Identification of stria vascularis cell type profile by single-cell RNA-sequencing in the adult mouse model of Pendred syndrome

The primary pathological alternations of SLC26A4-related hearing loss are endolymphatic pH acidification and luminal enlargement of the inner ear. However, the molecular cell type-specific influences remain poorly characterized. In this study, we confirmed a method of isolating single cells in the cochlea's stria vascularis (SV) from wild-type (WT) animals. We then applied it to characterize the distinct transcriptomes from mutant (Slc26a4+/- and Slc26a4-/-) samples. Single-cell RNA-sequencing (scRNA-seq) identified transcriptional profiles in Slc26a4-expressing (spindle) cells and Kcnj10-expressing (intermediate) cells of the SV. By Gene Set Enrichment Analysis (GSEA), we found that spindle cells contain extrinsic cellular components, a factor that enables cell-to-cell communication. In addition, the gene expression profile informed signaling pathways associated with pH regulation in spindle cells. Compared to WT control, the transcriptional profiles in the SV cells of Slc26a4-/- mice showed downregulation of extracellular exosome-related genes in spindle cells and GPI-anchor-related genes in the intermediate cells. Immunofluorescence studies in spindle cells of Slc26a4-/- mice validated the increased expression of the clathrin-mediated endocytosis-related protein. Overall, cell isolation of SV from WT and mutant (Slc26a4+/- and Slc26a4-/-) samples combined with cell type-specific transcriptomic analyses revealed pH-dependent alternations in spindle cells and intermediate cells, inspiring further studies into the dysfunctional role of SV cells in SLC26A4-related hearing loss. Stria vascularis cells of the the129S6 WT mice and mutant (Slc26a4+/- and Slc26a4-/-) mice were isolated and analyzed using scRNAseq.SLC26A4相关听力损失（SLC26A4-related hearing loss）的主要病理改变为内耳内淋巴液pH酸化及管腔扩张。然而，其细胞类型特异性的分子调控机制仍未得到充分阐明。本研究首先确立了从野生型（wild-type，WT）动物的耳蜗血管纹（stria vascularis，SV）中分离单细胞的方法，随后将该方法应用于突变型（Slc26a4+/-及Slc26a4-/-）样本的特异性转录组特征分析。单细胞RNA测序（Single-cell RNA-sequencing，scRNA-seq）分析了血管纹中表达Slc26a4的梭形细胞及表达Kcnj10的中间型细胞的转录谱。通过基因集富集分析（Gene Set Enrichment Analysis，GSEA），我们发现梭形细胞含有可介导细胞间通讯的外源性细胞组分；此外，基因表达谱分析揭示了梭形细胞中与pH调控相关的信号通路。与野生型对照组相比，Slc26a4-/-小鼠血管纹细胞的转录谱显示，梭形细胞中细胞外外泌体相关基因及中间型细胞中糖基磷脂酰肌醇锚定（GPI-anchor）相关基因的表达均出现下调。对Slc26a4-/-小鼠梭形细胞开展的免疫荧光实验证实，网格蛋白介导的内吞（clathrin-mediated endocytosis）相关蛋白的表达水平升高。综上，通过从野生型及突变型（Slc26a4+/-及Slc26a4-/-）样本中分离血管纹细胞，并结合细胞类型特异性转录组分析，本研究揭示了梭形细胞与中间型细胞中pH依赖性的改变，为进一步探究血管纹细胞在SLC26A4相关听力损失中的功能失调机制提供了研究方向。本研究对129S6品系野生型小鼠及突变型（Slc26a4+/-及Slc26a4-/-）小鼠的血管纹细胞进行了分离，并通过单细胞RNA测序（scRNA-seq）开展了相关分析。