STEM CELL ISOLATION CHARACTERIZATION AND AMPLIFICATION

骨髓、成人外周血和骨髓中的人造血干细胞 脐带血将通过一系列涉及SBA的步骤进行分离 凝集、免疫细胞黏附和免疫磁珠阳性 CD3+、CD33-Lin-人类白细胞抗原-DR-细胞阴性选择。其他内容 按生物物理标准进行分离，主要是密度，以及 免疫荧光和罗丹明123荧光强度的流式细胞仪检测 将进行分析。将进行干细胞鉴定 使用体外自我更新(Delta)试验，高增殖 潜在(HPP-CFU)集落试验--甲基纤维素中的原始集落试验 或在骨髓基质上，以及基于延长的 照射后CFU的重建和分化细胞的产生 骨髓基质。选择性生存、周期激活、自我更新和 早期干细胞的分化将通过 细胞因子与干细胞水平上已被证实的作用的组合- IL-1、IL-3、IL-6相互结合及与G-CSF、M-CSF 和GM-CSF。C-kit原癌基因配体(KL)在茎上的作用 将探索细胞增殖，以及刺激细胞增殖的潜力 一种IL-3-GM-CSF杂化分子。 逆转录病毒感染人脑脊液细胞因子基因的研究 将使用合适的骨髓基质细胞系来发展长期 维持长期重建干细胞的培养系统。 将选择用于持续生产各种G-CSF的品系， 粒-巨噬细胞集落刺激因子、IL-1、IL-6、IL-3和KL与细胞免疫功能的关系 选择性细胞黏附早期干细胞群体 生物淘金。干细胞潜在的负调控因子 增殖如TGFbeta-1、MIP-1a将被适当的 各种体外检测系统中的抗体。 茎的最终标准--长期、自我更新和 多潜能的保留将在照射后的基质上进行评估 细胞经持续造血维持具有较高的细胞比例 较长时间段的增量值。在体内，干细胞将是 接种于植入骨髓基质的SCID小鼠体内 建立人类在骨小骨内的造血将是 被监视着。

Human hematopoietic stem cells in marrow, adult peripheral blood and cord blood will be isolated by a combination of steps involving SBA agglutination, immunocytoadherence and immunomagnetic bead positive and negative selection for CD3+ , CD33- LIN- HLA-DR- cells. Additional separation by biophysical criteria, predominantly density, and immunofluorescence and rhodamine 123 fluorescence intensity by FACS analysis will be undertaken. Stem cell identification will be undertaken using an in vitro self-renewal (Delta) assay, a high proliferative potential (HPP-CFU) colony assay, a blast colony assay in methylcellulose or on marrow stroma, and a micro-assay based upon prolonged reconstitution of CFU and differentiated cell production on irradiated marrow stroma. Selective survival, cycle activation, self renewal and differentiation of early stem cells will be facilitated using combinations of cytokines with proven action at the stem cell level - IL-1, IL-3 and IL-6 .in combination with each other and with G-CSF, M-CSF and GM-CSF. The action of the c-kit protooncogene ligand (KL) on-stem cell proliferation will be explored, as will the stimulatory potential of an IL-3-GM-CSF hybrid molecule. The introduction of cytokine CSF genes by retroviral infection of appropriate marrow stromal cell lines will be used to develop long term culture systems for maintaining long term reconstituting stem cells. Lines will be selected for sustained production of, variously, G-CSF, GM-CSF, IL-1, IL-6, IL-3, and KL and retention of the ability to selectively cytoadhere early stem cell populations as determined by biological panning. Potential negative regulators of stem cell proliferation eg TGFbeta-1, MIP-la will be neutralized by appropriate antibodies in the various in vitro assay systems. The final criteria of stemness - long term, self-renewal and retention of pluripotentiality will be assessed on irradiated stromal cells by sustained hematopoiesis with maintenance of cells with a high delta value for prolonged periods. In vivo, stem cells will be inoculated into SCID mice bearing engrafted marrow stroma and establishment of human hematopoiesis within the bone ossicles will be monitored.