Engineering multi-lineage human inner ear organoids

摘要 内耳发育需要来自多个胚胎谱系的不同细胞的组装。上皮细胞， 内耳的神经元和神经胶质成分来自外胚层，而间充质成分来自外胚层 主要来源于中胚层。一个主要的工程挑战是建立多血统的内耳 研究人员可以用来研究人类听力和平衡相关疾病的体外组织 发展生物学问题，并评估有前途的治疗方法。患者派生的常规使用 内耳外植体的研究是不可行的，因为人类内耳很难活检。因此，我们的 长期目标是定义概括功能形成所需的化学和物理信号 人内耳组织体外来源于人多能干细胞(HPSCs)。该项目建立在最近的一项 本实验室报告的技术创新：一种多阶段3D内耳培养系统 含有感觉毛细胞和神经元的有机体。尽管取得了重大进展，但仍有一些 关于内耳有机化合物如何忠实地模仿正常胚胎发育的问题。此外，还有 可能限制内耳有机化合物集成到组织芯片药物发现平台的技术障碍。 具体地说，有机类物质的生产效率是可变的，有机类物质中的所有细胞类型都不清楚。 此外，我们实时跟踪内耳感觉细胞的发育或生理的能力也是有限的。 我们的研究计划将定义下一代内耳器官系统。对于目标1，我们将使用高- 通过单细胞分析生成发育中的内耳器官的细胞命运图。在目标2中，我们将 生成用于实时监测内耳器官感觉神经网络的双报告hPSC线。在……里面 目标3，我们将设计化学定义的内耳有机化合物，提高对哺乳动物的保真度 发展。最后，我们将验证一组四种人类诱导多能性的内耳器官的产生。 以确保我们结果的重复性。总之，这个项目的完成将加深我们的 表征人类内耳器官模型并促进将技术转让给其他人 研究实验室。未来的研究可能会继续探索未知的细胞信号机制，建立遗传模型 疾病，或将有机化合物集成到组织芯片系统中。我们预计我们的研究将提供广泛的 适用的见解，应有助于生产其他感官系统的有机化合物，并应提供 耳鼻喉科研究的有力工具。

ABSTRACT Inner ear development requires the assembly of diverse cells from multiple embryonic lineages. The epithelial, neuronal, and glial components of the inner ear are ectoderm-derived, whereas the mesenchymal components are predominantly mesoderm-derived. A major engineering challenge is to establish multi-lineage inner ear tissues in vitro, which researchers could use to study human hearing and balance-related diseases, investigate developmental biology questions, and evaluate promising therapeutics. The routine use of patient-derived inner ear explants for research is not feasible because the human inner ear is difficult to biopsy. Therefore, our long-term goal is to define the chemical and physical signals required to recapitulate formation of functional human inner ear tissue in vitro from human pluripotent stem cells (hPSCs). This project builds upon a recent technological innovation reported by our laboratory: a multi-stage 3D culture system for generating inner ear organoids that contain sensory hair cells and neurons. Despite significant progress, there are remaining questions about how faithfully inner ear organoids mimic normal embryonic development. Moreover, there are technical hurdles that may limit integration of inner ear organoids into tissue-chip drug discovery platforms. Specifically, organoid production efficiency is variable and the full range of cell types in organoids is unclear. Moreover, our ability to track the development or physiology of inner ear sensory cells in real-time is limited. Our research plan will define a next-generation inner ear organoid system. For Aim 1, we will use high- throughput single-cell analysis to generate a cell fate map of developing inner ear organoids. In Aim 2, we will generate dual-reporter hPSC lines for real-time monitoring of inner ear organoid sensorineural networks. In Aim 3, we will engineer chemically-defined inner ear organoids with improved fidelity to mammalian development. Finally, we will verify inner ear organoid production from a set of four human induced pluripotent stem cell lines to ensure the reproducibility of our results. Together, completion of this project will deepen our characterization of the human inner ear organoid model and facilitate transfer of the technology to other research laboratories. Future investigations could pursue unexplored cell signaling mechanisms, model genetic diseases, or integrate organoids into tissue-chip systems. We anticipate that our study will provide broadly applicable insights that should aid the production of organoids of other sensory systems and should provide a powerful tool for otolaryngology research.