A Fluorescence-Based High-Throughput Platform for Glycotyping the Hematopoietic Cell Lineage

基于荧光的造血细胞谱系糖分型高通量平台

• 批准号: 10248374
• 负责人: Hannes Erich Buelow
• 金额: $ 42.75万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248374
• 关键词: Address; Affinity; Antibodies; Binding; Biological Assay; Biological Markers; Blood; Blood Cells; Cell Adhesion; Cell Differentiation process; Cell Line; Cell Lineage; Cell surface; Cells; Characteristics; Chimeric Proteins; Communities; Complement; Complex; Coupled; Data; Development; Diagnostic; Differentiation Antigens; Disease; Engineering; Epitopes; Erythroid Cells; Evaluation; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; Future; Glycosaminoglycans; Goals; Growth; Hematopoiesis; Hematopoietic; Heparitin Sulfate; Immunoglobulin Fragments; Immunoglobulin G; Label; Laboratories; Libraries; Lymphoid Cell; Malignant Neoplasms; Methods; Modification; Molecular; Molecular Profiling; Mus; Myeloid Cells; Nature; Oligosaccharides; Organism; Pathology; Pattern; Physiology; Polysaccharides; Population; Positioning Attribute; Production; Proteins; Protocols documentation; Reagent; Research; Resolution; Scheme; Sorting - Cell Movement; Standardization; Structure; Surface; System; Techniques; Technology; Testing; Therapeutic; Tissues; Transplantation; Weight; analytical method; base; cell motility; cell type; computerized tools; experimental study; in vivo; insight; member; novel; novel diagnostics; novel therapeutic intervention; polysulfated glycosaminoglycan; population based; progenitor; software development; sortase; stem; tool

PIs: Buelow – Steidl Project Summary The surface characteristics of cells in multicellular organisms are a direct reflection of their function. Sensitive methods for detecting and defining novel surface characteristics of cells offer great opportunities for gaining new mechanistic insights and developing new therapeutic strategies. Heparan sulfate (HS) glycans are ubiquitous on the cell surface of metazoans and are expressed with extraordinary temporal and spatial resolution. Since HS are amongst the most diverse molecules in nature, as a result of complex modification patterns of the glycans, they could potentially provide the means to strongly discriminate between different cell and tissue types. However, current analytical methods for HS glycan structure determination are expensive, technically challenging and often low resolution, limiting their discriminatory power and widespread use. The development of novel tools to define and exploit the structural characteristics of HS on the surface of cells could open new avenues with important diagnostic and, possibly, therapeutic potential. We will develop a high- throughput, multiplexed fluorescence-based platform to define the HS surface characteristics using a set of HS-specific single chain variable fragment (scFvs) antibodies. We will first engineer the set of HS-specific single-chain variable fragment antibodies with various immunotags as well as directly label them with fluorescent tags using Sortase based approaches. Next, we will systematically determine the HS motifs that are recognized by the scFv antibodies using a combination of microarrays with defined heparan sulfate oligosaccharides and novel computational tools, which we will develop to define the HS motifs recognized by these reagents. Finally, in proof of principle experiments, we will apply our tool box of immuno reagents against defined HS motifs as an orthogonal system to the well-established cluster of differentiation (CD) system to characterize and define hematopoietic cell lineages. We will leverage our laboratories' unique capabilities in protein production and hematopoiesis research to generate, test, and disseminate the fluorescently labeled scFvs reagents and protocols to the research community. We anticipate that our robust, efficient and economical assays will lead to more widespread and standardized analyses of HS characteristics, which will lay the foundation for the development of novel diagnostic and therapeutic approaches in the blood and other systems in the future.

PI：Buelow – Steidl 项目概要 多细胞生物中细胞的表面特征是其功能的直接反映。 用于检测和定义细胞新表面特征的灵敏方法为 获得新的机制见解并开发新的硫酸乙酰肝素 (HS) 聚糖。 普遍存在于后生动物的细胞表面，并以非凡的时间和空间表达 由于 HS 是自然界中最多样化的分子之一，这是经过复杂修饰的结果。 聚糖的模式，它们可能提供强烈区分不同细胞的方法 然而，目前用于 HS 聚糖结构测定的分析方法非常昂贵， 技术上具有挑战性，而且分辨率通常较低，限制了其歧视性和广泛使用。 开发新工具来定义和利用细胞表面 HS 的结构特征 可以开辟具有重要诊断潜力和治疗潜力的新途径。 吞吐量，基于多重荧光的平台，使用一组定义 HS 表面特征 HS 特异性单链可变片段 (scFv) 抗体 我们将首先设计一组 HS 特异性抗体。 带有各种免疫标签的单链可变片段抗体以及直接标记它们 接下来，我们肯定会确定使用基于分选酶的方法的 HS 基序。 使用含有特定硫酸乙酰肝素的微阵列组合可被 scFv 抗体识别 寡糖和新颖的计算工具，我们将开发这些工具来定义 HS 基序 最后，在原理实验中，我们将应用我们的免疫试剂工具箱来对抗这些试剂。 将 HS 基序定义为与成熟的分化簇 (CD) 系统正交的系统 我们将利用我们实验室的独特能力来表征和定义造血细胞谱系。 蛋白质生产和造血研究，以生成、测试和传播荧光标记的 我们期望为研究界提供强大、高效和的 scFv 试剂和方案。 经济的检测将导致对 HS 特性进行更广泛和标准化的分析，这将 为血液和其他领域新型诊断和治疗方法的发展奠定基础 未来的系统。