Timing and Dosage of Notch Inhibition and Wnt Activation Enhanced Hair Cell-Like Cell Generation and Stereocilia Elongation in Otic Organoids

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The modulation of Notch and Wnt signaling was evaluated for its ability to promote the differentiation and maturation of hair cell (HC)-like cells in otic organoids derived from mouse embryonic stem cells (mESCs). Using R1/E mESCs, we generated otic organoids and treated them with the Notch pathway inhibitor (γ-secretase inhibitor, DAPT) and the Wnt signaling agonist (CHIR99021) during two-time windows: days (D) D12-D18 (early treatment) or D28-D34 (late treatment). Among the late treatment conditions, lower concentration (5 μM DAPT and 2.5 μM CHIR99021) yielded greater expression of MyoVIIa and Atoh1 compared with higher doses (10 and 5 μM, respectively). Ultrastructural analysis by transmission electron microscopy further demonstrated that late treatment with lower-dose DAPT and CHIR99021 generated HC-like cells harboring longer and more prominent stereocilia-like bundles than those observed in other conditions. In contrast, early treatment produced HCs arranged in a typical otic organoid pattern, resulting in a more morphologically stable but less robustly induced HC-like population. These findings indicate that temporally and dose-controlled modulation of Notch and Wnt signaling efficiently enhances HC-like cell differentiation and stereocilia maturation in mESC-derived otic organoids, providing a useful platform for future studies in inner ear regeneration and cell-based therapies.