Development of a model system to study human cerebellar neurons

开发研究人类小脑神经元的模型系统

• 批准号: 8954174
• 负责人: Mary Elizabeth Hatten
• 金额: $ 25.43万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8954174
• 关键词: Activities of Daily Living; Amyotrophic Lateral Sclerosis; Attention; Attention deficit hyperactivity disorder; Autistic Disorder; Benchmarking; Biological Assay; Biological Models; Brain; Brain imaging; Cell Differentiation process; Cell Line; Cell physiology; Cells; Cerebellar cortex structure; Cerebellum; Coculture Techniques; Complex; Data; Data Set; Databases; Derivation procedure; Development; Disease; Electrophysiology (science); Embryo; Equilibrium; Eye; Functional disorder; Gene Expression; Gene Expression Profiling; Generations; Genetic; Genetic Models; Goals; Human; Image; Imaging Techniques; Implant; In Vitro; Label; Language; Learning; Link; Measures; Membrane; Messenger RNA; Methodology; Methods; Modeling; Molecular; Motor; Motor Neurons; Movement; Mus; Neonatal; Neuraxis; Neurons; Parkinson Disease; Pathology; Patients; Physiology; Pluripotent Stem Cells; Population; Property; Protocols documentation; Purkinje Cells; RNA; RNA Sequences; Reporting; Research; Role; Saccades; Seminal; Staging; Stem cells; Techniques; Testing; Thyroid Hormones; Time; Tissues; Translating; Work; base; cell type; cognitive function; developmental disease; disease phenotype; dopaminergic neuron; embryonic stem cell; feeding; granule cell; human disease; human embryonic stem cell; implantation; in vivo; induced pluripotent stem cell; insight; migration; mouse model; nervous system disorder; neurodevelopment; neuron development; novel; progenitor; public health relevance; relating to nervous system; spatial memory; stem cell differentiation; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): The cerebellum has a critical role in motor coordination, balance and controlling eye sacchades, with recent evidence highlighting a role in feed-forward learning, visuo-spatial memory, attention, language, and other higher cognitive functions. Importantly, cerebellar pathology and dysfunction have been linked to developmental diseases such as autism and ADHD. While mouse models of such complex disorders have provided critical insights, mouse genetic models do not always model human disease phenotypes. There is therefore a critical need for a human model system to study cerebellar development and dysfunction. Excitingly, it is now possible to create human model systems of the central nervous system through the use of human pluripotent stem cells (hPSCs). While hPSC-based human model systems have been developed for disorders such as Parkinson's disease and Amyotrophic Lateral Sclerosis through the differentiation of dopaminergic or motor neuron subtypes, protocols for the generation of specific cerebellar neurons are lacking. The proposed research aims to develop methods to differentiate hPSCs into the two primary neurons of the cerebellum, the granule cell (GC) and the Purkinje cell (PC), and thoroughly characterize resulting cells. To assess gene expression, a novel genetic tool, the bacTRAP, will be employed to isolate translating mRNA specifically from EGFP-tagged GCs or PCs within a heterogeneous culture. Following RNA sequencing, results will be compared to datasets of various developmental stages of native mouse GCs and PCs already obtained in the lab. To assess physiology, basic membrane properties as well as GC and PC specific currents will be measured in vitro. To assess the ability to integrate into the cerebellar circuit, we will adapt methods we reported for mES cells to implant hPSC-derived GCs and PCs into the neonatal mouse cerebellum. Clarity or ClearT2 tissue clearing methods and novel whole brain imaging techniques will allow imaging of the development and integration of implanted neurons within the mouse cerebellar circuit. These assays will provide a detailed analysis of hPSC-derived GC and PC gene expression and functional capacity, against which patient-hPSC derived cerebellar neurons, as well as other neural subtypes, can be assessed. The Hatten lab has carried out seminal studies on cerebellar development and neuronal migration. In preliminary work, the Hatten lab has generated protocols for the differentiation of mouse ES cells into cerebellar neurons and utilized bacTRAP to obtain gene expression datasets of native mouse GCs and PCs. Importantly; we have adapted these differentiation protocols to hPSCs, generating definitive human GCs and PCs for the first time. The proposed research aims to refine these protocols to generate mature neurons, and to thoroughly characterize them through gene expression profiling, electrophysiology, and integration capacity into the mouse cerebellar circuit following implantation. These studies will create a critical new human model system of cerebellar development and dysfunction.

 描述（由申请人提供）：小脑在运动协调、平衡和控制眼跳方面发挥着关键作用，最近的证据强调了小脑在前馈学习、视觉空间记忆、注意力、语言和其他高级认知功能中的作用。重要的是，小脑病理学和功能障碍与自闭症和多动症等发育性疾病有关，尽管此类复杂疾病的小鼠模型提供了重要的见解，但小鼠遗传模型并不总是模拟人类疾病表型。研究小脑发育和功能障碍的人类模型系统的迫切需要令人兴奋的是，现在可以通过使用人类多能干细胞（hPSC）来创建中枢神经系统的人类模型系统。已经通过多巴胺能或运动神经元亚型的分化开发出针对帕金森病和肌萎缩侧索硬化症等疾病的治疗方案，但缺乏生成特定小脑神经元的方案。研究旨在开发将 hPSC 分化为小脑的两种主要神经元、颗粒细胞 (GC) 和浦肯野细胞 (PC) 的方法，并彻底表征所得细胞的基因表达，一种新型遗传工具 bacTRAP，将用于从异质培养物中特异地从带有 EGFP 标签的 GC 或 PC 中分离翻译 mRNA。RNA 测序后，结果将与已在其中获得的天然小鼠 GC 和 PC 的各个发育阶段的数据集进行比较。为了评估生理学，将在体外测量基本膜特性以及​​ GC 和 PC 特定电流，为了评估融入小脑回路的能力，我们将采用我们报道的 mES 细胞植入 hPSC 衍生 GC 的方法。 Clarity 或 ClearT2 组织透明化方法和新颖的全脑成像技术将允许对小鼠小脑回路内植入神经元的发育和整合进行成像。分析 hPSC 衍生的 GC 和 PC 基因表达和功能能力，可以评估患者 hPSC 衍生的小脑神经元以及其他神经亚型，Hatten 实验室开展了有关小脑发育和神经迁移的开创性研究。初步工作中，Hatten 实验室已制定了将小鼠 ES 细胞分化为小脑神经元的方案，并利用 bacTRAP 处理天然小鼠 GC 和 PC 的基因表达数据集。拟议的研究旨在完善这些协议以生成成熟的神经元，并通过基因表达谱、电生理学和小鼠小脑回路的整合能力来彻底表征它们。 植入后，这些研究将创建一个重要的新人类小脑发育和功能障碍模型系统。