limited capacity to regenerate lost auditory neurons. Understanding the molecular programs that sustain cochlear progenitor expansion and neurogenic competence could support neuronal regeneration and provide renewable models for auditory drug discovery. We previously established that WNT activation combined with dual SMAD inhibition (CHIR-DS) supports scalable expansion and neuronal differentiation of mouse auditory neuroprogenitors (ANPGs). Here, we characterize the transcriptional, chromatin and metabolic changes associated with the induced state. RNA-seq and ATAC-seq identified reduced Id1 and Id2 expression and increased accessibility at proneural basic helix-loop-helix motifs, alongside transcriptional enrichment of translation, mTOR and glucose-metabolism programs. Compared with growth-factor controls, CHIR-DS-treated ANPGs showed slight increase in glycolysis at early passage. However, after long-term expansion, both glycolysis and mitochondrial respiration were markedly enhanced. Human fetal ANPGs also responded to CHIR-DS with increased sphere growth and retained neural differentiation capacity, providing proof of concept beyond the mouse system. Finally, neurons derived from stemness-induced ANPGs (siANs) supported a 384-well reporter assay and a 2,560-compound pilot screen that nominated candidate neurite-outgrowth enhancers. Together, these findings validate the pathway basis of ANPG stemness induction, characterize the resulting molecular and metabolic state, and demonstrate human feasibility and a neuronal screening application.