Epigenetic Control of Neurogenesis in Different hESC lines

项目1：不同hESC系中神经发生的表观遗传控制 人类胚胎干细胞（human embryonic stem cells，hESCs）因其具有良好的自我再生能力而受到广泛关注。 具有理想的组织修复能力和分化潜力。然而，许多 使用具有不同人类遗传背景的胚胎建立了可用的hESC系， 广泛变化的培养程序。这种差异可能对遗传稳定性产生巨大影响， 表观遗传，并最终hESC的细胞特性，并因此影响这些细胞在 再生医学为了开始表征各种hESC系之间的差异，我们 显示虽然hESC系之一（HSF 1系）产生主要来自前脑的神经元， 其他（HSF 6）主要产生具有GABA能的中脑/后脑和脊髓起源的神经元， 多巴胺能、多巴胺能、血清素能和胆碱能神经递质特性。这些观察结果 提示这两个hESC系甚至在ESC阶段已经具有谱系分化偏好。这里我们 建议研究不同hESC系之间潜在的表观遗传预编程事件的性质 目的1、确定四种神经元亚型的分化特性， 目的2：利用mRNA， 作为microRNA表达阵列分析，定量RT-PCR和Western印迹方法来评估 这四种hESC系在ES细胞阶段和ES细胞阶段基因表达谱的差异， 在ES细胞转化为神经干/祖细胞以及有丝分裂后神经元之后;以及Aim 3，To 检查这四种不同的hESC系在它们的基因组和表观基因组（全基因组）上是否不同 DNA甲基化和组蛋白修饰模式）。通过定义机制， 这些常用的hESC系通过其优先分化成不同的神经元亚型， 我们的研究将为将来鉴定更多的hESC细胞系奠定基础并创造方法， 评估它们的分化潜力/偏差，这对于将来将hESC用于 再生医学，包括神经干细胞治疗和建立新的神经系统 使用hESC衍生的神经元的疾病模型，因为区域特异性是许多神经障碍的关键。

Project 1: Epigenetic control of neurogenesis in different hESC lines Human embryonic stem cells (hESCs) have garnered tremendous public interest for their promising selfrenewal capacities and differentiation potentials which are ideal for tissue repair. However, the many available hESC lines were established using embryos with different human genetic backgrounds and with widely varying culturing procedures. Such differences could have a huge influence on the genetic stability, epigenetic, and ultimately cellular properties of hESCs, and therefore influence the usage of these cells in regenerative medicine. To begin to characterize the differences between the various hESC lines, we have shown that while one of the hESC lines (the HSF1 line) produces neurons primarily of forebrain origin, the other (HSF6) primarily generates neurons of mid-/hind-brain and spinal cord origins with GABAergic, glutamatergic, dopaminergic, seratonergic, and cholinergic neuretransmitter traits. These observations suggest that these two hESC lines already have lineage differentiation bias even at the ESC stage. Here we propose to study the nature of the potential epigenetic pre-programing events among different hESC lines with three specific aims: Aim 1, To determine the neuronal subtype differentiation properties of four independent hESC lines, namely H1 and H9 in addition to HSF1 and HSF6; Aim 2, To employ mRNA as well as microRNA expression array analyses, quantitative RT-PCR, and Western blotting methods to evaluate the differences in gene expression profiles between these four hESC lines, both at the ES cell stage and after ES cells are converted into neural stem/progenitor cells as well as post-mitotic neurons; and Aim 3, To examine whether these four different hESC lines differ in their genomes and epigenomes (genome-wide DMA methylation and histone modification patterns) at ESC and NPC stages. By defining the mechanisms by which these commmonly used hESC lines preferentially diferentiate into different subtypes of neurons, our studies will set the stage and create methods for future characterization of additional hESC lines to assess their differentiation potentials/bias, which will be extremely valuable for any future use of hESCs in regenerative medicine, inlcuding neural stem cell therapy and the establishment of novel neurological disease models using hESC-derived neurons since regional specificity is key to many neurological disorders.