Generation of human inner ear organoids via genetic programming

项目摘要 听力损失和平衡障碍是最常见的两种残疾。全球超过6%的人遭受 有超过6%的人患有平衡障碍。负责声音和声音的细胞 运动检测是驻留在内耳的机械感觉毛细胞。因为头发的退化 细胞在哺乳动物中是不可逆转的，目前还没有被批准的用于感觉恢复的药物。在最近 多年来，已开发出从非耳源性细胞通过体外培养产生内耳细胞的衍生方法 阶梯式形态治疗或强制激活毛细胞转录因子(TF)。尽管提供了 前所未有的研究机会，目前的这些体外衍生方法都不适合 高通量治疗发现，由于难以扩大和相对较大的限制 低效，或缺乏有空间组织的感官上皮结构。为了克服这些限制，这 本研究旨在通过遗传转化聚合体人类来构建一种新的人类内耳器官模型 多能干细胞(PSC)通过基于CRISPR的耳祖细胞的激活而转化为耳祖细胞， 随后在3D培养中自组织细胞成熟。将进行CRISPR筛查以确定 可以提高血统转换效率的额外的TF。因为只需要一个处理步骤 并且派生协议是以种族血统为重点的，这种新的器类模型预计将具有更强的可扩展性 并且比目前的逐步形成剂治疗有机物模型更有效。此外，作为奥秘的祖先 已经被证明能够自主地产生适当组织的感觉上皮细胞类型 在体外，这种新的有机体模型有望在空间上组织起来容纳毛细胞和支持细胞 因此，与现有的直接毛细胞相比，更好地概括了天然感觉上皮结构 转换模型。由于这些优点，这种新的有机模型可能会成为一种 用于筛选化合物和测试听力损失的基因治疗治疗的治疗发现平台 和平衡功能障碍。除了建立新的模型，这项研究还将调查潜在的 通过鉴定耳科植物亚集的直接和间接下游基因来实现耳科分化机制 先祖TFS。调控细胞特性转换的转录网络的鉴定 成熟的感觉细胞类型的先祖细胞将极大地促进我们对人类的理解 内耳发育。总的来说，拟议的研究将在基础和 翻译内耳研究。

PROJECT ABSTRACT Hearing loss and balance disorders are the two most prevalent disabilities. Over 6% of people worldwide suffer from disabling hearing loss, and over 6% suffer from balance disorders. The cells responsible for sound and motion detections are the mechanosensory hair cells residing in the inner ear. As the degeneration of the hair cells is irreversible in mammals, there is currently no approved medications for sensory recovery. In recent years, derivation methods have been developed to generate inner ear cells from non-otic cells in vitro via stepwise morphogen treatment or forced activation of hair cell transcription factors (TFs). Despite providing unprecedented research opportunities, none of these current in vitro derivation approaches are suitable for high-throughput therapeutic discoveries due to limitations such as being difficult to scale up and relatively inefficient, or the lack of a spatially organized sensory epithelial structure. To overcome these limitations, this study aims to build a novel human inner ear organoid model by genetically converting aggregated human pluripotent stem cells (PSCs) into otic progenitor cells through CRISPR-based activation of otic progenitor TFs, followed by self-organized cellular maturation in 3D culture. A CRISPR screen will be performed to identify additional TFs that can enhance the lineage conversion efficiency. As only a single treatment step is required and the derivation protocol is otic lineage-focused, this new organoid model is expected to be more scalable and efficient than current stepwise morphogen treatment organoid models. Furthermore, as otic progenitors have been shown to be capable of autonomously generating properly organized sensory epithelium cell types in vitro, this novel organoid model is expected to harbor hair cells and supporting cells in a spatially organized manner, therefore better recapitulating the native sensory epithelium structures than the existing direct hair cell conversion models. Due to these advantages, this novel organoid model could potentially serve as a therapeutic discovery platform for screening compounds and testing gene therapy treatments for hearing loss and balance dysfunctions. In addition to establishing the new model, this study will investigate the underlying mechanism of otic differentiation by identifying the direct and indirect downstream genes of a subset of otic progenitor TFs. The identification of the transcription network that regulates the cellular identity transition from the otic progenitors towards mature sensory cell types will significantly advance our understanding of human inner ear development. Collectively, the proposed research will provide novel tools and insights in basic and translational inner ear research.