Otic progenitors via somatic cell nuclear reprogramming

描述（由申请人提供）：在本探索性研究资助申请中，拟研究体细胞核重编程在生成生殖祖细胞中的适用性。我们计划探索诱导多能干细胞是否可以定向分化为内耳细胞类型（Specific Aim 1），在第二组实验中，我们建议研究体细胞直接转化为能够分化为毛细胞标记表达细胞的祖细胞（Specific Aim 3）。利用基因芯片技术深入表征生成的细胞类型，并将纯化的胚胎和诱导多能干细胞生成的内耳祖细胞的转录谱与具有荧光耳基板细胞的转基因小鼠纯化的耳基板细胞的转录谱进行比较（Specific Aim 2）。通过体细胞重编程和干细胞引导获得的特定基因的潜在作用的知识将直接适用于关注受损哺乳动物器官Corti的毛细胞再生以及发育内耳生物学的研究。此外，拟议的实验与潜在的治疗高度相关，其中自体替代细胞来源非常可取，以避免对受体患者进行免疫抑制。成功证明体细胞核重编程在小鼠内耳细胞再生中的适用性，将为未来将这些实验扩展到患者来源的体细胞（如人成纤维细胞）提供概念基础。为了找到一种治疗听力损失的方法，建议探索从体细胞如成纤维细胞产生的诱导多能干细胞的潜力。具体来说，我们将测试诱导多能干细胞是否可以定向分化为耳蜗毛细胞类型，这可以用于未来的动物模型研究，以替代丢失的耳蜗毛细胞。这是一个高度相关的努力，因为利用自体来源的替代细胞是可取的，因为这些细胞可以在不抑制患者免疫的情况下使用。该研究是探索性的，因为它使用了最近开发的新型体细胞重编程技术，该技术尚未应用于内耳研究。同样，提出了一种将成纤维细胞等体细胞直接转化为内耳祖细胞的新概念。这种直接转化策略并不能将细胞完全转化为多能状态，而是将细胞转化为一种更受限制的状态，这种状态可能已经被赋予了生殖谱系。这种新策略可能比“传统”策略具有明显的优势，“传统”策略首先将细胞转换回胚胎干细胞状态，然后向前引导到特定的器官命运。如果成功，这种方法也可以应用于除内耳以外的其他器官。

DESCRIPTION (provided by applicant): In this exploratory research grant application, it is proposed to investigate the applicability of somatic cell nuclear reprogramming to generate otic progenitor cells. It is planned to explore whether induced pluripotent stem cells can be directed to differentiate into inner ear cell types (Specific Aim 1) and in a second set of experiments, it is proposed to investigate conversion of somatic cells directly into progenitors capable of differentiating into hair cell marker expressing cells (Specific Aim 3). An in-depth characterization of the generated cell types is proposed using gene chip technology and comparison of the transcriptional profile of purified embryonic and induced pluripotent stem cell-generated inner ear progenitors with the transcriptional profile of otic placode cells purified from transgenic mice with fluorescent otic placode cells (Specific Aim 2). Knowledge about the potential roles of specific genes gained with somatic cell reprogramming and stem cell guidance will be directly applicable and highly useful for studies focusing on hair cell regeneration in the damaged mammalian organ of Corti as well as for developmental inner ear biology. Moreover, the proposed experiments are highly relevant for potential treatment, where autologous sources of replacement cells are exceedingly desirable to avoid having to immunosuppress the recipient patient. Successful demonstration of applicability of somatic cell nuclear reprogramming to murine inner ear cell regeneration will provide the conceptual basis for future expansion of these experiments to patient-derived somatic cells such as human fibroblasts. Toward finding a treatment for hearing loss, it is proposed to explore the potential of induced pluripotent stem cells that can be generated from somatic cells such as fibroblasts. Specifically it will be tested whether induced pluripotent stem cells can be directed to differentiate into otic cell types, which could be used in future studies with animal models to replace lost cochlear hair cells. This is a highly relevant endeavor because utilization of autologous sources for replacement cells is desirable because these cells could be used without having to immuno-suppress the patient. The research is exploratory as it uses very recently developed novel somatic cell reprogramming technology, which has not been applied to inner ear research. Likewise, it proposes to explore a novel concept to directly convert somatic cells, such as fibroblasts, into inner ear progenitor cells. This direct conversion strategy does not convert the cells completely back into a pluripotent state, but rather into a more restricted state that is potentially already committed to the otic lineage. This novel strategy might bear distinct advantages over the "conventional" strategy, which first converts cells back into an embryonic stem cell state followed by a forward guidance toward a specific organ fate. If successful, this method might as well be applicable to other organs beside the inner ear.