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

项目总结/摘要 神经元板层的形成是哺乳动物大脑皮层区域的基本特征。这 该提案旨在生成一个定义的人类小脑神经元的3D系统，该系统忠实地再现了人类小脑神经元的原始结构。 小脑皮层的分层。小脑是研究的一个关键区域，作为一个典型的皮质结构， 作为一个大脑区域，既有感觉运动控制功能，也有非运动功能，包括认知， 情感和语言。我们将在哈滕实验室中使用两种协议来区分这两种主要的 来自人类多能干细胞（hPSC）的小脑神经元的类别和最先进的生物工程 方法来开发一个定义的三维层流培养系统（目标1）。然后我们将使用细胞类型的钙成像 特定的遗传编码的钙传感器，以确定神经元放电率的相关性作为第一步 在3D体外层压系统中记录人类小脑神经元的电路特性（Aim 2）， 针对天然发育组织的转录组学分析（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器，然而没有可用的方案来在体外衍生它们。我们 将开发第一个分化方案，以获得这些神经元，并将它们纳入 层流模型（目标4）。这项研究将联合收割机结合米伦实验室在人类小脑方面的专业知识， 这是哈滕实验室在从hPSC产生人小脑神经元方面的发展， 适当分层的人类小脑回路，用于疾病建模和药物筛选。

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.