Derivation of cerebral cortical GABAergic interneurons from human iPS cells

从人 iPS 细胞中衍生出大脑皮层 GABA 能中间神经元

• 批准号: 7837045
• 负责人: Stewart A Anderson
• 金额: $ 50万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7837045
• 关键词: Address; Affect; Alleles; Anxiety; Area; Autistic Disorder; Autopsy; Bacterial Artificial Chromosomes; Brain; Cell Cycle; Cell Line; Cells; Cerebral cortex; Cerebrum; Characteristics; Collecting Cell; Commit; Complement; Complex; Derivation procedure; Development; Disease; Electrophysiology (science); Embryo; Environment; Epilepsy; Etiology; Fibroblasts; Functional disorder; Future; GABA Receptor; Gene Expression; Gene Proteins; Genes; Genetic; Goals; Grant; Human; Interneurons; Knock-in Mouse; Knowledge; Memorial Sloan-Kettering Cancer Center; Mental disorders; Methods; Mitotic; Modeling; Modification; Morbidity - disease rate; Mus; Neurons; Patients; Physiological; Physiology; Pluripotent Stem Cells; Population; Population Control; Predisposition; Preparation; Prevention; Process; Prosencephalon; Protocols documentation; Reporter; Reporter Genes; Research Personnel; Risk; Risk Factors; Role; Sampling; Schizophrenia; Slice; Source; Specific qualifier value; Staging; Stem cells; System; Techniques; Telencephalon; Testing; Time; Tissues; Transgenic Mice; Transgenic Organisms; Transplantation; Xenograft procedure; base; cell type; clinically relevant; combinatorial; depression; developmental genetics; embryonic stem cell; experience; gene discovery; human embryonic stem cell; in utero; induced pluripotent stem cell; method development; migration; neurochemistry; neuron development; neuropsychiatry; nonhuman primate; patch clamp; patient population; postnatal; prevent; progenitor; prospective; protein expression; public health relevance; senescence; stem; stem cell biology; success; transcription factor

DESCRIPTION (provided by applicant): Broad challenge area (14), Stem Cells. Specific challenge topic 14-MH-101: Developing iPS cells for mental disorders. Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of morbidity). However, our lack of knowledge of how human interneurons develop and function, and how disease-related genes influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. In addition, since neuropsychiatric disorders such as schizophrenia and autism appear to largely result from the combinatorial effects of polygenic risk factors with a significant component of environmental influence (perhaps mainly in utero), techniques for addressing this complicated intermix of effects would be tremendously useful. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. Two specific examples of ways in which an iPSC based method could be used to study schizophrenia are provided in the Future Directions section below, but the ideal future objective would be to; 1) derive iPSCs from patients known to harbor particular risk alleles for the given neuropsychiatric illness together with unaffected and non-risk allele carrying controls, 2) insert a fluorescent reporter construct known to express at relevant stages of interneuron development (i.e. genesis, migration, elaboration of processes and connectivity, activity dependent refinement, mature function, senescence) that are implicated in how that risk allele affects the development