Development and optimization of neuronal reprogramming methods

精神分裂症是一种常见的精神障碍，会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病原体之间差异很大 分子、细胞生物学或神经回路水平上的机制知之甚少。然而，人类 遗传学研究表明，遗传因素是导致发病风险最高的因素。 疾病。遗传因素对疾病发展的重要性在该领域得到了广泛的认可。因为. 我们操纵和定义遗传背景和特定因素的能力(相对于潜在的 环境因素)在培养细胞中，人类大脑的关键疾病特征可能是 在以培养细胞为基础的模型中进行了概括。我们和其他人在表观遗传学领域的最新进展 重新编程和干细胞生物学使从 多能干细胞。因此，我们非常接近于审问人类疾病神经元的异常细胞- 以一种有意义的方式展示生物特征。项目1和项目2将开始利用现有的 方法：研究方法。然而，目前的技术存在一些缺陷，限制了充分的表型表征。 项目3(该项目)的目标是进一步开发和优化现有的干细胞方法，以使 以大幅增加待分析分析的光谱。随着协议的制定和实施 在项目3中可用时，它们将立即在项目1和2中实施和使用。 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 对有条件的和/或决定性的单基因和大型CNV突变进行基因工程的工具。(2)项目 将开发方法来开发确定的抑制性神经元亚型。连同我们已经存在的 方法来生成纯兴奋性神经元亚型，这将允许我们生成具有定义的 兴奋性/抑制性神经元成分，可用于表征抑制性突触 变速箱。此外，该项目还将开展两项非必要但非常受欢迎的议定书发展工作： (1)将制定大规模工业化生产神经元的方法，以改善一致的表型 分析并建立用于药物开发的人体细胞模型。(2)我们会 开发方法，消除目前需要的小鼠神经胶质细胞共培养，用已定义的 物质或人类细胞，因为使用含有支持性小鼠细胞的细胞模型可能会很复杂 供人体使用的治疗药物开发。我们相信，拟议的技术开发将 是对该领域的关键贡献，并最终能够产生真正的人类疾病细胞 模特们。

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.