ACTIVATION OF PURIFIED HEMOPOIETIC STEM CELLS

说明：在本修订提案中，研究人员要求三个 更多的资金来继续他们的工作， 表征造血干细胞。 在以前的研究中， 研究人员产生了针对两种新细胞的单克隆抗体 造血干细胞所展示的表面蛋白。 这两个标志物，CD 34和Thy-1，沿着其他细胞表面 用标记物对人骨髓、胎儿肝脏和脐带进行分类 将血液注入干细胞候选者的池中。 调查人员已 还描述了一个明显的端粒DNA缩短成年茎 细胞群或胎儿干细胞， 体外 根据这些先前的调查结果，调查人员计划 为了进一步表征造血干细胞， 获得纯群体和发展文化的目标 技术，将维持增殖和自我更新的高度 纯化的干细胞 提出了三个具体目标。 第一、 使用免疫磁性技术和多参数流式细胞术， 的 研究人员计划产生新的单克隆抗体， 筛选一大批抗体， 白细胞表面抗原研讨会。 希望新的试剂 这可能是另外有用的排序候选人 将干细胞群转化为更高纯度的细胞。 第二 具体目标是确定生存的培养条件 来自人的候选干细胞的活化、增殖和自我更新 个体发育不同阶段的造血组织。 既刺激 和抑制性细胞因子将测试它们对这些细胞的影响。 参数，研究者计划在体外（长期 培养起始细胞）和体内（SCID-hu）测定。 最后在 第三个具体目标，调查人员计划进一步 描述了细胞增殖潜能与 造血细胞及其端粒DNA的长度。 使用 端粒特异性探针，研究者将使用Southern印迹法 来计算端粒片段的平均长度。 这将是 与取自不同组织的细胞的增殖能力相关 造血来源。希望，随着可用性的 一种叫做端粒酶的酶，研究人员计划操纵 端粒长度在文化，从而测试假设，固定 遗传变化决定了干细胞的潜能。

DESCRIPTION: In this revised proposal, the Investigators request three additional years of funding to continue their work into characterizing hematopoietic stem cells. In previous studies, the Investigators generated monoclonal antibodies against two novel cell surface proteins which are displayed by hematopoietic stem cells. These two markers, CD34 and Thy-1, along with other cell surface markers were used to sort human bone marrow, fetal liver and cord blood into pools of stem cell candidates. The Investigators have also described an apparent shortening of telomeric DNA in adult stem cell populations or fetal stem cells which have been pass aged in vitro. On the basis of these previous findings, the Investigators plan to further characterize hematopoietic stem cells with a long-term goals of obtaining pure populations and developing the culture techniques that will sustain proliferation and self-renewal of highly purified stem cells. They propose three Specific Aims. First, using immunomagnetic techniques and multiparameter flow cytometry, the Investigators plan to generate new monoclonal antibodies and to screen a large panel of antibodies available through the 5th Workshop on Leukocyte Surface Antigens. Hopefully, new reagents will be obtained which may be additionally useful to sort candidate stem cell populations into more highly purified cells. The second Specific Aim is to define culture conditions for the survival activation, proliferation and self-renewal of candidate stem cells from hematopoietic tissues of various stages of ontogeny. Both stimulatory and inhibitory cytokines will be tested for their effects on these parameters, the Investigators plan to follow both in vitro (long-term culture initiating cell) and in vivo (SCID-hu) assays. Finally, in the third Specific Aim, the Investigators plan to further delineate the relationship between proliferative potential of hematopoietic cells and the length of their telomeric DNA. Using a telomere specific probe, the Investigators will use Southern blotting to calculate the mean length of telomeric segments. This will be correlated with proliferative capacity of cells taken from various hematopoietic sources.Hopefully, with the availability of an enzyme called telomerase, the investigators plan to manipulate telomere length in culture, and thereby test the hypothesis that fixed genetic changes dictate stem cell potential.