Direct conversion of fibroblasts into neurons: A novel approach to study neuropsy

描述(申请人提供)：最近发现的转录因子(TF)可以诱导成纤维细胞转化为多能干细胞(IPS)，这使得从成纤维细胞生成患者特有的神经元成为可能。然而，这样产生的神经元很难获得。目前的项目建立在初步结果的基础上，在这些结果中，我们展示了成人成纤维细胞直接转化为神经元，称为“诱导神经元细胞”(在细胞内)，而不需要iPS中间体。由此产生的细胞具有神经元的功能特性，包括电生理学检测的形成功能性突触的能力。因此，细胞内技术为产生和研究人类神经元提供了一种新的、更方便的方法，并为研究人类疾病过程开辟了一条新的途径。然而，目前In cell技术仅适用于小鼠成纤维细胞，有关In细胞转化过程和分子同一性的基本问题尚未确定，人成纤维细胞产生In细胞的可能性尚未建立，最重要的是，In cell技术用于研究影响神经功能的疾病的可行性尚未得到证实。在这个项目中，我们建议以一种跨学科的方法来应对这些重要的挑战，利用维尔尼希和S|dhof实验室的综合专业知识。我们提出的实验将系统地研究In细胞的细胞和分子特性，并开发从成纤维细胞诱导特定神经元亚群的方案。然后，这些方案将被用于在细胞内模拟小鼠的遗传病。此外，我们将把我们的发现扩展到人类成纤维细胞，长期目标是建立神经精神疾病的细胞模型。我们的目标将通过组织培养实验、细胞生物学、分子生物学和电生理学相结合来实现。我们相信，我们提出的实验有可能从根本上改变现有的细胞分化和表观遗传基因调控的范式，并可能为研究各种脑部疾病患者的人类神经元提供一个新的平台。 与公共卫生相关：这项应用将开发直接从非神经细胞产生神经元的方法，允许从人类患者的皮肤成纤维细胞产生神经元。然后，这些方法将被用于测试与神经精神障碍相关的突变对神经生物学的影响，长期目标是更好地了解这些疾病在人类神经元中的病理机制。

DESCRIPTION (provided by applicant): The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (iPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results in which we demonstrate direct conversion of adult fibroblasts cells into neurons, referred to as 'induced neuronal cells' (iN cells), without an iPS intermediate. The resulting iN cells have the functional properties of neurons, including the ability to form functional synapses as assayed by electrophysiology. Thus, the iN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to investigating human disease processes. However, at this point the iN cell technology has only been developed for mouse fibroblasts, fundamental questions regarding the conversion process and the molecular identity of iN cells were not determined, the generation of iN cells from human fibroblasts has not yet been established, and most importantly, the feasibility of the iN cell technology to study diseases affecting neuronal function has not been demonstrated. In this project, we propose to address these important challenges in an interdisciplinary approach capitalizing from the combined expertises of the Wernig and S|dhof laboratories. We propose experiments that will systematically investigate the cellular and molecular identity of iN cells, and develop protocols to induce specific neuronal subpopulations from fibroblasts. These protocols will then be employed to model genetic diseases in mouse iN cells. Furthermore, we will extend our findings to human fibroblasts, with the long term goal to establish a cell model for neuropsychiatric diseases. Our goals will be pursued by a combination of tissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. We believe our proposed experiments have the potential to fundamentally change existing paradigms of cellular differentiation and epigenetic gene regulation, and could provide a novel platform to study human neurons from patients suffering from a variety of brain diseases. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. These methods will then be used to test the effects of mutations associated with neuropsychiatric disorders on neuronal biology, with the long-term goal of establishing a better understanding of the pathomechanism of these diseases in human neurons.