Optimal Generation and Characterization of iPS Cell Lines from Healthy and ADHD S

描述（由申请人提供）：了解神经精神疾病的分子和细胞基础是21世纪神经科学中最大和最重要的挑战之一。注意缺陷多动障碍（ADHD）是最常见的儿童精神障碍，其特征是注意力不集中、注意力不集中、冲动和/或存在多动。尽管环境因素对ADHD的发病机制也有重要影响，但许多实验室已经表明ADHD具有高度遗传性（近似遗传率为0.8）。为了进一步了解ADHD的分子发病机制，需要建立具有ADHD遗传背景和健康受试者的多能干细胞进行生物学和分子比较。2006年，Shinya Yamanaka和他的同事发表了突破性的研究成果，表明通过逆转录病毒转导四种转录因子（即Oct4、Sox2、Klf4和c-Myc），体细胞可以产生多能干细胞，即所谓的“诱导多能干细胞（iPS）”。引人注目的是，包括Yamamaka的研究小组在内的几个研究小组很快证明，人类iPS细胞可以用类似的方法生成。虽然这种iPS技术仍处于早期阶段，需要进一步发展，但基于这些开创性的研究和我们自己的初步结果，我们假设可以通过生成ADHD特异性iPS细胞并使用体外分化研究来研究ADHD的潜在致病机制，并将其与来自健康受试者的iPS细胞进行比较。为了实现这一长期目标，在项目的R21阶段，我们建议(1)从健康和ADHD受试者中建立多个iPS细胞系，(2)通过细胞、分子和分化分析验证iPS细胞系，以及(3)确定RAG1/RAG2 VDJ重组酶系统是否可用于筛选和/或监测人类iPS细胞的身份。此外，在R33期，我们提出(1)表征单个iPS细胞系向可扩展的神经祖细胞/前体的分化，(2)表征其向相关神经元亚型（如多巴胺能和去甲肾上腺素能神经元）的分化，以及(3)比较健康和ADHD受试者的iPS细胞系的细胞和分化特性，以寻找潜在的致病机制。

DESCRIPTION (provided by applicant): Understanding the molecular and cellular basis of neuropsychiatric disorders is one of the biggest and most significant challenges for the 21st century in neuroscience. Attention deficit hyperactivity disorder (ADHD) is the most common childhood psychiatric disorder and is characterized by inattention, distractibility, impulsivity, and/or the presence of hyperactivity. Numerous laboratories have shown that ADHD is highly heritable (approximate heritability is 0.8) although environmental factors also importantly influence its pathogenesis. To further advance our understanding of ADHD's molecular pathogenesis it is desirable to establish multipotent stem cells with the genetic backgrounds of ADHD and healthy subjects for biological and molecular comparisons. In 2006, Shinya Yamanaka and his colleagues published their groundbreaking work showing that pluripotent stem cells, so called "induced pluripotent stem (iPS) cells, can be generated from somatic cells by retroviral transduction of four transcription factors (i.e., Oct4, Sox2, Klf4 and c-Myc). Strikingly, several groups, including Yamamaka's, soon demonstrated that human iPS cells could be generated by similar methods. Although this iPS technology is still at an early stage and needs further development, based on these pioneering studies and our own preliminary results, we hypothesize that potential pathogenic mechanisms of ADHD can be investigated by generating ADHD-specific iPS cells and by using in vitro differentiation studies, comparing them with those of iPS cells derived from healthy subjects. Toward this long-term goal, during the R21 phase of the project, we propose (1) to establish multiple iPS lines from healthy and ADHD subjects, (2) to validate iPS cell lines by cellular, molecular, and differentiation analyses, and (3) to address whether the RAG1/RAG2 VDJ recombinase system can be used to screen and/or monitor human iPS cells' identity. In addition, during the R33 phase, we propose (1) to characterize the differentiation of individual iPS cell lines to expandable neural progenitors/precursors, (2) to characterize their differentiation to relevant neuronal subtypes such as dopaminergic and noradrenergic neurons, and (3) to compare cellular and differentiation properties of iPS cell lines from healthy and ADHD subjects for potential pathogenic mechanisms. PUBLIC HEALTH RELEVANCE: Recent groundbreaking work by Shinya Yamanaka and his colleagues introduced the "induced pluripotent stem (iPS) cell" technology, a tantalizing new method generating genetically matched pluripotent stem cells without embryo destruction. To fully utilize this revolutionary technique, we propose to further advance this method by establishing a human iPS screening/monitoring system, and to generate and characterize multiple iPS lines from healthy and ADHD subjects. This approach will advance the iPS cell technology and provide a platform for investigating the biological and molecular mechanisms underlying ADHD pathogenesis.