Cell fate determination in the early development of the otic lineage in human inner ear organoids

项目摘要/摘要 大多数先天性听力损失是感觉神经性耳聋(SNHL)，至少有一半与 基因缺陷。不幸的是，再生医学和内耳疾病的基因治疗普遍 没有在人类患者身上实现，因为无法在大量人类身上进行伦理实验 以科学严谨的方式进行胚胎移植。这也意味着在总体上存在关键的知识差距 对人类感觉器官早期发育的理解。就内耳而言，众所周知， 适当的细胞命运对耳道胎盘的承诺对内耳的形成至关重要。既有感觉上皮，又有 毛细胞位于耳蜗处，前庭耳蜗神经来自内陷的耳囊。 来自耳道胎盘；毛细胞和/或前庭耳蜗神经的缺陷导致SNHL。它很难研究。 早期耳道发育，特别是在胎盘前外胚层(PPE)的诱导和形成期间， 耳聋的安慰剂来自哺乳动物的胚胎(更不用说人类胚胎了)。本研究旨在 1)研究WNT信号通路在耳聋早期发育中的作用，重点是PPE；2) 确定PPE中的细胞命运决定如何最终导致人类内耳耳聋谱系的起源 有机化合物。在目标1中，我们将研究WNT信号对耳廓发育的影响，目标是 确定WNT的最佳水平，以最大限度地诱导内耳有机物质。单细胞RNA测序 (scRNA-seq)分析人类内耳器官系统将揭示早期耳廓谱系进展和 WNT介导的过程的潜在机制。在目标2中，有机物的空间基因表达谱 以最佳的WNT调制水平处理将被创建。在将这些空间数据与 来自Aim 1的临时scRNA-seq数据将是人类内耳器官中早期耳聋谱系的细胞图谱 已经成立了。这些时空转录数据随后将被用来创建早期的基因蓝图 人类听觉发育，重点是描述对细胞命运至关重要的基因调控网络 承诺是大脑感觉器官正常发育的基础。这样的数据将使人类 发育生物学，2)生成一个分子数据库，用于了解与之相关的先天性疾病 外胚层衍生物(包括内耳)，以及3)再生医学和基因疗法的进展 治疗内耳疾病。

PROJECT SUMMARY/ABSTRACT The majority of congenital hearing loss cases are sensorineural (SNHL), and at least half are associated with genetic defects. Unfortunately, regenerative medicine and gene therapy for inner ear diseases have generally not been realized in human patients due to the inability to ethically experiment on large numbers of human embryos in a scientifically rigorous manner. This also means that key knowledge gaps exist in the overall understanding of the early development of human sensory organs. In the case of the inner ear, it is known that proper cell fate commitment to the otic placode is essential for inner ear formation. Both sensory epithelia, where hair cells are located, and the vestibulocochlear nerve are derived from the otic vesicle, which is invaginated from the otic placode; defects in hair cells and/or the vestibulocochlear nerve result in SNHL. It is difficult to study early otic development, specifically during the induction and patterning of the pre-placodal ectodermal (PPE), where otic placodes are derived from, in mammalian embryos (not to mention human embryos). This study aims to 1) examine the role of WNT signaling pathways in early otic development, with a focus on the PPE, and 2) determine how cell fate decisions in the PPE culminate in the origin of the otic lineage in human inner ear organoids. In Aim 1, the effects of WNT signaling on otic development will be examined, with the goal of identifying the optimal level of WNT that maximizes inner ear organoid induction. Single-cell RNA sequencing (scRNA-seq) analyses in the human inner ear organoid system will reveal the early otic lineage progression and mechanisms underlying the processes mediated by WNT. In Aim 2, spatial gene expression profiles of organoids treated with the optimal level of WNT modulation will be created. Upon integrating these spatial data with the temporal scRNA-seq data from Aim 1, a cell atlas of the early otic lineage in human inner ear organoids will be established. These spatio-temporal transcriptomic data will then be used to create a genetic blueprint for early human otic development, with a focus on characterizing the gene regulatory networks that are critical to cell fate commitments underlying proper cranial sensory organ development. Such data will 1) advance human developmental biology, 2) generate a molecular database for understanding congenital disorders associated with ectodermal derivatives (including the inner ear), and 3) progress regenerative medicine and gene therapy for inner ear diseases.