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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：小脑在运动协调，平衡和控制眼acchades中具有关键作用，最近的证据突显了在馈送前进学习，视觉空间记忆，注意力，语言和其他更高的认知功能中的作用。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表型。因此，人类模型系统迫切需要研究小脑发育和功能障碍。令人兴奋的是，现在可以通过使用人类多能干细胞（HPSC）来创建中枢神经系统的人类模型系统。尽管基于HPSC的人类模型系统是通过通过分化多巴胺能或运动神经元亚型的分化来用于帕金森氏病和肌萎缩性侧索硬化症等疾病的，但缺乏生成特定小脑神经元的方案。拟议的研究旨在开发将HPSC区分为小脑的两个主要神经元，颗粒细胞（GC）和Purkinje细胞（PC）的方法，并彻底表征了所得细胞。为了评估基因表达，将采用一种新型的遗传工具，即BacTrap，用于分离RNA测序后的翻译mRNA，将结果与在实验室中已经获得的天然小鼠GC和PC的各种发育阶段的数据集进行比较。为了评估生理，将在体外测量基本的膜特性以及GC和PC特定电流。为了评估整合到小脑回路中的能力，我们将适应我们报告的MES细胞的方法，以将HPSC衍生的GC和PC植入新生儿小鼠小脑中。清晰度或Cleart2组织清除方法和新型的全脑成像技术将允许成像小鼠小脑回路中植入神经元的发展和整合。这些评估将对HPSC衍生的GC和PC基因表达和功能能力进行详细分析，可以评估患者HPSC衍生的小脑神经元以及其他神经元亚型的患者。哈滕实验室已经对小脑发育和神经元迁移进行了第二项研究。在初步工作中，Hatten Lab生成了将小鼠ES细胞分化为小脑神经元的方案，并利用BACTRAP获得了天然小鼠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调整为HPSC，首次生成确定的人GC和PC。拟议的研究旨在完善这些方案以产生成熟的神经元，并通过基因表达分析，电生理学和整合能力彻底表征它们，并将其整合到小鼠小脑电路中植入后。这些研究将创建一个关键的小脑发育和功能障碍的新型人类模型系统。