Novel Method for Profiling Megakaryocytes

分析巨核细胞的新方法

• 批准号: 7481803
• 负责人: Yajuan Jiang
• 金额: $ 14.11万
• 依托单位: ONE CELL SYSTEMS, INC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481803
• 关键词: Biological Assay; Blood Cells; Blood Platelets; CD34 gene; Cell Differentiation process; Cell Proliferation; Cell Separation; Cell surface; Cells; Cellular Morphology; Cellular biology; Colony-forming units; Control Groups; Cultured Cells; Development; Drug Formulations; Encapsulated; Enzyme-Linked Immunosorbent Assay; Evaluation; Flow Cytometry; Frequencies; Gel; Gene Expression; Giemsa stain; Government; Hematopoietic; Hematopoietic stem cells; Human; IL8 gene; In Vitro; Influentials; Interleukin-3; Legal patent; Marrow; Measures; Megakaryocytes; Methods; Microspheres; Mononuclear; Numbers; Persons; Phase; Phenotype; Play; Population; Process; Production; Proteins; Public Health; Purpose; Research; Role; Sepharose; Signal Transduction; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Sorting - Cell Movement; Stem cells; Surface; System; Techniques; Technology; adult stem cell; cell type; clinical application; cytokine; day; extracellular; gel microdrop encapsulation; human cord blood CD34+ cell; improved; interest; novel; novel strategies; progenitor; programs; secretory protein; self-renewal; stem; stem cell therapy; transcription factor

DESCRIPTION (provided by applicant): Stem cells represent unique cell populations that have the ability to undergo both self-renewal and differentiation. Although "adult" stem cells may develop into only a limited number of cell types, in contrast, early repopulating stem cells have the ability to initiate lineage-specific differentiation programs and to form numerous cell types in vitro. Megakaryocytopoiesis involves the commitment of hematopoietic stem cells, and the proliferation, maturation, and terminal differentiation of megakaryocytic progenitors. Characterization of megakaryocyte cytokines and regulators are important not only for improving our understanding of megakaryocytopoiesis and stem cell biology, but also for potential clinical applications. However, characterization of megakaryocytopoiesis, and examination of cellular development, gene expression or protein composition within the hematopoietic microenvironment have been difficult because of the relatively low cell frequency of megakaryoctyes: megakaryocytes represent only 0.015% to 0.03% of the total number of nucleated cells in human marrow. Furthermore, analysis of the role of endogenously secreted regulators by ELISA and other conventional bioassay techniques, which detect supernatant from heterogeneous cell populations, is difficult and imprecise because endogenous proteins are sometimes secreted at very low levels. To aid the study of endogenous secreted proteins and the process of megakaryocyte differentiation, this SBIR will use single cell gel microdrop (GMD) encapsulation technology and flow cytometry to develop a multi-parameter assay to detect cell surface marker expression and cytokine secretion simultaneously on single, viable hematopoietic stem/progenitor cells. This assay will facilitate sensitive determination of secretion at the single cell level together with clear cut identification of each immunostained, functionally active megakaryocyte. PUBLIC HEALTH RELEVANCE: The proposed single cell assay will facilitate the study of endogenous secreted proteins and the process of megakaryocyte differentiation, and aid the development of stem cell therapies.

描述（由申请人提供）：干细胞代表具有自我更新和分化能力的独特细胞群。虽然“成体”干细胞可能只发育成有限数量的细胞类型，但相反，早期再生干细胞具有启动谱系特异性分化程序并在体外形成多种细胞类型的能力。巨核细胞生成涉及造血干细胞的定向分化，以及巨核祖细胞的增殖、成熟和终末分化。巨核细胞细胞因子和调节因子的特性不仅对提高我们对巨核细胞生成和干细胞生物学的理解很重要，而且对潜在的临床应用也很重要。然而，由于巨核细胞的细胞频率相对较低：巨核细胞仅占人骨髓中有核细胞总数的0.015%至0.03%，因此巨核细胞生成的表征以及造血微环境中细胞发育、基因表达或蛋白质组成的检查一直很困难。此外，通过ELISA和其他常规生物测定技术（其检测来自异质细胞群体的上清液）分析内源性分泌的调节剂的作用是困难的和不精确的，因为内源性蛋白质有时以非常低的水平分泌。为了帮助内源性分泌蛋白和巨核细胞分化过程的研究，该SBIR将使用单细胞凝胶微滴（GMD）包封技术和流式细胞术来开发多参数测定，以同时检测单个活造血干/祖细胞上的细胞表面标志物表达和细胞因子分泌。该试验将有助于在单细胞水平上灵敏地测定分泌物，并明确鉴定每个免疫染色的功能活性巨核细胞。公共卫生关系：单细胞检测方法的建立将有助于研究内源性分泌蛋白和巨核细胞分化过程，并有助于干细胞治疗的发展。