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学控制 人类胚胎干细胞(HESCs)因其良好的自我更新能力而引起公众的极大兴趣。 能力和分化潜能，是组织修复的理想选择。然而，许多人 利用具有不同人类遗传背景的胚胎建立了可用的hESC系 培养程序千差万别。这种差异可能会对遗传稳定性产生巨大影响， 表观遗传学，并最终影响hESCs的细胞特性，因此影响这些细胞在 再生医学。为了开始描述不同hESC系之间的差异，我们有 研究表明，虽然其中一个hESC系(HSF1系)产生的神经元主要来自前脑，但 其他(HSF6)主要产生起源于中/后脑和脊髓的神经元， 谷氨酸能、多巴胺能、血清能和胆碱能神经递质特性。这些观察结果 提示这两个hESC系在胚胎干细胞发育阶段就已经存在谱系分化偏向。在这里我们 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 有三个具体目的：目标1，确定四种神经元亚型的分化特性 独立的hESC系，即除了HSF1和HSF6之外的H1和H9；目标2，也使用mRNA 作为microRNA表达阵列分析、定量RT-PCR和Western blotting方法的评估 这四个hESC系在ES细胞期和ESC期的基因表达谱差异 在胚胎干细胞转化为神经干细胞/祖细胞以及有丝分裂后神经元后；以及目标3，至 检查这四个不同的hESC株在基因组和表观基因组(全基因组)上是否存在差异 DNA甲基化和组蛋白修饰模式)在ESC和NPC阶段。通过定义机制 通过这些常用的hESC系优先分化为不同亚型的神经元， 我们的研究将为未来更多hESC品系的鉴定奠定基础并创造方法 评估它们的分化潜能/偏向，这对未来人类胚胎干细胞的任何使用都将非常有价值 再生医学，包括神经干细胞治疗和建立新的神经学 使用hESC来源的神经元的疾病模型，因为区域特异性是许多神经疾病的关键。

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.