Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.

毛细胞变性是感音神经性听力损失的主要原因。哺乳动物耳蜗中的毛细胞不会自发再生，这对恢复听力构成了巨大挑战。在这里，我们建立了一个强大的、高通量的耳蜗类器官平台，该平台可促进耳蜗祖细胞的 3D 扩增和毛细胞以时间调节的方式分化。对 FDA 批准的药物库进行的高通量筛选确定了 regorafenib（一种 VEGFR 抑制剂）是一种用于毛细胞分化的有效小分子。Regorafenib 还促进正常和新霉素损伤的耳蜗外植体中毛细胞的重编程和成熟。从机制上讲，VEGFR 的抑制通过 MEK 通路抑制 TGFB1 的表达，而 TGFB1 的下调直接介导了瑞戈非尼对毛细胞重编程的影响。