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: 7943095
• 负责人: Stewart A Anderson
• 金额: $ 49.92万
• 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/7943095
• 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 Depression; 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; 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）：广泛的挑战区域（14），干细胞。特定挑战主题14-MH-101：为精神障碍的IPS细胞开发。大脑皮层的GABA能中神经元功能障碍与多种主要神经精神疾病有关，包括精神分裂症，自闭症，焦虑和癫痫病（抑郁症是发病率的主要来源）。但是，我们缺乏对人中间神经元如何发展和功能的知识，以及与疾病相关的基因如何影响这一过程，极大地阻碍了我们理解，预防或治疗与中间神经中有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于多基因风险因素的组合作用带来的，具有重要的环境影响的重要组成部分（也许主要是在子宫内），因此解决这一复杂的效果中的技术将非常有用。诱导的多能干细胞（IPSC）是人类中间神经元的重要潜在来源，可用于解决对中间神经元发育和功能的遗传影响，并在此过程中研究遗传环境相互作用。下面的未来方向部分提供了两个可以使用基于IPSC的方法来研究精神分裂症的特定示例，但理想的未来目标是： 1）从已知的患者中得出IPSC，以具有给定的神经精神疾病具有特定风险等位基因，以及不受影响和非风险等位基因携带控制的患者，2）插入一个已知在中间神经元间发育相关阶段表达的荧光记者的构造（即，迁移，迁移和连接性，依赖的方式，依赖于依赖的方式，依赖于依据疾病的发展，3）将IPSC的分化指导为中间神经元发育的特定阶段，以及4）在该阶段通过FACS收集细胞，以研究与疾病相关的基因和蛋白质表达，神经元发育或功能，对环境损害模型的敏感性以及预防或纠正剂的测试。作为朝着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类成纤维细胞衍生的IPSC中生成，隔离和研究假定的皮质中间神经元的方法。该提案的基础是基于Anderson和Studer Labs在i）使用经过改进的细菌性人造染色体（BAC）获得转基因小鼠以及小鼠和人类胚胎干细胞中报告基因的组织特异性表达的基础LHX6-GFP报告基因分离针对中间神经元的IPS细胞的特定目的2。使用NKX2.1-GFP报道器分离针对Interneuron lineagy nkx2.1的IPS细胞分离出有丝分裂的IPS细胞在有丝分裂中的内神经元中表达的，而在隔离的循环中，在某些循环中表达了一些循环，并在某些过程中表达了一些循环。通过将IPSC引导到假定的中间神经元祖细胞命运（NKX2.1+），我们可以在这个关键阶段前瞻性地分离细胞，用于遗传和移植研究，以定义其命运潜力。当大多数人的NKX2.1下调时，在表达LHX6的阶段的分离细胞将得到这些研究的补充。我们可以再次进行遗传和命运潜在的研究，这些研究特有在这些中间神经元发育的后期阶段。尽管使用基于IPS的方法来研究精神疾病，但必须克服一些主要的挑战才能实现这一诺言。关键问题包括：首先，从IPSCS中得出类似细胞的中间神经元促进剂的最佳方法是什么？其次，我们如何将它们区分为不同的中间神经元类型，我们可以使用哪种系统来定义人类大脑之外的这些类型？第三，当比较了从同一人类来源得出的多个神经元间分化的样品时，该方法中存在多少可变性？必须解决这些问题，以实现使用IPSC研究与中间神经元间疾病的重要效用。来自同一来源的多个样本中的高度可变性将使患者和控制衍生的样本之间的比较毫无意义，甚至误导。为了应对这些挑战，我们在项目的每个层面上都组建了一个具有丰富经验的团队； 1）胚胎和IPS衍生的干细胞生物学（Co-Pi Dr. Studer），2）皮质间神经内发育（Pi-Dr。Anderson），3）前脑切片中的电生理学（Goldstein博士的共同投资者）和4）人类脑皮质和脑膜的脑皮质和病理学学杂志（咨询）（咨询型）。实现该项目目标的成功将使我们和许多其他群体能够实现大量的未来研究，该研究致力于与疾病相关的基因与环境的相互作用，涉及中间神经元的起源，成熟和功能。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的重要作用产生重大影响。 公共卫生相关性：大脑皮层中GABA能中间神经元功能障碍与各种主要神经精神疾病有关，包括精神分裂症，自闭症，焦虑症和癫痫病（抑郁症是主要的痛苦来源）。但是，我们普遍缺乏关于人间神经元如何发展和功能的知识，以及我们对与疾病有关的基因如何影响这一过程的具体知识，极大地阻碍了我们理解，预防或治疗与中间神经元间有关的精神疾病的能力。诱导的多能干细胞（IPSC）是人类中间神经元的重要潜在来源，可用于解决对中间神经元发育和功能的遗传影响，并在此过程中研究遗传环境相互作用。该提案的目的是开发方法和方案，以从人IPSC的功能抑制性中间神经元持续推导，以对抑制性中间神经元在神经精神疾病中的作用进行批判性研究。