Growth, Differentiation and Genetic Alteration of Human ES Cells

描述(申请人提供)：人类胚胎干细胞(HES)在培养中分化为多个谱系，为研究人类发育的早期阶段和产生大量特定类型的细胞用于细胞治疗提供了前所未有的机会。实现这一潜力的关键一步是提供支持人类胚胎干细胞研究的基础设施。挑战包括细胞的生长和分化必须变得标准化，以使来自不同学科的研究人员能够轻松地利用这一体外系统。这些努力的中心将是一个核心设施，旨在开发HES细胞技术，并支持和培训西奈山的研究社区。此外，我们还将讨论与HES细胞生物学和HES细胞技术相关的基本问题。为了充分开发HES细胞的潜力，重要的是要开发出允许引入影响其分化的基因以及那些可以防止其后代在移植到受体后发生排斥反应的基因的方法。我们将通过分别使用腺相关病毒(AAV)和噬菌体整合酶来开发对HES细胞进行位置特异性遗传修饰的方法来解决这些问题。我们将利用初步研究中记录的HES细胞的红系分化潜力，解决与人类珠蛋白基因表达调控机制有关的问题。我们将研究FOXO转录因子和氧化应激在HES细胞生长和红系特异性分化中的作用。这些研究的结果将提供多个HES细胞系的生长和分化潜力的比较、这些细胞的位置特异性遗传修饰的新方法、对FOXO蛋白在HES细胞生长和分化中的作用的洞察以及关于人类红系发育早期阶段珠蛋白基因转换调控的新信息。我们建议使用以下ES细胞系：ES01、ES02、ES03、ES04、ES05、ES06、T3-03、TE-04、TE-06、UC01、UC06、MI01、BG01、BG02、BG03、WA01、WA07、WA09、WA13、WA14。

DESCRIPTION (provided by applicant): The differentiation of human embryonic stem (hES) cells to multiple lineages in culture offers unprecedented opportunities to investigate the earliest stages of human development and to generate large numbers of specific cell types for cell-based therapy. An essential step toward realizing this potential is the availability of an infrastructure that supports human ES cell research. Challenges include that the growth and differentiation of cells must become standardized to enable investigators from different disciplines to easily utilize this in vitro system. At the center of these efforts will be a Core facility that is designed to develop hES cell technologies and to support and train the research community at Mount Sinai. In addition,we will address fundamental questions relating to hES cell biology and hES cell-based technologies. To fully exploit the potential of hES cells, it is important to develop approaches allowing for the introduction of genes that can impact their differentiation as well as those that can prevent the rejection of their progeny, following transplantation into recipients. We will address these issues by developing approaches for site-specific genetic modification of the hES cells, through the use of adeno-associated viruses (AAV) and bacteriophage integrases, respectively. We will take advantage of the erythroid differentiation potential of hES cells as documented in the Preliminary Studies, to address questions relating to the mechanisms of the regulation of human globin gene expression. We will investigate the roles of FOXO transcription factors and oxidative stress on hES cell growth and erythroid specific differentiation. The outcome of these studies will provide a comparison of the growth and differentiation potential of multiple hES cell lines, new approaches for site-specific genetic modification of these cells, insights into the role of FOXO proteins on hES cell growth and differentiation and new information on the regulation of globin gene switching during the early stages of human erythroid development. We propose to use the following ES cells lines: ES01, ES02, ES03, ES04, ES05, ES06, T3-03, TE-04, TE-06, UC01, UC06, MI01, BG01, BG02, BG03, WA01, WA07, WA09, WA13, WA14.