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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.