Development of Genetic and Stem Cell Approaches in the Cochlear Lateral Wall

描述(由申请人提供)：耳蜗侧壁维持正常耳蜗生理学所必需的离子代谢，耳蜗壁侧壁功能的破坏--由遗传或环境侮辱--导致听力损失。尽管这种结构很重要，但人们对其正常生长发育知之甚少，一旦受损，目前还没有办法修复这种结构。在这里，我们建议开发必要的遗传学和基于干细胞的方法，以加快对侧壁的一个组成部分--螺旋韧带的研究，螺旋韧带的完整性对侧壁功能至关重要。这项提议的最终目标是开发基于干细胞的治疗方法，以纠正螺旋韧带功能障碍的动物模型Brn4/Pou3f3突变的听力损失。为了有效地实施和优化干细胞的使用，必须对螺旋韧带的生长和分化的基本生物学有一个基本的了解。特别是，对支持螺旋韧带纤维细胞生长和维持的细胞信号通路的了解将是优化干细胞方法的关键。因此，在资助期的早期，我们建议对正常调节螺旋韧带纤维细胞的生长因子进行表征。随后，一旦我们在小鼠身上建立了干细胞治疗的工作模型，我们就可以利用我们对纤维细胞生长因子调节的知识来设计合理的方法来优化螺旋韧带中干细胞的靶向和生长。此外，我们建议开发一种强大的、通用的遗传工具来在螺旋韧带中表达基因，包括相关的生长因子。使用先前鉴定的Brn4基因的听觉增强子，我们将在耳蜗纤维细胞中表达四环素诱导基因表达系统的Tet-on版本。这些工具/方法中的每一种都有很大的潜力来加强我们对螺旋韧带的发育、生理和功能的了解。然而，这些工具/方法将协同作用，加快对耳蜗壁的实验，并优化我们使用干细胞疗法补充听力损失动物模型中螺旋纤维细胞的能力。 与公共卫生相关：干细胞疗法在重建或替换因遗传或环境侮辱而受损的组织方面具有巨大的前景。在这里，我们建议开发干细胞方法来逆转螺旋韧带的损伤，螺旋韧带是维持内耳适当的离子环境所必需的结构。通过开发强大的实验工具，我们将加快有助于治愈或改善听力损失的研究。

DESCRIPTION (provided by applicant): The lateral cochlear wall maintains the ionic metabolism necessary for normal cochlear physiology, and disruption of lateral cochlear wall function - by genetic or environmental insults - leads to hearing loss. Despite the importance of this structure, little is known about its normal growth and development, and there are currently no means to repair this structure once it is damaged. Here, we propose to develop genetic and stem cell-based approaches necessary to accelerate research in a component of the lateral wall, the spiral ligament, whose integrity is vital for lateral wall function. The ultimate goal of this proposal is to develop stem cell-based therapies to correct hearing loss in an animal model of spiral ligament dysfunction, the Brn4/Pou3f3 mutant. To effectively implement and optimize the use of stem cells, a fundamental understanding of the basic biology of growth and differentiation of the spiral ligaments is necessary. In particular, knowledge of the cell signaling pathways that support the growth and maintenance of spiral ligament fibrocytes will be key to optimizing stem cell approaches. Therefore, early in the grant-funding period, we propose to characterize the growth factors that normally regulate spiral ligament fibrocytes. Subsequently, once we have developed a working model of stem cell therapy in mice, we can use our knowledge of fibrocyte growth factor regulation to design rational approaches to optimize the targeting and growth of stem cells in the spiral ligament. Additionally, we propose to develop a powerful, versatile genetic tool to express genes, including the relevant growth factors, in the spiral ligament. Using a previously characterized otic enhancer from the Brn4 gene, we will express the Tet- On version of the tetracycline-inducible gene expression system in cochlear fibrocytes. Each of these tools/approaches alone has great potential to enhance our understanding of the development, physiology and function of the spiral ligament. However, in conjunction, these tools/approaches will synergize to accelerate experimentation on the lateral cochlear wall, and optimize our ability to replenish spiral fibrocytes in animal models of hearing loss using stem cell therapy. PUBLIC HEALTH RELEVANCE: Stem cell therapies hold tremendous promise for reconstructing or replacing tissue damaged by genetic or environmental insults. Here, we propose to develop stem cell approaches to reverse damage of the spiral ligament, a structure that is necessary to maintain the proper ionic environment in the inner ear. By developing powerful experimental tools, we will accelerate research that can help to cure or ameliorate hearing loss.