Cellular therapy for the inner ear

描述（由申请人提供）：耳蜗神经元和毛细胞的丧失是感音神经性听力损失的主要原因。一种潜在的治疗方法是外源性祖细胞的移植。我们开发内耳疾病细胞疗法的策略是研究Corti去传入器官的神经再生。我们采用这种方法是因为1)只有一种细胞类型，即螺旋神经节细胞，必须被替换才能看到功能恢复；2)在其他神经退行性疾病（如帕金森氏症）的动物模型中，通过细胞替代实现受损组织的部分再生已经成功，这表明，如果引入适当的祖细胞，成人神经系统中的神经回路可以重建。3)了解成人耳中毛细胞神经连接重建的机制对任何治疗感音神经性听力损失的方法都很重要。具体目标包括四个相互关联的实验，以探索可能影响移植神经祖细胞成功移植毛细胞神经的关键变量。在第一期研究中，我们将采用免疫组织化学的方法研究螺旋神经节神经元中几个神经引导分子家族的发育表达。在Aim 2中，我们使用去传入耳蜗外植体来研究移植物祖细胞对毛细胞的再神经支配，在体外，通过改变供体细胞的特征（例如沉默选定的引导分子的表达或控制分化程度）或宿主环境（例如外植体的发育年龄或初始去传入的病因）。在目标3中，我们以体外实验结果为基础，选择供体细胞进行体内移植到化学去传入沙鼠耳中，其中感觉细胞完好无损，但螺旋神经节细胞被破坏。在这个模型系统中，可以从功能和组织学上分析神经再生的成功。在Aim 4中，我们使用了一种报告细胞实验，通过在一定的时间序列中添加特定的生长因子，以及在通往感觉神经元的通路中过度表达转录因子，来确定将干细胞偏向分化为与感觉神经元相对应的谱系所需的条件。在该目的下分离的克隆细胞系将作为在Aims 3和4中研究的额外供体细胞类型。

DESCRIPTION (provided by applicant): Loss of cochlear neurons and hair cells is a major cause of sensorineural hearing loss. One approach to potential treatments is the transplantation of exogenous progenitor cells. Our strategy for developing cell based therapies for inner ear disorders is to study the reinnervation of a de-afferented organ of Corti. We take this approach because 1) only a single cell type, the spiral ganglion cell, must be replaced to see functional recovery; 2) partial regeneration of damaged tissue by cell replacement has been successful in animal models of other neurodegenerative diseases, such as Parkinson's, suggesting that neuronal circuits in the adult nervous system can be reconstituted if appropriate progenitor cells are introduced and 3) understanding the mechanisms underlying re-formation of neural connections to hair cells in the adult ear is important to any therapeutic approach to sensorineural hearing loss. The Specific Aims comprise four inter-related experiments to probe key variables likely to influence the success of hair cell reinnervation by transplanted neural progenitor cells. In Aim 1 the developmental expression by spiral ganglion neurons of several families of neuronal guidance molecules will be studied by immunohistochemistry. In Aim 2 we use de-afferented cochlear explants to study the reinnervation of hair cells by grafted progenitors, in vitro, by modifying characteristics of the donor cells (e.g. silencing expression of selected guidance molecules or controlling degree of differentiation) or the host environment (e.g. developmental age of the explant or etiology of the initial de-afferentation). In Aim 3 we build on the in vitro results to choose donor cells for in vivo transplantation into a chemically de-afferented gerbil ear, in which the sensory cells are intact but spiral ganglion cells are destroyed. In this model system, success of reinnervation can be assayed both functionally and histologically. In Aim 4 we use a reporter assay to determine conditions needed to bias differentiation of stem cells to a lineage corresponding to sensory neurons by adding specific growth factors in a timed sequence, and by over expressing transcription factors in the pathway to sensory neurons. Clonal cell lines isolated under this Aim will serve as additional donor cell types to be studied in Aims 3 and 4.