Engineering multi-lineage human inner ear organoids

摘要 内耳的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是外胚层来源的，而间充质成分是外胚层来源的。 主要来源于中胚层。一个主要的工程挑战是建立多谱系内耳 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， 发育生物学问题，并评估有前途的治疗方法。常规使用患者源性 用于研究的内耳外植体是不可行的，因为人的内耳很难进行活组织检查。所以我们的 长期目标是确定所需的化学和物理信号，以概括功能的形成， 从人多能干细胞（hPSC）体外获得人内耳组织。该项目建立在最近的 我们实验室报告的技术创新：用于生成内耳的多阶段3D培养系统 含有感觉毛细胞和神经元的类器官。尽管取得了重大进展， 内耳类器官如何忠实地模仿正常的胚胎发育。而且还有 技术障碍可能会限制内耳类器官整合到组织芯片药物发现平台中。 具体而言，类器官的生产效率是可变的，类器官中的全部细胞类型尚不清楚。 此外，我们实时跟踪内耳感觉细胞的发育或生理的能力是有限的。 我们的研究计划将定义下一代内耳类器官系统。对于目标1，我们将使用高- 通过单细胞分析来产生发育中的内耳类器官的细胞命运图。在目标2中，我们将 产生用于实时监测内耳类器官感觉神经网络的双报告基因hPSC系。在 目标3，我们将工程化化学定义的内耳类器官，提高对哺乳动物的保真度， 发展最后，我们将验证内耳类器官的生产从一组四个人诱导多能 干细胞系，以确保我们的结果的可重复性。总之，该项目的完成将加深我们的 表征人类内耳类器官模型，并促进该技术向其他人的转移。 研究实验室。未来的研究可以探索未探索的细胞信号传导机制，模型遗传学， 疾病，或将类器官整合到组织芯片系统中。我们预计，我们的研究将提供广泛的 适用的见解，应该有助于其他感觉系统的类器官的生产，并应提供一个 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.