Data_Sheet_1_Frizzled-9+ Supporting Cells Are Progenitors for the Generation of Hair Cells in the Postnatal Mouse Cochlea.docx

Lgr5+ cochlear supporting cells (SCs) have been reported to be hair cell (HC) progenitor cells that have the ability to regenerate HCs in the neonatal mouse cochlea, and these cells are regulated by Wnt signaling. Frizzled-9 (Fzd9), one of the Wnt receptors, has been reported to be used to mark neuronal stem cells in the brain together with other markers and mesenchymal stem cells from human placenta and bone marrow. Here we used Fzd9-CreER mice to lineage label and trace Fzd9+ cells in the postnatal cochlea in order to investigate the progenitor characteristic of Fzd9+ cells. Lineage labeling showed that inner phalangeal cells (IPhCs), inner border cells (IBCs), and third-row Deiters’ cells (DCs) were Fzd9+ cells, but not inner pillar cells (IPCs) or greater epithelial ridge (GER) cells at postnatal day (P)3, which suggests that Fzd9+ cells are a much smaller cell population than Lgr5+ progenitors. The expression of Fzd9 progressively decreased and was too low to allow lineage tracing after P14. Lineage tracing for 6 days in vivo showed that Fzd9+ cells could also generate similar numbers of new HCs compared to Lgr5+ progenitors. A sphere-forming assay showed that Fzd9+ cells could form spheres after sorting by flow cytometry, and when we compared the isolated Fzd9+ cells and Lgr5+ progenitors there were no significant differences in sphere number or sphere diameter. In a differentiation assay, the same number of Fzd9+ cells could produce similar amounts of Myo7a+ cells compared to Lgr5+ progenitors after 10 days of differentiation. All these data suggest that the Fzd9+ cells have a similar capacity for proliferation, differentiation, and HC generation as Lgr5+ progenitors and that Fzd9 can be used as a more restricted marker of HC progenitors.

Lgr5+ 鼠耳蜗支持细胞（SCs）已被报道为毛细胞（HC）祖细胞，具有在新生小鼠耳蜗中再生毛细胞的能力，这些细胞受Wnt信号通路调控。Frizzled-9（Fzd9），作为Wnt受体之一，已被报道与其它标记物一同用于标记大脑中的神经元干细胞，以及来自人胎盘和骨髓的间充质干细胞。在本研究中，我们利用Fzd9-CreER小鼠对出生后耳蜗中的Fzd9+细胞进行谱系标记和追踪，以研究Fzd9+细胞的祖细胞特性。谱系标记显示，内指骨细胞（IPhCs）、内边界细胞（IBCs）和第三排Deiters细胞（DCs）为Fzd9+细胞，但在出生后第3天（P3）时并非内支柱细胞（IPCs）或更大上皮隆起（GER）细胞，这表明Fzd9+细胞群体远小于Lgr5+祖细胞。Fzd9的表达随着时间逐渐减少，在P14后降至无法进行谱系追踪的程度。在体内的6天谱系追踪显示，Fzd9+细胞也能产生与Lgr5+祖细胞相似数量的新毛细胞。球形形成实验表明，经过流式细胞术分离的Fzd9+细胞能够形成球形，且与Lgr5+祖细胞相比，在球形数量或直径上无显著差异。在分化实验中，Fzd9+细胞在分化10天后能产生与Lgr5+祖细胞相似的Myo7a+细胞数量。所有这些数据表明，Fzd9+细胞在增殖、分化和毛细胞生成方面的能力与Lgr5+祖细胞相似，并且Fzd9可作为更严格的毛细胞祖细胞的标记。