A Fully Patterned Human Neural Tube Model Using Microfluidics

项目摘要 基于微流控技术的全图形化人体神经管模型 脊椎动物中枢神经系统的发育始于神经管的形成 以及其区域模式，以沿着吻侧（R）-尾侧（C）和背侧（D）产生神经元亚型， 腹（V）轴。人类NT的区域模式是一个严格调节的过程，偏离这个过程， 导致神经发育障碍，并可能导致不同的神经和精神疾病后， 生活人类NT的区域模式仍然不完全理解，这是由于对人类NT的有限访问。 胚胎组织动物模型在理解人类中枢神经系统的发育方面起着重要作用 和相关的疾病。然而，它们在揭示发展的一些基本方面是有限的， 遗传学、病理学和人类特有的疾病机制。干细胞体外模型 人类神经系统发育，包括神经类器官和生物工程NT发育模型， 正在成为有前途的实验工具。然而，目前没有一种基于干细胞的神经 发育模型能够沿着3D管状结构中的两个正交轴沿着概括神经模式 几何形状，NT在体内图案化的标志。此外，现有的神经发育模型仅 概括了人类大脑或脊髓区域发育的某些方面，但不是两者都有。 在我们的初步研究中，我们成功地利用了发展潜力和自我- 人类多能干细胞（hPSC）的组织特性与微流体技术相结合， 这是第一个合成的、完全有图案的人类NT模型。利用这个微流体平台， 可以建立沿沿着两个正交轴的形态发生剂梯度， 微流体人类NT样结构沿着R-C和D-V轴，在脑样和脊髓样 地区这种微流体图案化的人类NT样结构表现出NT发育的许多特征， 包括管状几何形状、由神经元祖细胞包围的单个连续中心腔 包括HOX基因在内的典型R-C和D-V区域标记的模式化表达，以及 神经中胚层祖细胞和峡部组织者。因此，微流体人体的发展 NT样结构紧密模仿NT发育，首次提供体内样组织结构 具有一致的时空细胞分化和组织。 这项R 01研究的目标是开发这种令人兴奋的微流体人类NT样模型（Aim 1）， 利用其技术优势，研究不同外源性形态发生信号在神经细胞中的作用， 图案化（目标2和3）。将进行遗传扰动和谱系追踪试验，以研究人类 神经中胚层祖细胞发育（目标2）。在目标3中，我们进一步的目标是实现D-V模式化的人类。 NT样结构与前脑或脊髓的身份，并使用这些结构重演 区域间的细胞相互作用和兴奋抑制的相互作用在皮质发育。

Project Summary A Fully Patterned Human Neural Tube Model Using Microfluidics The development of vertebrate central nervous system (CNS) begins with the formation of neural tube (NT) and its regional patterning to generate neuronal subtypes along the rostral (R)-caudal (C) and dorsal (D)- ventral (V) axes. Regional patterning of the human NT is a tightly regulated process, deviation from which can result in neurodevelopmental disorders and may lead to distinct neurological and psychiatric diseases later in life. Regional patterning of the human NT remains incompletely understood due to limited access to human embryonic tissues. Animal models have been instrumental in understanding the development of human CNS and associated disorders. However, they are limited in revealing some fundamental aspects of development, genetics, pathology, and disease mechanisms that are unique to humans. Stem cell-based in vitro models of human nervous system development, including neural organoids and bioengineered NT development models, are emerging as promising experimental tools. However, none of the current stem cell-based neural development models is capable of recapitulating neural patterning along two orthogonal axes in a 3D tubular geometry, the hallmark of NT patterning in vivo. Furthermore, the existing neural development models only recapitulate certain aspects of the development of either human brain or spinal cord regions but not both. In our preliminary study, we have successfully leveraged the developmental potential and self- organizing property of human pluripotent stem cells (hPSCs) in conjunction with microfluidics to develop the first of its kind, synthetic, fully patterned human NT model. Using this microfluidic platform, exogenous morphogen gradients along two orthogonal axes can be established to achieve regional patterning of the microfluidic human NT-like structure along both the R-C and D-V axes, in both brain-like and spinal cord-like regions. This microfluidic patterned human NT-like structure exhibits many hallmarks of NT development, including a tubular geometry, a single continuous central lumen enclosing by neuronal progenitor cells, patterned expression of canonical R-C and D-V regional markers including HOX genes, and the emergence of neural mesodermal progenitors and the isthmic organizer. Thus, the development of the microfluidic human NT-like structure closely mimics NT development, offering for the first time an in vivo-like tissue architecture with consistent spatiotemporal cell differentiation and organization. The goal of this R01 research is to develop this exciting microfluidic human NT-like model (Aim 1) and leverage its technical advantages to study the roles of different exogenous morphogen signals in neural patterning (Aim 2 & 3). Genetic perturbations and lineage tracing assays will be conducted to study human neural mesodermal progenitor development (Aim 2). In Aim 3 we further aim to achieve D-V patterned human NT-like structures with either forebrain or spinal cord identities and use these structures to recapitulate interregional cellular interactions and excitation-to-inhibition interplays during cortical development.