Effects of Lead on Neuronal Differentiation in Human Embryonic Stem Cells

描述（由申请人提供）：本提案的主要目标是确定铅（Pb）暴露对人胚胎干细胞（hESC）神经和神经元分化的表观遗传和形态学影响。铅中毒在美国仍然是一个严重的健康问题。在全国范围内，约有25万名1-5岁儿童的血铅水平超过每分升血液10微克铅（目前CDC的行动水平）。众所周知，铅暴露会导致儿童的神经系统问题，如注意力缺陷障碍和精细运动协调的丧失，但铅诱导的神经毒性机制和铅如何导致神经缺陷的表观遗传基础尚不清楚。在人类中，大脑在受孕后大约三周开始形成，并继续发育和成熟到生命的最初几年。早期脑发育是一个非常敏感的时期，在此期间暴露于有毒物质，如铅，可以影响进一步的脑发育和成熟。胚胎干细胞在体外分化为神经前体细胞（NPC）重演了人类大脑发育的早期阶段，为了解铅对人类大脑形成的影响提供了独特的机会。这里提出的实验的意义是，他们将允许一个独特的外观铅的分化和成熟的NPC的影响，并在与铅暴露相关的全球DNA甲基化模式的潜在变化。这些差异将揭示铅神经毒性的新机制，也将建立一个试探性的表观遗传纲要，可用作铅暴露的指标。NPC衍生的神经元将用于了解导致铅诱导的神经系统改变的基因组DNA CpG甲基化状态的微小变化。对来自hESC的NPC的初步研究表明，Pb是一种表观遗传干扰物，并导致全球DNA甲基化水平的快速（24小时内）重编程。该假说是铅引起特定CpG位点的DNA甲基化的变化，并且hESC和NPC将具有与hESC衍生的神经前体和神经元的形态学、分子和功能变化相关的表观遗传特征的变化。提出了三个目标：目标1。确定慢性铅暴露对人胚胎干细胞来源的NPC表观遗传特征的影响。目标二。研究铅介导的候选基因DNA甲基化变化对神经元分化和基因表达的影响。成果：该项目将使人们了解铅暴露如何导致神经损伤，以及DNA甲基化结构如何导致铅的神经毒性作用。这将是一个原型研究的毒理基因组学的新兴领域的应用，人类大脑的发展，其中的概念可以用来设计各种致畸毒性剂的其他类型的细胞系以及体外测定。

DESCRIPTION (provided by applicant): The principal goal of this proposal is to identify the epigenetic and morphological effects of lead (Pb) exposure on the neural and neuronal differentiation of human embryonic stem cells (hESCs). Lead poisoning remains a significant health problem in the United States. Nationally, about 250,000 children aged 1-5 years have blood lead levels greater than 10 ¿g of lead per deciliter of blood (current CDC action level). It s known that Pb exposure causes neurological problems in children, such as attention deficit disorder and loss of fine-motor coordination, but the mechanisms of Pb-induced neurotoxicity and the epigenetic basis for how Pb causes neurological deficits is not known. In humans, the brain starts forming at about three weeks after conception and continues to develop and mature well into the first years of life. Early brain development is a very sensitive time during which exposure to toxicants such as Pb can affect further brain development and maturation. In vitro differentiation of embryonic stem cells into neural precursor cells (NPCs) recapitulates the early stages of brain development in humans, giving one the unique opportunity to understand the effects of Pb on the formation of the human brain. The significance of the experiments proposed here is that they will allow one to get a unique look at the impact of Pb on the differentiation an the maturation of NPCs and at the potential changes in the global DNA methylation patterns associated with Pb exposure. These differences will reveal novel mechanisms of Pb neurotoxicity and will also establish a tentative epigenetic outline that can be used as an index for Pb exposure. NPC-derived neurons will be used to understand even subtle changes in genomic DNA CpG methylation status leading to the neurological alterations induced by Pb. Preliminary studies with NPCs derived from hESCs indicate that Pb is an epigenetic disruptor and causes rapid (within 24 hours) reprogramming of global DNA methylation levels. The hypothesis is that Pb causes changes in DNA methylation at specific CpG sites and that hESCs and NPCs will have changes in epigenetic signature that correlate with morphological, molecular, and functional changes in the hESC-derived neural precursors and neurons. Three aims are proposed: Aim 1. Determine the effects of chronic Pb exposure on the epigenetic profile of hESC- derived NPCs. Aim 2. Examine the influence that Pb-mediated DNA methylation changes in candidate genes have on neuronal differentiation and gene expression. Outcomes: This project will allow one to understand how exposure to Pb leads to neural impairment and how the architecture of a DNA methylome leads to the neurotoxic effects of Pb. It will be a prototype study for the application of the emerging field of toxicogenomics to human brain development, where the concepts could be used to devise in vitro assays for a variety of teratogenic toxic agents for other types of cell lineages as well.