Development and optimization of neuronal reprogramming methods

神经元重编程方法的开发和优化

• 批准号: 9116018
• 负责人: Marius Wernig
• 金额: $ 44.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9116018
• 关键词: 16p11.2; 22q11.2; Abnormal Cell; Alleles; Astrocytes; Biological; Biological Assay; Biological Models; Brain; Cell Culture Techniques; Cell Line; Cell model; Cells; Chemicals; Coculture Techniques; Complex; Conditioned Culture Media; Copy Number Polymorphism; Cultured Cells; Data; Dependovirus; Development; Disease; Disease model; Drug Industry; Engineering; Environmental Risk Factor; Epigenetic Process; Gene Targeting; Generations; Genes; Genetic; Genetic Engineering; Genetic Identity; Genetic study; Goals; Grant; Human; Human Genetics; Infection; Interneurons; Libraries; Mediating; Mental disorders; Methods; Modeling; Modification; Molecular; Mus; Mutation; Neuroglia; Neurons; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pluripotent Stem Cells; Preclinical Drug Evaluation; Production; Protocols documentation; Psychotic Disorders; Reproducibility; Risk; Risk Factors; Schizophrenia; Severities; Solid; Specific qualifier value; Staging; Stem cells; Synapses; Synaptic Transmission; System; Technology; Therapeutic; Therapeutic Agents; Tissues; Transgenes; Work; base; cell type; conditional mutant; disease phenotype; drug development; high risk; human disease; human subject; improved; in vivo; induced pluripotent stem cell; neuronal circuitry; novel therapeutics; protocol development; research study; stem cell biology; suicide rate; synaptogenesis; technology development; tool; trait; transcription factor

Schizophrenia is a frequent psychotic disorder leading to severe human suffering and increased suicide rates. Quality and severity of disease phenotypes vary dramatically between patients and the pathogenetic mechanisms on the molecular, cell biological, or neuronal circuit level are poorly understood. However, human genetics studies have demonstrated that genetic factors contribute to the highest risk factors to develop disease. The importance of genetic factors for disease development is widely accepted in the field. Because of our ability to manipulate and define genetic backgrounds and specific factors (as opposed to potential environmental factors) in cultured cells, it is possible that critical disease traits of the human brain can be recapitulated in cultured cell-based models. Recent advances by us and others in the field of epigenetic reprogramming and stem cell biology has made it possible to generate fully functional human neurons from pluripotent stem cells. We are therefore very close to interrogate human disease neurons for abnormal cell- biological traits in a meaningful way. Projects 1 and 2 will begin to analyze disease-specific traits using existing methods. However, current technology has several shortcomings limiting the full phenotypic characterization. The goal of Project 3 (this project) is to further develop and optimize existing stem cell methods that will allow to substantially increase the spectrum of assays to be analyzed. As the protocols are being developed and become available in Project 3, they will be immediately implemented and utilized in Projects 1 and 2. In particular, Project 3 will develop two critical components for the overall consortium grant: (1) It will provide the tools to genetically engineer conditional and/or definitive single gene and large CNV mutations. (2) The project will develop methods to develop defined inhibitory neuronal subtypes. Together with our already existing method to generate pure excitatory neuronal subtypes this will allow us to generate mixed cultures with defined excitatory/ inhibitory neuronal components which will allow the characterization of inhibitory synaptic transmission. In addition, the project will work on two non-essential but highly desired protocol developments: (1) Methods will be devised for industrial generation of neurons in large scale to improve consistent phenotypic analyses and enable the establishment of human cell models for pharmaceutical drug development. (2) We will develop ways to eliminate the currently required co-culture of mouse glia to be replaced with defined substances or human cells because use of cell models containing supportive mouse cells may complicated therapeutic drug development for human use in vivo. We believe the proposed technology developments will be critical contributions to the field and ultimately enable the generation of authentic human disease cell models.

精神分裂症是一种常见的精神障碍，导致严重的人类痛苦和自杀率增加。 疾病表型的质量和严重程度在患者和致病者之间变化很大。 分子、细胞生物学或神经元回路水平上的机制知之甚少。但人类 遗传学研究表明，遗传因素有助于最高风险因素的发展 疾病遗传因素对疾病发展的重要性在该领域被广泛接受。因为 我们操纵和定义遗传背景和特定因素的能力（而不是潜在的 环境因素）在培养的细胞中，有可能人类大脑的关键疾病特征可以是 在基于培养细胞的模型中重现。我们和其他人在表观遗传学领域的最新进展 重编程和干细胞生物学使得从神经元中产生功能齐全的人类神经元成为可能。 多能干细胞。因此，我们非常接近于询问人类疾病神经元的异常细胞- 有意义的生物学特征。项目1和项目2将开始利用现有的遗传学方法分析疾病特异性特征。 方法.然而，目前的技术有几个缺点，限制了完整的表型表征。 项目3（本项目）的目标是进一步开发和优化现有的干细胞方法， 以显著增加待分析的化验谱。随着协议的制定， 一旦项目3中的信息可用，它们将立即在项目1和2中得到实施和利用。在 特别是，项目3将为整个财团赠款开发两个关键组成部分：（1）它将提供 基因工程工具的条件和/或确定的单基因和大CNV突变。(2)项目 将开发方法来开发定义的抑制性神经元亚型。加上我们现有的 方法来产生纯兴奋性神经元亚型，这将使我们能够产生混合培养物， 兴奋性/抑制性神经元成分，这将允许表征抑制性突触 传输此外，该项目将致力于两个非必要但非常需要的协议开发： (1)将设计大规模工业化生产神经元的方法，以提高表型的一致性 分析并能够建立用于药物开发的人类细胞模型。(2)我们将 开发方法以消除目前所需的小鼠神经胶质的共培养物， 因为使用含有支持性小鼠细胞的细胞模型可能会使 用于人体内使用的治疗药物开发。我们认为，拟议的技术发展将 对该领域做出重要贡献，并最终能够产生真正的人类疾病细胞 模型