Modeling Inner Ear Differentiation with Pluripotent Stem cells

用多能干细胞模拟内耳分化

• 批准号: 8696409
• 负责人: Eri Hashino
• 金额: $ 45.87万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696409
• 关键词: Acquired Deafness; Address; Adult; Affect; Afferent Neurons; American; Americas; Auditory system; Binding; Biochemical; Biological Assay; Biological Models; Biology; Cell Differentiation process; Cells; Child; Complex; Data; Development; Disease Progression; Ear; Ectoderm; Electrophysiology (science); Embryonic Development; Epithelium; Equilibrium; Event; Exhibits; Fibroblast Growth Factor; Fibroblasts; Generations; Genes; Glutamates; Hair Cells; Health; Hearing problem; Histones; Human; Imaging Techniques; In Vitro; Inherited; Investigation; Kinocilium; Labyrinth; Lead; Mechanoreceptor Cell; Mediating; Membrane; Membrane Proteins; Methylation; Modeling; Molecular; Myosin ATPase; Neuronal Differentiation; Neurons; Nucleic Acid Regulatory Sequences; Organ; Otic Placodes; Outcome; Pathogenesis; Patients; Phenotype; Pluripotent Stem Cells; Process; Property; Proteins; Protocols documentation; Public Health; Recombinant Proteins; Relative (related person); Resolution; Seminal; Sensory; Sensory Hair; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Skin; Stem cells; Stereocilium; Surface; Synapses; System; Testing; Time; Transcriptional Activation; Vesicle; Work; base; cell injury; cell type; chromatin immunoprecipitation; deafness; embryonic stem cell; equilibration disorder; hearing impairment; high throughput screening; histone methyltransferase; induced pluripotent stem cell; inhibitor/antagonist; inner ear diseases; neurotransmitter release; novel; novel strategies; optogenetics; progenitor; promoter; public health relevance; regenerative; scale up; small molecule; stem cell biology

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.

描述（由申请人提供）：先天性和后天性耳聋是影响超过 3600 万美国人的主要公共卫生问题。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由多能干细胞的聚集体形成。在这些开创性研究的推动下，我们建立了一种新颖的 3D 培养系统，可以使用 3D 培养忠实地再现内耳感应。我们已经证明，通过精确控制 BMP、TGF¿值得注意的是，在自我引导的过程中，含有前感觉细胞的囊泡从假定的耳基板中出现，并产生带有毛细胞的细胞。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细胞相似。首先优化我们的体外系统，以便正确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会改变耳祖细胞的相对数量。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）。基于 ChIP 的生化检测，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节，以及核心组蛋白的组成型甲基化是否会增加干细胞来源的耳祖细胞的数量，从而产生前感觉细胞，从而产生毛细胞此外，我们将结合单细胞电生理学来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（目标 3）。利用光遗传学和高分辨率成像技术，我们将测试干细胞衍生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，并与感觉神经元建立突触连接。通过实现这些目标，我们不仅会取得进展。我们对毛细胞发育生物学的理解，同时还建立了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。