Dissecting the mechanism of direct neural induction through genomic interrogation

该项目涵盖3个主题领域：应用基因组学和其他高通量技术，将基础科学发现转化为新的和更好的治疗方法，重振生物医学研究社区。体细胞在成年动物中是高度稳定的，这是由于强大的基因表达模式，这是稳定的表观遗传机制。然而，诱导多能干细胞（iPS）的开创性发明提供了令人惊讶的结论，即分化状态可以通过简单表达四种转录因子（TF）来逆转。这一发现证明，即使是被认为稳定的基因表观遗传修饰，也基本上是由TF控制的。我们问这个概念是否可以扩展到一种细胞类型到另一种的转分化，最近成功地将小鼠成纤维细胞直接转化为功能性神经元，称为诱导神经元（iN）细胞，通过过度表达只有三个谱系特异性TF。我们的研究结果表明，TF不仅足以逆转特定的分化途径，而且还可以将细胞中的转录调控网络重定向到完全不同的途径。这一基本结果回答了该领域的一个关键开放问题，也是当前提案的基础。除了记录TF对表观遗传修饰的优势之外，iN细胞可以代表从皮肤成纤维细胞衍生患者特异性神经元的有吸引力的方式。这可用于模拟各种神经系统疾病或用于细胞移植治疗。该提案旨在在分子水平上表征iN细胞生成的过程，期望获得对潜在转分化过程的生物学的基本见解。除了确定成纤维细胞向神经元转化的分子事件外，本研究还将特别评估iN细胞状态的表观遗传稳定性以及其潜在致瘤性的安全性，这是iN细胞技术临床应用的关键先决条件。我们的多学科方法需要最先进的高通量测序技术，用于表观遗传状态和转录的基因组水平询问，新开发的微流体装置能够对小细胞群体进行全基因组分析，以及在单细胞水平上进行多重基因表达，从而能够确定细胞异质性，电生理学和神经发育技术。 公共卫生相关性：该申请将开发直接从非神经元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。患者来源的神经元可用于神经系统疾病的建模或作为细胞移植物来治疗神经退行性疾病，如帕金森病。

DESCRIPTION (provided by applicant): This project covers 3 thematic areas: Applying Genomics and Other High Throughput Technologies, Translating Basic Science Discoveries into New and Better Treatments and Reinvigorating the Biomedical Research Community. Somatic cells are highly stable in adult animals due to robust gene expression patterns, which are stabilized by epigenetic mechanisms. The seminal invention of induced pluripotent stem (iPS) cells, however, provided the surprising conclusion that the differentiated state can be reversed by simple expression of four transcription factors (TFs). This finding proved that even supposedly stable epigenetic modifications of genes are essentially controlled by TFs. We asked whether this concept can be extended to trans-differentiation of one cell type into another, and recently succeeded in converting mouse fibroblasts directly into functional neurons, referred to as induced neuronal (iN) cells, by overexpression of only three lineage-specific TFs. Our findings indicate that TFs suffice to not only reverse a particular pathway of differentiation, but also to redirect the transcriptional regulatory network in a cell into a completely different pathway. This fundamental result answered one of the key open questions in the field, and is the basis of the current proposal. Apart from documenting the dominance of TFs over epigenetic modifications, iN cells could represent an attractive way to derive patient- specific neurons from skin fibroblasts. This may be used to model various neurological diseases or for cell transplantation therapy. This proposal aims to characterize the process of iN cell generation on the molecular level, with the expectation to gain fundamental insights into the biology of the underlying trans-differentiation process. In addition to identifying the molecular events underlying the fibroblast-to-neuron conversion, this study will in particular assess the epigenetic stability of the iN cell state as well as their safety with respect to their potential tumorigenicity, key prerequisites for clinical application of iN cell technologies. Our multidisciplinary approach entails state-of-the art high-throughput sequencing technologies for genome-level interrogation of epigenetic states and transcription, newly developed microfluidic devices enabling genome- wide analyses of small cell populations as well as multiplex gene expression on the single cell level allowing the determination of cellular heterogeneity, electrophysiology, and neurodevelopmental techniques. 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. Patient-derived neurons could be used for modeling neurological diseases or as cell grafts to treat neurodegenerative diseases like Parkinson's disease.