목적: Inner ear organoid (IEO) is an in vitro artificial organ that mimics the inner ear, serving as an essential tool for hair cell regeneration research. Currently the established IEO protocol has enabled the generation of cochlear organoid through ventralization, but the role of retinoic acid (RA) as a morphogen related to the cochlea has not been explored. Based on this, we aimed to develop an IEO with cochlear characteristics and, in the process, improved the protocol using RA. 방법:IEOs were generated from mouse embryonic stem cells (mESCs) and tested by applying RA treatment at two time points: one time point (early treatment) before the development of the otic placode and the other time point (late treatment) when the otic vesicles were developed. Immunohistochemistry (IHC) and Fluorescence Activated Cell Sorting (FACS) were conducted using MYO7A, SOX2, TUJ1, GBX2 and GATA3. RT-qPCR and Single-cell RNA sequencing (scRNA-seq) were performed. 결과:RT-qPCR results showed that Gata3 expression was significantly increased in all groups applying RA treatment. In the early treatment, Pax2 and Pax8 expression tended to increase depending on RA concentration. Otx2 expression was significantly decreased in the early treatment and significantly increased in the late treatment. FACS results indicated that increased both MYO7A-positive cells and MYO7A/GATA3 double stained cells in the early treatment. scRNA-seq analysis identified transcriptional identities related to specification of spiral ganglion neurons in the early treatment. 결론:RA treatments showed similar effects in promoting the specification of spiral ganglion neurons and cochlear hair cells in IEO. This approach could help develop IEO platforms for subdividing neuronal lineage specification.