of disease, 3) direct the differentiation of the iPSCs to the particular stage of interneuron development, and 4) collect cells at that stage by FACS for the study of disease-related gene and protein expression, neuronal development or function, susceptibility to models of environmental insults, and testing of preventative or corrective agents. As a first step towards this idealized objective, the goal of this proposal is to develop and validate methods of generating, isolating, and studying putative cortical interneurons from human fibroblast derived iPSCs. The proposal builds on the progress in the Anderson and Studer labs at i) using modified bacterial artificial chromosomes (BACs) to obtain tissue-specific expression of reporter genes in transgenic mice as well as in mouse and human embryonic stem cells ii) developing highly efficient protocols for the conversion of human ES cells and human iPSCs into neural cells with forebrain identities, We propose the following Aims: Specific Aim 1. Use of an Lhx6-GFP reporter to isolate iPS cells directed to interneuron lineages Specific Aim 2. Use of an Nkx2.1-GFP reporter to isolate iPS cells directed to interneuron lineages Nkx2.1 is expressed in mitotic interneuron progenitors, whereas Lhx6 is expressed by the some of the same progenitors at the time of cell cycle exit and maintained during subsequent development. By directing iPSCs to putative interneuron progenitor fates (Nkx2.1+), we can prospectively isolate cells at this critical stage for genetic and transplantation studies aimed at defining their fate potential. These studies will be complemented by the isolating cells at Lhx6-expressing stages, when most of them have down-regulated Nkx2.1. We can again perform genetic and fate potential studies particular to these later stages of interneuron development. Despite the tremendous potential for using iPS-based approaches to study mental illness, several key challenges must be overcome to realize this promise. Among the key questions are: First, what is the optimal method for deriving interneuron-progenitor like cells from iPSCs? Second, how do we differentiate them into distinct interneuron types, and what system can we use to define those types outside of the human brain? Third, how much variability is present in this method when multiple interneuron-differentiated samples derived from the same human source are compared? These questions must be addressed to achieve significant utility in the use of iPSCs to study interneuron-related disease. High variability within multiple samples from the same source will render comparisons between patient and control-derived samples meaningless, or even misleading. To address these challenges we have assembled a team with considerable experience at each level of the project; 1) embryonic and iPS-derived stem cell biology (Co-PI Dr. Studer), 2) cortical interneuron development (PI-Dr. Anderson), 3) electrophysiology in forebrain slices (Co-Investigator Dr. Goldstein), and 4) human cerebral cortex/interneuron and pathobiology of schizophrenia (Consultant David Lewis). Success in achieving the goals of the project will enable a wealth of future studies by our and many other groups, directed at the interaction of disease-related genes and environment on interneuron