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（hESCs）神经和神经元分化的表观遗传学和形态学影响。在美国，铅中毒仍然是一个严重的健康问题。在全国范围内，约有250,000名1-5岁儿童的血铅水平超过每分升血铅10克（目前的疾病预防控制中心行动水平）。已知铅暴露会导致儿童神经系统问题，如注意缺陷障碍和精细运动协调能力丧失，但铅诱导的神经毒性机制和铅导致神经功能障碍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，并在生命的最初几年继续发育和成熟。早期大脑发育是一个非常敏感的时期，在此期间暴露于铅等有毒物质会影响大脑的进一步发育和成熟。胚胎干细胞向神经前体细胞（npc）的体外分化概括了人类大脑发育的早期阶段，为了解铅对人脑形成的影响提供了独特的机会。这里提出的实验的意义在于，它们将使人们能够以独特的方式观察Pb对npc分化和成熟的影响，以及与Pb暴露相关的全球DNA甲基化模式的潜在变化。这些差异将揭示新的铅神经毒性机制，并将建立一个初步的表观遗传学大纲，可作为铅暴露的指标。npc衍生的神经元将用于理解基因组DNA CpG甲基化状态的细微变化，从而导致Pb诱导的神经系统改变。对hESCs衍生的npc的初步研究表明，Pb是一种表观遗传干扰物，可导致全球DNA甲基化水平的快速（24小时内）重编程。假设Pb引起特定CpG位点DNA甲基化的变化，hESCs和npc将发生表观遗传特征的变化，这些变化与hesc衍生的神经前体和神经元的形态、分子和功能变化相关。提出了三个目标：目标1。确定慢性铅暴露对hESC衍生npc表观遗传谱的影响。目标2。探讨铅介导的候选基因DNA甲基化变化对神经元分化和基因表达的影响。结果：该项目将使人们了解暴露于铅如何导致神经损伤，以及DNA甲基化组的结构如何导致铅的神经毒性作用。这将是将毒物基因组学这一新兴领域应用于人类大脑发育的一个原型研究，其中的概念也可用于设计用于其他类型细胞谱系的各种致畸毒性物质的体外分析。

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.