Effects of Lead on Neuronal Differentiation in Human Embryonic Stem Cells

描述(由申请人提供)：这项建议的主要目标是确定铅暴露对人类胚胎干细胞(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25万名1-5岁儿童的血铅水平超过每分升血铅10？克(当前疾病控制与预防中心的行动水平)。S知道，铅暴露会导致儿童的神经问题，如注意力缺陷障碍和精细运动协调能力丧失，但铅导致神经毒性的机制和铅如何导致神经功能障碍的表观遗传学基础尚不清楚。在人类中，大脑在受孕后大约三周开始形成，并在生命的头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，暴露在铅等有毒物质中会影响大脑的进一步发育和成熟。胚胎干细胞在体外分化为神经前体细胞(NPC)的过程概括了人类大脑发育的早期阶段，这给了人们了解铅对人脑形成的影响的独特机会。这里提出的实验的意义在于，它们将使人们能够独特地观察铅对鼻咽癌细胞分化和成熟的影响，以及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，并将建立一个初步的表观遗传学轮廓，可用作铅暴露的指标。鼻咽癌来源的神经元将被用来了解基因组DNA CpG甲基化状态的细微变化，从而导致铅引起的神经改变。对来自hESCs的npC的初步研究表明，铅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)重新编程。假设铅引起特定CpG位点DNA甲基化的改变，hESCs和NPC的表观遗传特征将发生变化，这与hESC衍生的神经前体和神经元的形态、分子和功能变化有关。目的1.确定慢性铅暴露对人胚胎干细胞来源的鼻咽癌细胞表观遗传学的影响。目的2.检测铅介导的候选基因DNA甲基化变化对神经元分化和基因表达的影响。结果：这个项目将使人们了解铅暴露是如何导致神经损伤的，以及DNA甲基组的结构如何导致铅的神经毒性效应。这将是将毒素基因组学这一新兴领域应用于人脑发育的原型研究，其中这些概念也可以用于为其他类型的细胞系设计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.