genesis, maturation, and function. Such iPSC-based approaches could have a major impact on understanding the tremendously complex, and difficult to study, role of interneurons in neuropsychiatric diseases. PUBLIC HEALTH RELEVANCE: Dysfunction of GABAergic interneurons of the cerebral cortex has been implicated in a variety of major neuropsychiatric illnesses, including schizophrenia, autism, anxiety, and epilepsy (where depression is a major source of suffering). However, both our general lack of knowledge regarding how human interneurons develop and function, and our specific lack of knowledge of how disease-related genes may influence this process, greatly hinder our ability to understand, prevent or to treat interneuron-related mental illness. Induced pluripotent stem cells (iPSCs) are an important potential source of human interneurons that could be used to address both genetic influences on interneuron development and function, and to study genetic-environmental interactions in this process. The goal of this proposal is to develop methods and protocols for the consistent derivation of function inhibitory interneurons from human iPSCs, to lay critical ground word for future studies on the role of inhibitory interneurons in neuropsychiatric disease.

描述（由申请人提供）：广泛的挑战领域（14），干细胞。具体挑战主题 14-MH-101：开发治疗精神障碍的 iPS 细胞。大脑皮层 GABA 能中间神经元的功能障碍与多种主要的神经精神疾病有关，包括精神分裂症、自闭症、焦虑症和癫痫（其中抑郁症是发病的主要来源）。然而，我们对人类中间神经元如何发育和发挥作用，以及疾病相关基因如何影响这一过程缺乏了解，极大地阻碍了我们理解、预防或治疗与中间神经元相关的精神疾病的能力。此外，由于精神分裂症和自闭症等神经精神疾病似乎很大程度上是由多基因危险因素与环境影响的重要组成部分（可能主要在子宫内）的组合效应引起的，因此解决这种复杂的混合影响的技术将非常有用。诱导多能干细胞（iPSC）是人类中间神经元的重要潜在来源，可用于解决遗传对中间神经元发育和功能的影响，并研究该过程中遗传与环境的相互作用。下面的“未来方向”部分提供了两个基于 iPSC 的方法可用于研究精神分裂症的具体示例，但理想的未来目标是： 1) 从已知携带给定神经精神疾病的特定风险等位基因的患者以及未受影响和非风险等位基因携带对照中获得 iPSC，2) 插入已知在中间神经元发育的相关阶段（即发生、迁移、过程和连接的详细阐述、活动依赖的细化、成熟功能、衰老）表达的荧光报告构建体，这些阶段与风险等位基因如何影响有关 疾病的发展，3) 引导 iPSC 分化至中间神经元发育的特定阶段，4) 通过 FACS 收集该阶段的细胞，用于研究疾病相关基因和蛋白质表达、神经元发育或功能、对环境损害模型的易感性以及预防或纠正剂的测试。作为实现这一理想化目标的第一步，该提案的目标是开发和验证从人成纤维细胞衍生的 iPSC 中生成、分离和研究假定的皮质中间神经元的方法。该提案建立在Anderson和Studer实验室的进展基础上：i）使用改良的细菌人工染色体（BAC）在转基因小鼠以及小鼠和人类胚胎干细胞中获得报告基因的组织特异性表达ii）开发将人类ES细胞和人类iPSC转化为具有前脑特性的神经细胞的高效方案。我们提出以下目标：具体目标1.使用 Lhx6-GFP 报告基因分离针对中间神经元谱系的 iPS 细胞 具体目标 2. 使用 Nkx2.1-GFP 报告基因分离针对中间神经元谱系的 iPS 细胞 Nkx2.1 在有丝分裂中间神经元祖细胞中表达，而 Lhx6 在细胞周期退出时由一些相同的祖细胞表达，并在随后的发育过程中维持。通过将 iPSC 引导至假定的中间神经元祖细胞命运 (Nkx2.1+)，我们可以在这一关键阶段前瞻性地分离细胞，用于旨在确定其命运潜力的遗传和移植研究。这些研究将得到 Lhx6 表达阶段分离细胞的补充，此时大多数细胞的 Nkx2.1 下调。我们可以再次进行针对中间神经元发育后期阶段的遗传和命运潜力研究。尽管使用基于 iPS 的方法研究精神疾病具有巨大潜力，但要实现这一承诺，必须克服几个关键挑战。关键问题包括：首先，从 iPSC 中获取中间神经元祖细胞样细胞的最佳方法是什么？其次，我们如何将它们区分为不同的中间神经元类型，以及我们可以使用什么系统来定义人脑之外的这些类型？第三，当比较来自同一人类来源的多个中间神经元分化样本时，该方法存在多少变异性？必须解决这些问题，才能在使用 iPSC 研究中间神经元相关疾病方面发挥重要作用。来自同一来源的多个样本的高变异性将使患者样本和对照样本之间的比较变得毫无意义，甚至产生误导。为了应对这些挑战，我们组建了一支在项目各个层面都拥有丰富经验的团队； 1) 胚胎和 iPS 衍生干细胞生物学（Co-PI Dr. Studer），2）皮质中间神经元发育（PI-Dr. Anderson），3）前脑切片电生理学（共同研究员 Goldstein 博士），以及 4）人类大脑皮层/中间神经元和精神分裂症病理学（顾问 David Lewis）。成功实现该项目的目标将使我们和许多其他小组能够开展大量未来研究，这些研究旨在研究疾病相关基因和环境对中间神经元发生、成熟和功能的相互作用。这种基于 iPSC 的方法可能会对理解中间神经元在神经精神疾病中极其复杂且难以研究的作用产生重大影响。 公共卫生相关性：大脑皮层 GABA 能中间神经元的功能障碍与多种主要的神经精神疾病有关，包括精神分裂症、自闭症、焦虑症和癫痫症（抑郁症是其中的主要痛苦来源）。然而，我们普遍缺乏关于人类中间神经元如何发育和功能的知识，以及我们对疾病相关基因如何影响这一过程的具体知识缺乏，极大地阻碍了我们理解、预防或治疗与中间神经元相关的精神疾病的能力。诱导多能干细胞（iPSC）是人类中间神经元的重要潜在来源，可用于解决遗传对中间神经元发育和功能的影响，并研究该过程中遗传与环境的相互作用。该提案的目标是开发从人类 iPSC 中一致衍生功能抑制性中间神经元的方法和方案，为未来研究抑制性中间神经元在神经精神疾病中的作用奠定关键基础。