Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution. Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human ES cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analysis with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that hair cells arise primarily from supporting cells, rather than bi-fated transitional cells in organoids. Moreover, ion channel- and ion transporter-related gene sets were enriched in supporting cells vs. prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells vs. supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss and balance disorders. Overall design: Yoshitomo, Ueda

内耳感觉上皮包含机械敏感性毛细胞（mechanosensitive hair cells）与支持细胞（supporting cells）。两类细胞均起源于表达SOX2的前感觉细胞（prosensory cells），但目前该类细胞谱系分化的潜在机制仍未明确。为阐明前感觉细胞的转录轨迹，我们借助CRISPR/Cas9技术构建了SOX2-2A-ntdTomato人类胚胎干细胞（human ES）系，并对分化第20天至第60天不同时间点从内耳类器官中分离得到的SOX2阳性细胞开展单细胞RNA测序分析。我们的拟时间分析结果显示，类器官中的毛细胞主要由支持细胞分化而来，而非双命运过渡细胞。此外，与离子通道、离子转运蛋白相关的基因集在支持细胞中较前感觉细胞显著富集，而与Wnt信号通路（Wnt signaling）相关的基因集则在毛细胞中较支持细胞富集。本研究结果为阐明人类内耳发育过程中前感觉细胞如何分化为毛细胞与支持细胞提供了宝贵见解，或可为促进听力损失及平衡障碍患者体内常驻支持细胞向毛细胞再生提供新思路。整体实验设计：Yoshitomo、Ueda