Modeling Otic Neurogenesis in Human Stem Cell-Derived Organoids

项目摘要 感音神经性听力损失是最常见的听力损失形式，由变性、功能障碍或 内耳感觉毛细胞或螺旋神经节神经元畸形。而螺旋线的规格 神经节神经元在发育过程中对将听觉信息传递到大脑至关重要，我们缺乏 完全了解它们被诱导的机制并建立突触连接。 多能干细胞(PSCs)是一种很有前途的内耳发育模型工具。 用于细胞治疗的替代感觉细胞。然而，人类内耳细胞的受控来源 PSCS仍然是一项重大挑战。我们最近描述了一种从内耳中提取有机化合物的方法 小鼠PSCs的三维培养体系。模仿正常发育，有机化合物一开始是 耳泡和逐渐形成的神经元和含有毛细胞的感觉上皮细胞。在这项研究中，我们将 在一种新的人体器官培养系统的初步研究基础上，研究神经机制 来自异种祖细胞的诱导。在这里，我们假设动态的Wnt、Notch和成纤维细胞生长因子 (成纤维细胞生长因子)信号活性对耳神经前体细胞的特性至关重要。在目标1中，我们将定义原点和 人类耳祖细胞产生的内耳神经元类型，响应Wnt的激活，通过跟踪 发育阶段。在目标2中，我们将可视化和量化干细胞来源的神经祖细胞。 利用CRISPR/Cas9基因编辑技术构建新型报告细胞系。使用这种报告细胞系， 我们将系统地研究Notch和Fgf信号通路在神经前体细胞中的作用 规格。总之，我们期望这些目标的完成将建立一种新的体外模型 人类内耳的神经发生。此外，我们的长期目标是应用优化的技术方法 在这项研究中建立了未来关于询问机制和转录调控的研究 控制听觉回路的形成。

PROJECT ABSTRACT Sensorineural hearing loss, the most common form of hearing loss, is caused by degeneration, dysfunction, or malformation of sensory hair cells or spiral ganglion neurons in the inner ear. While specification of spiral ganglion neurons during development is critical for transmitting auditory information to the brain, we lack a complete understanding of the mechanisms by which they are induced and establish synaptic connections. Pluripotent stem cells (PSCs) serve as a promising tool to model inner ear development and produce replacement sensory cells for cellular therapy. However, the controlled derivation of inner ear cells from human PSCs remains a significant challenge. We recently described a method for deriving inner ear organoids from mouse PSCs in a three-dimensional culture system. Mimicking normal development, the organoids started as otic vesicles and progressively formed neurons and sensory epithelia containing hair cells. In this study, we will build on preliminary studies of a novel human organoid culture system to investigate the mechanisms of neural induction from otic progenitors. Here, we hypothesize that dynamic Wnt, Notch, and fibroblast growth factor (FGF) signaling activity is critical for otic neural progenitor specification. In Aim 1, we will define the origin and type of inner ear neurons produced by human otic progenitors, in response to Wnt activation, by tracking the developmental stages. In Aim 2, we will visualize and quantify neural progenitors in stem cell-derived otic epithelia by using CRISPR/Cas9 gene editing to engineer a novel reporter cell line. Using this reporter cell line, we will systematically examine the roles of Notch and FGF signaling pathways in neural progenitor specification. Together, we anticipate that the completion of these aims will establish a novel in vitro model of human inner ear neurogenesis. Moreover, our long-term goal is to apply the optimized technical approach established in this study to future studies on interrogating the mechanisms and transcriptional regulators governing auditory circuit formation.