ACTIVATION OF PURIFIED HEMOPOIETIC STEM CELLS

描述：在这份修订后的提案中，调查人员要求 额外的几年资金，以继续他们的工作 造血干细胞的特征。在以前的研究中， 研究人员研制出了针对两种新细胞的单抗 由造血干细胞显示的表面蛋白。 这两个标记CD34和Thy-1以及其他细胞表面 使用标记对人的骨髓、胎肝和脐带进行分选 将血液注入候选干细胞池中。调查人员已经 还描述了成体茎中端粒DNA的明显缩短 已经老化的细胞群体或胎儿干细胞 体外培养。根据这些先前的发现，调查人员计划 进一步鉴定造血干细胞的长期特性 获得纯净人口和发展文化的目标 将维持高度繁殖和自我更新的技术 纯化的干细胞。他们提出了三个具体目标。第一, 利用免疫磁学技术和多参数流式细胞术， 研究人员计划制造新的单抗和 为了筛查一大批抗体，直到5号 关于白细胞表面抗原的讲习班。希望，新的试剂 将获得可能对对候选对象进行排序额外有用的 干细胞群体转化为更高纯度的细胞。第二 明确的目标是确定生存的文化条件 人骨髓间充质干细胞的激活、增殖和自我更新 个体发育不同阶段的造血组织。两者都具有刺激性 抑制性细胞因子将测试它们对这些细胞因子的影响 参数，研究人员计划在体外跟踪这两个参数(长期 培养启动细胞)和体内(SCID-HU)检测。最后，在 第三个具体目标，调查人员计划进一步 勾勒出细胞增殖潜能与细胞增殖能力的关系 造血细胞及其端粒DNA的长度。使用 端粒特异性探针，调查人员将使用Southern blotting 计算端粒片段的平均长度。这将是 与不同来源细胞的增殖能力相关 造血源。希望，随着一种 一种名为端粒酶的酶，研究人员计划操纵 端粒的长度，从而验证了固定端粒长度的假设 基因变化决定了干细胞的潜力。

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.