Modeling Inner Ear Differentiation with Pluripotent Stem cells

描述(申请人提供)：先天性和获得性耳聋是一个主要的公共卫生问题，影响着超过3600万美国人。干细胞生物学最近的突破揭示，在3D培养中，由多能干细胞聚集而成的具有所有神经元亚型的复杂感觉器官，在不久前似乎还很遥远和未来。在这些开创性研究的推动下，我们建立了一种新的3D培养系统，以忠实地概括使用小分子抑制剂和重组蛋白的组合进行内耳诱导。我们已经证明，通过对骨形态发生蛋白、转化生长因子�和成纤维细胞生长因子信号的精确时间控制，干细胞聚集体可以顺序地转化为非神经性、胎盘前和耳道胎盘样上皮细胞。值得注意的是，在一个自我引导的过程中，含有前感觉细胞的囊泡从假定的安慰剂中出现，并产生带有立体纤毛和动纤毛的毛细胞。这些干细胞来源的毛细胞在结构上和生化上与前庭上皮中的毛细胞相似。在这项研究中，我们将首先优化我们的体外系统，以便适当地模拟整个内耳结构的形成和分化，包括耳蜗细胞类型(目标1)。我们将测试Wnt和Shh信号通路的操纵是否会改变分别来自多能干细胞的耳蜗祖细胞和耳蜗细胞类型的相对数量。此外，通过利用我们的高通量培养系统，我们将开始破译毛细胞分化的分子机制(目标2)。使用基于芯片的生化分析，我们将测试前感觉基因的表达是否受到Pax2的遗传和表观遗传调控，以及核心组蛋白的结构性甲基化是否增加了干细胞衍生的先祖细胞的数量，从而产生前感觉细胞，从而产生毛细胞。此外，我们将验证这些干细胞来源的毛细胞的功能特性，并确定毛细胞表型的身份(目标3)。我们将结合单细胞电生理学、光遗传学和高分辨率成像技术，测试干细胞来源的毛细胞是否表现出内耳天然感觉毛细胞的结构和功能特性，并与感觉神经元进行突触连接。通过这些目标的实现，我们不仅将促进我们对毛细胞发育生物学的理解，还将建立一个有效的模型系统，用于研究各种形式的遗传性耳聋和平衡障碍的发病机制。

DESCRIPTION (provided by applicant): Congenital and acquired deafness is a major public health problem affecting more than 36 million American people. Recent breakthroughs in stem cell biology have revealed that a complex sensory organ with all neuronal subtypes can be formed from aggregates of pluripotent stem cells in 3D culture, which seemed remote and futuristic not long ago. Spurred by these seminal studies, we have established a novel 3D culture system to faithfully recapitulate inner ear induction using a combination of small molecule inhibitors and recombinant proteins. We have demonstrated that, by precise temporal control of BMP, TGF� and FGF signaling, stem cell aggregates transform sequentially into non-neural, pre-placodal and otic placode-like epithelia. Remarkably, in a self-guided process, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia and a kinocilium. These stem cell-derived hair cells are structurall and biochemically comparable to those in the vestibular epithelia. In this study, we will first optimize our in vitro system in order to appropriately model the formation and differentiation of the entire inner ear structures, including cochlear cell types (Aim 1). We will test whether manipulation of Wnt and Shh signaling pathways alter the relative number of otic progenitor cells and cochlear cell types, respectively, derived from pluripotent stem cells. In addition, by taking advantage of our high-throughput culture system, we will begin to decipher the molecular mechanisms underlying hair cell differentiation (Aim 2). Using ChIP-based biochemical assays, we will test whether expression of prosensory genes is genetically and epigenetically regulated by Pax2 and whether constitutive methylation of a core histone protein increases the number of stem cell-derived otic progenitors giving rise to prosensory cells, and consequently hair cells. Furthermore, we will validate functional properties of these stem cell-derived hair cells and define the identity of hair cell phenotypes (Aim 3). Using a combination of single-cell electrophysiology, optogenetics and high-resolution imaging techniques, we will test whether stem cell-derived hair cells exhibit structural and functional properties of native sensory hair cells in the inner ear and make synaptic connections with sensory neurons. By accomplishing these aims, we will not only advance our understanding of the biology of hair cell development, but also establish a potent model system with which to investigate pathogenesis of various forms of hereditary deafness and balance disorders.