Modeling Inner Ear Differentiation with Pluripotent Stem cells

先天性和后天性耳聋是一个主要的公共卫生问题，影响超过3600万美国人。干细胞生物学的最新突破表明，具有所有神经元亚型的复杂感觉器官可以在3D培养中由多能干细胞聚集体形成，这在不久前似乎是遥远和未来的。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，使用小分子抑制剂和重组蛋白的组合忠实地再现内耳诱导。我们已经证明，通过对BMP、TGF β和FGF信号的精确时间控制，干细胞聚集体依次转化为非神经、前基板和耳基板样上皮。值得注意的是，在一个自我指导的过程中，泡含有prosensory细胞出现从假定的耳基板，并产生毛细胞轴承静纤毛和动纤毛。这些干细胞衍生的毛细胞在结构和生物化学上与前庭上皮中的毛细胞相当。在这项研究中，我们将首先优化我们的体外系统，以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信号通路的操作是否分别改变来自多能干细胞的耳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ChIP的生化分析，我们将测试是否prosensory基因的表达是遗传和表观遗传调节Pax2和核心组蛋白的组成性甲基化是否增加干细胞衍生的耳祖细胞的数量，从而产生prosensory细胞，并因此毛细胞。此外，我们将验证这些干细胞衍生的毛细胞的功能特性，并定义毛细胞表型的身份（目的3）。使用单细胞电生理学，光遗传学和高分辨率成像技术的组合，我们将测试干细胞衍生的毛细胞是否表现出内耳中天然感觉毛细胞的结构和功能特性，并与感觉神经元建立突触连接。通过实现这些目标，我们不仅将推进我们对毛细胞发育生物学的理解，而且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.