ACTIVATION AND PROLIFERATION OF HEMOPOIETIC STEM CELLS

血细胞来源于一种自我更新的造血干细胞群体 产生祖细胞的细胞，这些细胞不可逆转地致力于 各种分化谱系中的一种。最近在老鼠身上进行的研究已经 表明这些造血干细胞可以在同种情况下分离出来 条件。纯化的人类造血干细胞提供了巨大的 同种异体和自体骨髓的治疗潜力 移植手术。此外，这种电池的可用性将使 对正常和异常干细胞调节的关键研究以及 基因治疗方案的发展。这个项目的长期目标是 是定义将维持核扩散和自我更新的条件 高纯度的人类造血干细胞。这样做的具体目的是 建议有三个方面。第一个是提炼小规模干细胞 纯化程序采用流式细胞术和细胞分选。为了这个 目的制备细胞表面抗原的详细图谱。 利用直接标记的ANIT-CD34在共表达CD34的细胞上表达 单抗和多参数流式细胞术及细胞分选。 选定的细胞组分将在集落形成试验中进行分析，并长期- 定期骨髓培养。干细胞将在操作上被定义为 能够启动长期骨髓培养的细胞，如图所示 培养5周后产生集落形成细胞。这个 第二个具体目标是建立小型和大规模的干细胞 使用免疫吸收技术的纯化程序。这是 重要的是因为流式细胞术和细胞分选可能不能提供 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双特异性抗-HBs的用途 CD34 x抗去雄四聚体抗体复合物和柱，含有 用于纯化表达细胞的镀膜玻璃微珠 CD34。这一领域的研究有可能产生相对简单的 小尺度和大尺度(高达1010个单元格/列)的程序 表达基因的(亚)细胞群的纯化程序 CD34用于实验室和临床研究。最后一个具体目标是 确定生存、激活和增殖的培养条件 已排序的干细胞(亚)种群。对于这些研究，数字较低或 单个细胞将被直接分选到培养板的孔中 含有不同的饲养层细胞、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 使用允许延时视频的细胞分析仪系统进行分析 显微图像的记录。培养检测和单抗 将被用来表征从培养的分选细胞中获得的细胞 细胞。 综合这些对干细胞互补区域的研究 研究将产生可直接应用于临床的技术 医药。这一结果将使人们更好地理解干细胞。 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.