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: 9066826
• 负责人: Mary Elizabeth Hatten
• 金额: $ 21.19万
• 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/9066826
• 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; Health; 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; 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; 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、平衡和控制眼球跳动方面起着关键作用，最近的证据强调了它在前馈学习、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，小脑病理和功能障碍与自闭症和ADHD等发育疾病有关。虽然这种复杂疾病的小鼠模型提供了关键的见解，但小鼠遗传模型并不总是模拟人类疾病的表型。因此，迫切需要一个人体模型系统来研究小脑的发育和功能障碍。令人兴奋的是，现在可以通过使用人类多能干细胞(HPSCs)来创建人类中枢神经系统的模型系统。虽然已经开发了基于hPSC的人类模型系统，通过分化多巴胺能或运动神经元亚型来治疗帕金森病和肌萎缩侧索硬化症等疾病，但缺乏产生特定小脑神经元的方案。本研究旨在开发将hPSCs分化为小脑的两个初级神经元，即颗粒细胞(GC)和浦肯野细胞(PC)的方法，并对所产生的细胞进行彻底的鉴定。为了评估基因的表达，一种新的遗传工具，bacTRAP，将被用来从异质培养中的EGFP标记的GC或PC中分离出特异的翻译mRNA。在RNA测序之后，结果将与实验室中已经获得的天然小鼠GC和PC的不同发育阶段的数据集进行比较。为了评估生理学，我们将在体外测量膜的基本性质以及GC和PC的比电流。为了评估整合到小脑回路的能力，我们将采用我们报道的MES细胞将hPSC来源的GC和PC植入新生小鼠小脑的方法。Clarity或ClearT2组织清除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的神经元的发育和整合进行成像。这些检测将提供hPSC来源的GC和PC基因表达和功能能力的详细分析，据此可以评估患者hPSC来源的小脑神经元以及其他神经亚型。哈腾实验室对小脑发育和神经元迁移进行了开创性的研究。在初步工作中，Hatten实验室已经制定了将小鼠ES细胞分化为小脑神经元的方案，并利用bacTRAP获得了本地小鼠GC和PC的基因表达数据集。重要的是，我们已经将这些分化方案适应于hPSC，首次产生了确定的人类GC和PC。这项拟议的研究旨在完善这些方案，以产生成熟的神经元，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关键的新的人类小脑发育和功能障碍的模型系统。

项目成果

N-cadherin provides a cis and trans ligand for astrotactin that functions in glial-guided neuronal migration.

• DOI: 10.1073/pnas.1811100115
• 发表时间: 2018-10-16
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Horn Z;Behesti H;Hatten ME
• 通讯作者: Hatten ME

Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells.

• DOI: 10.7554/elife.67074
• 发表时间: 2021-11-29
• 期刊: eLife
• 影响因子: 7.7
• 作者: Behesti H;Kocabas A;Buchholz DE;Carroll TS;Hatten ME
• 通讯作者: Hatten ME