A Fully Patterned Human Neural Tube Model Using Microfluidics

项目摘要 基于微流控技术的全模式化人体神经管模型 脊椎动物中枢神经系统的发育始于神经管的形成。 和它的区域模式，沿着吻侧(R)-尾侧(C)和背侧(D)-产生神经元亚型- 腹侧(V)轴。人类NT的区域模式是一个严格控制的过程，偏离这一过程可能会 导致神经发育障碍，并可能导致不同的神经和精神疾病 生活。由于接触人类的机会有限，人类NT的区域模式仍未完全了解 胚胎组织。动物模型有助于理解人类中枢神经系统的发育 以及相关的疾病。然而，它们在揭示发展的一些基本方面方面受到限制， 人类独有的遗传学、病理学和疾病机制。以干细胞为基础的体外模型 人类神经系统发育，包括神经有机体和生物工程NT发育模型， 正在成为一种很有前途的实验工具。然而，目前还没有一种基于干细胞的神经细胞 开发模型能够在3D管状结构中沿两个正交轴重现神经模式 几何学，活体NT图案化的标志。此外，现有的神经发育模型仅 概括一下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 人多能干细胞(HPSCs)的组织特性与微流控相结合开发 第一个，合成的，完全有图案的人类NT模型。利用这个微流控平台，外源 可以建立沿两个正交轴的形态梯度，以实现区域图案 沿R-C轴和D-V轴的微流控人NT样结构，在类脑和类脊髓中 地区。这种微流控图案化的人NT样结构显示出NT发展的许多特征， 包括管状结构，单个连续的中央管腔被神经元前体细胞包围， 包括HOX基因在内的规范R-C和D-V区域标记的模式表达以及 神经中胚层前体细胞和峡部组织者。因此，微流控人类的发展 类NT结构与NT的发育密切相关，首次提供了体内类组织结构 具有一致的时空细胞分化和组织。 这项R01研究的目标是开发这种令人兴奋的微流控类人NT模型(目标1)和 利用其技术优势研究不同外源性形态信号在神经中的作用 构图(目标2和3)。将进行遗传扰动和谱系追踪分析来研究人类 神经中胚层前体细胞发育(目标2)。在目标3中，我们的进一步目标是实现D-V模式的人类 具有前脑或脊髓特征的NT样结构，并使用这些结构来概括 大脑皮层发育过程中的区域间细胞相互作用和兴奋抑制相互作用。

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.