ACTIVATION AND PROLIFERATION OF HEMOPOIETIC STEM CELLS

血细胞起源于造血干细胞的自我更新群体 这些细胞产生祖细胞，这些祖细胞不可逆地致力于 各种分化谱系之一。 最近在老鼠身上的研究 表明这些造血干细胞可以分离在同质 条件 纯化的人类造血干细胞提供巨大的 在同种异体和自体骨髓中的治疗潜力 移植 此外，这种电池的可用性将使 对正常和异常干细胞调节的重要研究， 基因治疗方案的发展。 这个项目的长期目标是 是确定维持核武器扩散和自我更新的条件， 高度纯化的人类造血干细胞。 具体目标是 建议有三个方面。 第一是提炼小规模的干细胞 使用流式细胞术和细胞分选进行纯化程序。 为此 目的制备细胞表面抗原的详细图谱 使用直接标记的抗-CD34在共表达CD34的细胞上表达 单克隆抗体和多参数流式细胞术和细胞分选。 将在集落形成试验中测定选定的细胞级分，并进行长- 骨髓培养。 干细胞将被定义为 能够启动长期骨髓培养的细胞，如 在培养5周后它们的集落形成细胞的产生。 的 第二个具体目标是建立小型和大型干细胞 使用免疫吸附技术进行纯化程序。 这是 重要的是，因为流式细胞术和细胞分选可能无法提供 临床用纯化干细胞制备的实用方法 目的 这些研究将进一步探索使用双特异性抗- CD34 x抗去铁蛋白四聚体抗体复合物和含有其的柱 去铁蛋白包被的玻璃珠用于纯化表达 CD34。 这一领域的研究有可能产生相对简单的 小规模和大规模（高达1010个细胞/柱）的程序 纯化表达的细胞（亚）群的方法 CD34用于实验室和临床研究。 最后一个具体目标是 定义用于存活、活化和增殖的培养条件， 分选的干细胞（亚）群体。 在这些研究中， 将单个细胞直接分选到培养板的威尔斯孔中 含有不同的饲养细胞、生长因子、细胞外基质 蛋白质或抗体。 细胞在培养过程中的命运将是 使用细胞分析仪系统进行分析， 显微镜图像的记录。 培养测定和单克隆抗体 将用于表征来自培养的分选细胞的细胞 细胞 综合这些在干细胞互补领域的研究， 研究将产生直接适用于临床的技术， 药 这些结果将有助于更好地了解干细胞 生物学和进一步研究的新方向。

Blood cells originate from a self-renewing population of hemopoietic stem cells which generate progenitor cells that are irreversibly committed to one of the various differentiation lineages. Recent studies in mice have shown that these hemopoietic stem cells can be isolated in homogeneous conditions. Purified human hemopoietic stem cell offer enormous therapeutic potential in allogeneic and autologous bone marrow transplantations. Furthermore the availability of such cells will enable crucial studies of normal and abnormal stem cell regulation as well as the development of gene therapy protocols. The long-term goal of this project is to define conditions that will sustain proliferation and self-renewal of highly purified human hemopoietic stem cells. The specific aims of this proposal are threefold. The first is to refine small scale stem cell purification procedures using flow cytometry and cell sorting. For this purpose it is proposed to prepare detailed maps of cell surface antigens expressed on cells that co-express CD34 using directly labelled anit-CD34 monoclonal antibodies and multiparameter flow cytometry and cell sorting. Selected cell fractions will be assayed in colony-forming assays and long- term bone marrow culture. Stem cells will be operationally defined as cells capable of initiating long-term bone marrow cultures as shown by their production of colony-forming cells after 5 weeks in culture. The second specific aim is to establish small and large scale stem cell purification procedures using immunoabsorption techniques. This is important because flow cytometry and cell sorting may not provide a practical approach for the preparation of purified stem cells for clinical purposes. These studies will further explore the use of bispecific anti- CD34 x anti-desferal tetrameric antibody complexes and columns containing desferal coated glass beads for the purification of cells that express CD34. Research in this area has the potential to yield relative simple procedures for both small and large scale (up to 1010 cells/column) procedures for the purification of (sub)populations of cells that express CD34 for laboratory and clinical studies. The last specific aim is to define culture conditions for the survival, activation and proliferation of sorted stem cell (sub)populations. For these studies low numbers or individual cells will be directly sorted into the wells of culture plates containing different feeder cells, growth factors, extracellular matrix proteins or antibodies. The fate of the cells during culture will be analyzed using a Cell Analyzer System that allows time lapse video recordings of microscopic images. Culture assays and monoclonal antibodies will be used to characterize the cells derived from the cultured sorted cells. Taken together these investigations in complementary areas of stem cell research will yield technology that is directly applicable in clinical medicine. The results will result in a better understanding of stem cell biology and new directions for further study.