Novel Method for Profiling Megakaryocytes

描述（由申请人提供）：干细胞代表独特的细胞群，具有自我更新和分化的能力。尽管“成体”干细胞可能只发育成有限数量的细胞类型，相反，早期再生干细胞有能力启动谱系特异性分化程序，并在体外形成多种细胞类型。巨核细胞生成涉及造血干细胞的承诺，以及巨核细胞祖细胞的增殖、成熟和终末分化。巨核细胞细胞因子和调节因子的表征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，巨核细胞仅占人类骨髓中有核细胞总数的0.015%至0.03%，因此，在造血微环境中表征巨核细胞生成以及检查细胞发育、基因表达或蛋白质组成一直很困难。此外，通过ELISA和其他传统的生物测定技术（从异质细胞群中检测上清）分析内源性分泌调节因子的作用是困难和不精确的，因为内源性蛋白质有时分泌水平非常低。为了帮助研究内源性分泌蛋白和巨核细胞分化过程，该SBIR将使用单细胞凝胶微滴（GMD）包埋技术和流式细胞术开发一种多参数检测方法，同时检测单个活的造血干细胞/祖细胞的细胞表面标志物表达和细胞因子分泌。该试验将促进单细胞水平分泌的敏感测定，并明确识别每个免疫染色，功能活跃的巨核细胞。公共卫生相关性：提出的单细胞试验将促进内源性分泌蛋白和巨核细胞分化过程的研究，并有助于干细胞治疗的发展。

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.