Lenz Danielle R, Gunewardene Niliksha, Abdul-Aziz Dunia E, Wang Quan, Gibson Tyler M, Edge Albert S B
Department of Otolaryngology, Harvard Medical School, Boston, MA, United States.
Eaton-Peabody Laboratories, Massachusetts Eye and Ear, Boston, MA, United States.
Front Cell Dev Biol. 2019 Feb 19;7:14. doi: 10.3389/fcell.2019.00014. eCollection 2019.
The mouse cochlea contains approximately 15,000 hair cells. Its dimensions and location, and the small number of hair cells, make mechanistic, developmental and cellular replacement studies difficult. We recently published a protocol to expand and differentiate murine neonatal cochlear progenitor cells into 3D organoids that recapitulate developmental pathways and can generate large numbers of hair cells with intact stereociliary bundles, molecular markers of the native cells and mechanotransduction channel activity, as indicated by FM1-43 uptake. Here, we elaborate on the method and application of these Lgr5-positive cochlear progenitors, termed LCPs, to the study of inner ear development and differentiation. We demonstrate the use of these cells for testing several drug candidates, gene silencing and overexpression, as well as genomic modification using CRISPR/Cas9. We thus establish LCPs as a valuable tool for the analysis of progenitor cell manipulation and hair cell differentiation.
小鼠耳蜗大约包含15,000个毛细胞。其尺寸、位置以及毛细胞数量较少，使得进行机制、发育和细胞替代研究变得困难。我们最近发表了一种方案，可将小鼠新生耳蜗祖细胞扩增并分化为三维类器官，这些类器官重现了发育途径，并且能够产生大量具有完整静纤毛束、天然细胞分子标记以及机械转导通道活性（通过FM1-43摄取来表明）的毛细胞。在此，我们详细阐述这些被称为LCPs的Lgr5阳性耳蜗祖细胞在研究内耳发育和分化中的方法及应用。我们展示了利用这些细胞来测试几种候选药物、进行基因沉默和过表达，以及使用CRISPR/Cas9进行基因组修饰。因此，我们将LCPs确立为分析祖细胞操作和毛细胞分化的宝贵工具。
