목적: The dynamic interplay of signalling pathways, namely Fgf2, Notch, Wnt, and Sonic Hedgehog (SHH), intricately guides the development of the inner ear. Fgf2 supplementation can enhance otic progenitor cell differentiation while manipulating Notch signalling pathways allows for precise control over sensory cell generation. Wnt activation is crucial for recapitulating organoid morphogenesis, and SHH modulation guides the spatial arrangement of sensory structures. These insights have been applied to embryonic stem cells and induced pluripotent stem cells to form inner ear organoids. This study investigated the effects of Fgf2, Notch, Wnt, and SHH signalling in LGR5-positive progenitor cells to develop mature inner ear cochlear hair cells (HCs). 방법:Building on our previous RNA sequencing results comparing the manual isolation to Magnetic-Activated Cell Sorting (MACS), we used the MACS- isolated LGR5-positive cells from postnatal P1-4 mice cochlea and induced HC differentiation using agonists and inhibitors for Fgf2, Notch, Wnt, and SHH signalling pathways. The organoids were closely observed for 24 days, and subjected to various analyses, including immunofluorescence, RNA sequencing, electrophysiology and transmission electron microscopy. 결과:MACS-isolated LGR5-postive progenitor cells were successfully formed organoids and differentiated into putative HCs with Notch inhibition and Wnt agonist treatment however, the HCs were non-functional thus deemed immature. FGF2 receptor inhibition led to smaller-sized spheroids but did not improve hair cell characteristics in MACS-derived organoids. Meanwhile, increased Notch inhibition exhibited enhanced mechanotransduction channel functionality, while SHH activation resulted in enhanced cochlear hair cell prevalence and functionality. 결론:The orchestrated interplay of Notch inhibition, Wnt signalling activation, and FGF2 inhibition is pivotal in driving LGR5-positive supporting cell differentiation into inner ear hair cells. These findings contribute to our understanding of auditory system development and hold implications for regenerative medicine interventions aimed at harnessing the regenerative potential of LGR5- positive supporting cells.