Applied Stem Cell Glycomics and Glycoproteomics

应用干细胞糖组学和糖蛋白质组学

• 批准号: 8382722
• 负责人: MICHAEL TIEMEYER
• 金额: $ 31.41万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8382722
• 关键词: Aneuploidy; Antibodies; Appearance; Biology; Cell Count; Cell Culture Techniques; Cell Differentiation process; Cell Separation; Cell surface; Cells; Ceramides; Chemicals; Complex; Cultured Cells; Detection; Development; Embryonic Development; Endoderm; Fatty Acids; Fingerprint; Glycoconjugates; Glycolipids; Glycoproteins; Glycosphingolipids; Goals; Heterogeneity; Kinetics; Label; Lectin; Link; Lipids; Maps; Mediating; Mesoderm; Metabolic; Methods; Minor; Molecular Profiling; Mus; Peptides; Polysaccharides; Population; Proteins; Reagent; Reporting; Resolution; Role; Sensitivity and Specificity; Site; Sphingosine; Stem cells; Structure; Techniques; Testing; Therapeutic; Transcriptional Regulation; base; candidate marker; cell type; combinatorial; genetic pedigree; glycosylation; human embryonic stem cell; instrumentation; interest; macromolecule; nerve stem cell; novel strategies; polypeptide; relating to nervous system; self-renewal; stem cell differentiation; tool

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.

该提案的具体目的检验了中心假设，即细胞聚糖的表达谱可以 用作独特而敏感的指纹，能够定义特定的细胞类型或报告细胞状态 在文化期间。用于纯化细胞特定亚种群的标记和试剂的开发是 促进hESC的治疗和研究用途的高度优先级。尽管他们主要是 在细胞表面的定位，糖缀合物的靶向为这种用途。这种缺陷 反映了缺乏可访问的技术或专业知识来表征这些复杂的技术 大分子。我们已经开发了一套复杂的工具，用于定量分析，并使用 从少量材料中分离出来的前所未有的深度，聚糖，糖蛋白和糖脂。 我们已经成功地应用了这些工具来评估与 小鼠ESC的区分，现在建议应用我们开发的糖蛋白酶 对于人类ESC生物学的基本重要性问题的技术。我们的工具将使我们能够筛选 对于全球或特定分子的糖基化变化，细胞培养 条件，通过数，细胞谱和分化。在特定的目标1中，我们将定义糖胶质 hESC线的指纹和hESC与确定的内胚层，中胚层和神经区分开 前体人群。非整倍性和细胞培养条件的影响也将得到评估。在 特定的目标2，我们将绘制确定的确定的聚糖标记，将hESC或分化的​​细胞命运定义为 蛋白质和脂质核的特定位点。在特定目标3中，我们将生成用于检测和丰富的工具 基于特定糖基因和/或 糖蛋白酶标记。这些目标的完成不仅会提供表征的新方法， 定义并丰富特定的细胞类型，但也将为探索糖基化的作用提供基础 hESC自我更新和差异化。