Applied Stem Cell Glycomics and Glycoproteomics

这项提案的具体目的是检验中心假设，即细胞多糖的表达谱可以 作为唯一且敏感的指纹，能够定义特定的细胞类型或报告细胞状态 在文化过程中。用于纯化特定细胞亚群的标记和试剂的发展是一种 推进人类胚胎干细胞治疗和研究用途的高度优先事项。尽管他们占主导地位 为了定位到细胞表面，糖偶联物已经成为这类用途的靶标。这一缺陷 反映了缺乏可广泛使用的技术或专业知识来表征这些复杂的 大分子。我们已经开发了一套复杂的定量分析工具， 前所未有的深度，从少量物质中分离出多糖、糖蛋白和糖脂。 我们已经成功地应用这些工具来评估与糖链相关的糖链表达变化 小鼠胚胎干细胞的分化，并建议应用我们开发的糖和糖蛋白质组 对人类胚胎干细胞生物学具有根本重要性的问题的技术。我们的工具将允许我们筛选 对于非整倍体导致的整体或特定分子上糖基化的变化，细胞培养 条件、传代次数、细胞谱系和分化。在具体目标1中，我们将定义血糖 HESC系的指纹图谱和hESCs分化为最终的内胚层、中胚层和神经 前身种群。还将评估非整倍体和细胞培养条件的影响。在……里面 具体目标2，我们将识别的定义hESCs或分化细胞命运的糖链标记映射到 蛋白质和脂核上的特定位置。在具体目标3中，我们将生成用于检测和丰富 基于特定血糖和/或表达的hESCs和分化细胞亚群 糖蛋白组学标记。完成这些目标不仅将提供表征的新方法， 定义和丰富特定的细胞类型，但也将提供一个基础，探索糖基化在 HESC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.