Table_3_Characterizing Adult Cochlear Supporting Cell Transcriptional Diversity Using Single-Cell RNA-Seq: Validation in the Adult Mouse and Translational Implications for the Adult Human Cochlea.DOCX

Hearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell (HC) loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate HCs. Such therapy would require a recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature HCs and supporting cells (SCs). Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear SC transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear SCs from the LfngEGFP adult mouse, in which SCs express GFP. We demonstrate that adult cochlear SCs are transcriptionally distinct from their perinatal counterparts. We establish cell-type-specific adult cochlear SC transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and quantitative polymerase chain reaction (qPCR) of adult cochlear SCs. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult SCs and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal SCs.

听力损失是影响大量成年人群体的常见健康问题。因强噪声暴露、化疗及衰老导致的耳蜗毛细胞（Cochlear hair cell，HC）丢失，是听力损失的主要潜在诱因。此类患者中，相当一部分对现有的治疗方案（包括助听器与人工耳蜗植入）并不满意。而恢复听力的另一可行途径，便是实现毛细胞的再生。此类治疗方案需要复刻柯蒂氏器（organ of Corti）的复杂结构，因此需要同时实现成熟毛细胞与支持细胞（supporting cells，SCs）的再生。耳蜗成熟细胞类型的转录组谱，可为最终的再生治疗提供评估标准。为助力该领域研究，本研究首次对成年小鼠耳蜗支持细胞的转录组进行单细胞表征分析。我们以LfngEGFP成年小鼠为研究对象，该模型的支持细胞可表达绿色荧光蛋白（Green Fluorescent Protein，GFP），并通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting，FACS）纯化得到成年小鼠耳蜗支持细胞，随后对其进行单细胞RNA测序。研究证实，成年小鼠耳蜗支持细胞在转录层面与围产期的同类细胞存在显著差异。本研究构建了细胞类型特异性的成年小鼠耳蜗支持细胞转录组图谱，并通过荧光免疫组化、原位杂交共定位实验以及成年耳蜗支持细胞的实时定量聚合酶链反应（quantitative polymerase chain reaction，qPCR）联合验证了该表达图谱的准确性。此外，我们通过人类颞骨组织病理学的免疫荧光实验，证实了该转录组图谱与成年人类耳蜗的相关性。最后，我们检测了成年支持细胞中细胞周期调控因子的表达情况，并通过通路分析筛选出可促进成年哺乳动物耳蜗有丝分裂再生（包括细胞增殖、分化及最终的组织再生）的潜在作用机制。本研究结果凸显了对成熟而非围产期支持细胞进行表征分析的重要性。