Applied Stem Cell Glycomics and Glycoproteomics

该提案的具体目的是检验核心假设，即细胞聚糖的表达谱 作为唯一和敏感的指纹，能够定义特定的细胞类型或报告细胞状态 在培养过程中。用于纯化特定细胞亚群的标记物和试剂的开发是一项重要的技术。 高度优先推进hESC的治疗和研究用途。尽管他们占主导地位 由于糖缀合物定位于细胞表面，因此糖缀合物在此类用途中的靶向不足。这一缺陷 反映了缺乏广泛可用的技术或专门知识来表征这些复杂的 大分子我们开发了一套复杂的定量分析工具， 在前所未有的深度，聚糖，糖蛋白和糖脂分离少量的材料。 我们已经成功地应用这些工具来评估聚糖表达的变化， 分化的小鼠胚胎干细胞，现在提出应用我们开发的糖和糖蛋白质组学 技术对人类ESC生物学具有根本重要性的问题。我们的工具将允许我们筛选 对于由于非整倍性而在整体上或在特定分子上发生的糖基化的变化， 条件、传代次数、细胞谱系和分化。在具体目标1中，我们将定义糖组学 hESC系的指纹，以及向定形内胚层、中胚层和神经内胚层分化的hESC。 前体人口。还将评估非整倍性和细胞培养条件的影响。在 具体目标2，我们将定位确定的聚糖标志物，其定义hESC或分化的细胞命运， 蛋白质和脂质核心上的特定位点。在具体目标3中，我们将生成用于检测和丰富的工具 hESC和分化细胞的亚群，其基于特定糖组蛋白和/或 糖蛋白质组学标记这些目标的完成不仅将提供表征的新方法， 定义和富集特定的细胞类型，但也将为探索糖基化在 人胚胎干细胞自我更新和分化。

The specific aims of this proposal test the central hypothesis that expression profiles of cellular glycans can serve as unique and sensitive fingerprints, capable of defining specific cell types or reporting cell status during culture. The development of markers and reagents for purifying specific sub-populations of cells is a high priority for advancing the therapeutic and investigational uses of hESCs. Despite their predominant localization to the cell surface, glycoconjugates have been under targeted for such uses. This deficiency reflects the lack of broadly accessible techniques or expertise for characterizing these complex macromolecules. We have developed a sophisticated suite of tools for quantitatively analyzing, with unprecedented depth, the glycans, glycoproteins, and glycolipids isolated from small amounts of material. We have successfully applied these tools to assess glycan expression changes associated with the differentiation of mouse ESCs and now propose to apply our developed glycomic and glycoproteomic techniques to questions of fundamental importance for human ESC biology. Our tools will allow us to screen for changes in glycosylation that occur globally or on specific molecules as a result of aneuploidy, cell culture conditions, passage number, cell pedigree, and differentiation. In Specific Aim 1, we will define the glycomic fingerprints of hESC lines, and hESCs differentiated toward definitive endoderm, mesoderm, and neural precursor populations. The impact of aneuploidy and cell culture conditions will also be assessed. In Specific Aim 2, we will map identified glycan markers, which define hESCs or differentiated cell fates, to specific sites on proteins and lipid cores. In Specific Aim 3, we will generate tools for detecting and enriching sub-populations of hESCs and differentiated cells based on the expression of specific glycomic and/or glycoproteomic markers. Completion of these aims will not only provide novel approaches for characterizing, defining, and enriching specific cell types but will also provide a basis for exploring the role of glycosylation in hESC self renewal and differentiation.