Development of a model system to study human cerebellar neurons

 描述（由申请人提供）：小脑在运动协调、平衡和控制眼囊方面具有关键作用，最近的证据强调了其在前馈学习、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等发育性疾病有关。虽然这种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。因此，迫切需要一个人类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（hPSC）来创建中枢神经系统的人类模型系统。虽然已经通过多巴胺能或运动神经元亚型的分化开发了用于诸如帕金森病和肌萎缩性侧索硬化症的病症的基于hPSC的人模型系统，但是缺乏用于产生特定小脑神经元的方案。 该研究旨在开发将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。为了评估基因表达，一种新的遗传工具，bacTRAP，将被用来分离翻译mRNA特异性从EGFP标记的GC或PC内的异质性培养。在RNA测序后，将结果与实验室中已经获得的天然小鼠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特异性电流。为了评估整合到小脑回路中的能力，我们将调整我们报道的用于mES细胞的方法，以将hPSC衍生的GC和PC植入新生小鼠小脑中。Clarity或ClearT 2组织清除方法和新型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。这些测定将提供hPSC衍生的GC和PC基因表达和功能能力的详细分析，可以针对其评估患者-hPSC衍生的小脑神经元以及其他神经亚型。 哈滕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，哈滕实验室已经生成了小鼠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适用于hPSC，首次产生了确定的人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，并通过基因表达谱，电生理学和整合能力将其彻底表征到小鼠小脑回路中 植入后。这些研究将创造一个重要的新的人类模型系统的小脑发育和功能障碍。

 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.