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

项目摘要/摘要 神经层的形成是哺乳动物大脑皮质区域的一个基本特征。这 该提案旨在生成一种定义的3D人类小脑神经元系统，该系统能够忠实地复制本地人 小脑皮质的层状结构。小脑是一个需要研究的关键区域，作为一种原型皮质结构和 作为一个既有感觉运动控制功能，又有包括认知在内的非运动功能的大脑区域， 情感和语言。我们将使用Hatten Lab中的两个协议来区分这两个主节点 人多能干细胞(HPSCs)的小脑神经元分类及生物工程研究现状 方法建立三维层流培养系统(目标1)。然后我们将使用细胞类型的钙成像 作为第一步，用特定的基因编码的钙传感器来确定神经元放电率的相关性 关于在3D体外层叠系统中记录人小脑神经元的电路特性(Aim 2)和 针对天然发育组织的转录图谱分析(目标3)。在小脑中间神经元之间，单极刷状突起 细胞是小脑回路的重要调节器，但在体外还没有可用的方案来衍生它们。我们 将开发第一个分化方案来衍生这些谷氨酸能神经元并将它们整合到 层流模型(目标4)。这项研究将结合米伦实验室在人类小脑方面的专业知识 与Hatten Lab共同开发从hPSC培养人小脑神经元的研究提供了第一个 为疾病建模和药物筛选的目的，适当地对人类小脑回路进行分层。

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.