A Bioengineering Approach to Develop a Laminar 3D Cerebellar Neuronal Circuit for Modeling Human Cerebellum

开发用于模拟人类小脑的层状 3D 小脑神经元回路的生物工程方法

• 批准号: 10604377
• 负责人: Mary Elizabeth Hatten
• 金额: $ 61.08万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604377
• 关键词: 3-Dimensional; ATAC-seq; Address; Algorithms; Architecture; Area; Biological Assay; Biomedical Engineering; Brain; Brain region; Brush Cell; Calcium; Calcium Spikes; Cell Density; Cell Nucleus; Cells; Cerebellar Cortex; Cerebellum; Cognition; Cytoplasmic Granules; Data Set; Development; Diameter; Disease model; Drug Screening; Emotions; Functional disorder; Gene Expression; Genetic Transcription; Glass; Glutamates; Human; Image; In Vitro; Interneurons; Language; Light; Lip structure; Methodology; Methods; Microscopy; Modeling; Neurons; Organoids; Pharmacotherapy; Property; Protocols documentation; Purkinje Cells; RNA; Reproducibility; Research; Rhodopsin; Scanning; Sensory; Signal Pathway; Sorting; Structure; System; Testing; Thick; Tissues; calcium indicator; cell type; cellular imaging; comparative; design; differentiation protocol; drug discovery; experimental study; fetal; granule cell; human model; human pluripotent stem cell; in vivo; motor control; neuronal circuitry; novel; novel therapeutics; optogenetics; progenitor; redshift; self organization; sensor; single cell analysis; single-cell RNA sequencing; three dimensional cell culture; three-dimensional modeling; transcriptome; transcriptome sequencing; transcriptomic profiling; transcriptomics

PROJECT SUMMARY/ ABSTRACT The formation of neuronal laminae is a fundamental feature of cortical regions of the mammalian brain. This proposal aims to generate a defined 3D system of human cerebellar neurons that faithfully reproduces the native lamination of the cerebellar cortex. The cerebellum is a key area to study, as an archetypal cortical structure and as a brain region that functions both in sensory-motor control and in non-motor functions, including cognition, emotion and language. We will use two protocols in place in the Hatten Lab to differentiate the two primary classes of cerebellar neurons from human pluripotent stem cells (hPSCs) and state of the art bioengineering methods to develop a defined 3D laminar culture system (Aim 1). We will then use calcium imaging of cell-type specific genetically encoded calcium sensors to determine correlations in neuronal firing rates as a first step toward recording circuit properties of human cerebellar neurons in a 3D in vitro laminated system (Aim 2) and transcriptomic profiling against native developing tissue (Aim 3). Among cerebellar interneurons, unipolar brush cells are important modulators of the cerebellar circuit, yet no protocols are available to derive them in vitro. We will develop the first differentiation protocols to derive these glutamatergic neurons and incorporate them into laminar models (Aim 4). This research will combine the expertise of the Millen Lab in human cerebellar development with that of the Hatten Lab in generating human cerebellar neurons from hPSCs to provide the first properly stratified human cerebellar circuit for the purposes of disease modeling and drug screening.

项目摘要/摘要 神经元层的形成是哺乳动物大脑皮质区域的基本特征。这 提案旨在产生人类小脑神经元的定义的3D系统，该系统忠实地再现了本地 小脑皮层的层压。小脑是研究的关键领域，作为原型皮质结构和 作为在感觉运动控制和非运动功能（包括认知）中起作用的大脑区域 情绪和语言。我们将在Hatten Lab中使用两个协议来区分这两个主要 来自人类多能干细胞（HPSC）的小脑神经元和艺术生物工程状态 开发定义的3D层流培养系统的方法（AIM 1）。然后，我们将使用细胞类型的钙成像 特定的遗传编码钙传感器，以确定神经元放电速率的相关性作为第一步 要记录3D体外层压系统中人小脑神经元的电路特性（AIM 2）和 针对天然发展组织的转录组分析（AIM 3）。在小脑中间神经元中，单极刷 细胞是小脑回路的重要调节剂，但是没有任何方案可以在体外得出它们。我们 将制定第一个分化方案来得出这些谷氨酸能神经元并将其纳入 层流型号（AIM 4）。这项研究将结合米伦实验室在人类小脑中的专业知识 通过Hatten Lab的开发，从HPSC产生人类小脑神经元以提供第一个 适当分层的人小脑回路，以进行疾病建模和药物筛查。