利用猕猴胚胎干细胞神经分化过程建立灵长类神经管缺陷体外三维模型的研究

Neural tube defects (NTDs) are one of the most common congenital malformations with a worldwide prevalence of more than 1 per 1000 births. Studies of the etiology of NTDs have involved animal models such as Xenopus laevis, chickens, mice and zebrafish. However, due to disparities in anatomy and neurogenesis patterns between these models and primates, mechanisms underlying human NTDs remain elusive. Neural differentiation from ESCs is a promising approach in developmental neurobiology, by providing models of neural development. Remarkably, researchers have generated neural tube-like (rosette) structures that are definitive neuroepithelia from primate ES cells. These rosette neural stem cells (R-NSCs) exhibit plasticity toward generating a broad range of neuronal types in response to appropriate developmental signals. Therefore, this neural differentiation system is helpful for exploring mechanism of normal or abnormal neural development in vitro. In our previous study, rhesus monkey ES cells (RESCs) were differentiated into R-NSCs, and FA concentrations were controlled either by using FA-free basic medium or by adding methotrexate (MTX), the primary FA antagonist. Neural conversion, neuronal differentiation, cell proliferation and apoptosis were measured. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1 or Pax-6, among neural progenitors obtained from different FA concentration treatment and with the FA antagonist-methotrexate (MTX). However, FA depletion decreased cell proliferation, affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that ESCs system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates. In this project, we are willing to establish a three-dimensional (3D) neural differentiation system to mimic in vivo neural development. The possible methods to set up the 3D system include persistently suspending EBs, 3D culture cells in ECM such as Matrigel, culture R-NSCs as neural sphere, or use similar method as Studer's lab to simulate time-space pattern of neurogenesis. The 3D system will be used to explore neural tube closure, polarity formation and neural differentiation potential in rhesus monkey. The aim of our study is to found an in vitro 3D model of primate neural tube defects.

神经管缺陷是最常见的出生缺陷之一，发病率高于千分之一。人类对该疾病的研究主要利用爪蟾、鸡、小鼠和斑马鱼等动物模型，目前还没有该疾病的灵长类动物模型，因而人类神经管缺陷的发病机理还不清楚。灵长类胚胎干细胞向神经分化的过程与体内胚胎发育具有极其类似的时间模式，可能是研究体内神经发育的较好途径。我们在猕猴胚胎干细胞体外分化的研究中发现，叶酸缺乏会影响神经管样神经干细胞（R-NSCs）的花环结构的形成，还会影响神经元的分化能力。本项目拟在前期工作基础上通过EB持续悬浮培养、ECM三维培养、神经球培养法及Studer时空模式法等途径建立三维的神经分化体系，模拟体内空间结构研究叶酸对神经管闭合、极性形成和神经分化潜能等方面的影响，为深入研究神经管缺陷的发病机理建立体外立体模型，以对人类神经管缺陷的发病机理提供可靠证据。

神经管缺陷（Neural tube defects，NTDs）是由于胚胎早期发育阶段神经管闭合不全造成的严重的先天性畸形。该病的起因非常复杂，包括环境、营养和遗传等因素以及这些因素之间的相互作用。该病全球发病率约为千分之一，在我国甚至高达千分之五左右，严重影响优生优育及人口健康。研究该病的发病机理、探索预防及治疗方案对提高我国人民健康水平至关重要。现已发现，补充叶酸可以有效预防70%左右的NTDs的发生，说明环境及营养因素是导致NTDs的重要因素。然而，叶酸补充不能防止全部NTDs的发生，提示遗传缺陷是另一个导致NTDs发生的重要因素。人类神经管在胚胎发育的第28 天左右闭合，而多数情况下，母亲此时基本还不知道自己已经怀孕，因此对预防和治疗此类疾病造成困难，也不利于开展早期体内研究。灵长类胚胎干细胞（ESCs）向神经分化的过程与体内胚胎发育具有极其类似的时间模式，因此，利用灵长类ESCs 在体外模拟情况下研究体内胚胎发育过程意义重大。由于体内神经管是一个具有三维结构、典型空间极性的组织，而体外分化一般在培养皿中进行，很难模拟体内环境，这给我们试图通过体外分化研究神经发育带来困难。.本项目计划目标为建立体外三维培养体系，研究叶酸在神经管发育中的作用，体外条件下探索神经管缺陷的发病机理，为建立体内外神经管缺陷动物模型提供理论及技术依据。现已建立并优化了拟胚体三维体外分化及培养体系，通过转录组分析探讨了叶酸代谢通路在神经分化过程中的可能作用途径，并在此基础上筛选了在神经管闭合中发挥重要调控作用的分子，将通过基因修饰进一步研究神经管发育中核心调控基因在灵长类神经管畸形中的分子调控机制。项目实施期间共发表SCI论文6篇，中文核心期刊论文1篇，培养博硕士研究生及专业技术人员10余人。项目执行期间积极开展学术交流，共参加国内外学术会议12次。