CORE--STEM CELL

干细胞中心将提供一个中心设施，用于从鼠和人来源大规模分离造血干细胞群体。来源将包括正常鼠骨髓（Balb/c）、来自正常供体的人骨髓、来自某些人群（地中海贫血、G6 PD缺乏症）的骨髓、来自正常供体的G-CSF动员的外周血和脐带血。标准人制备物将是通过FACS分析纯度为90-95%并通过阳性免疫磁性选择获得的CD 34+群体。用于更专门研究的第二代产品，例如CD 34 + CD 38-或CD 34 +/Thy 1+，将通过无菌FACS分选和/或使用免疫磁性微珠的阳性选择从最初选择的CD 34+细胞获得。这些产物的实例是AC 133 +/CD 34+、KDR++ CD 34+、KDR+/AC 133+、CD 34 + CD 38-、CD 34 + Thy 1+。对于一些研究，将获得单独的“谱系阴性”细胞（Lin-），用于用针对各种分化抗原的一组抗体进行免疫珠粒消耗，伴随或不伴随CD 34+消耗（Lin-CD 34-）。用于获得富集的干细胞的“替代”程序将包括来自鼠和人骨髓或血液的Hoechst染料SP级分的FACS分选，基于醛脱氢酶表达或多药物流出MDR-1基因的表达（罗丹明染料排除）选择细胞。将通过SCA 1+、Lin-、c-Kit+、Thy-1低和罗丹明低表型选择鼠干细胞。将通过标准体外测定（在某些情况下通过NOD-SCID植入测定）、SDF-1趋化性、TRAP测定和端粒长度来评估制剂的干细胞和祖细胞含量。Core还将向项目4提供人树突状细胞群，这些细胞来源于经奎宁刺激的CD 34+细胞或从正常血沉棕黄层制备物中获得的血液单核细胞。将测定每份制备液在MLC中的FACS表征和刺激活性。体外生成的人B细胞等同于扁桃体幼稚B细胞群，将通过体外培养脐带血CD 34+细胞生成。

The Stem Cell Core will provide a central facility for the large-scale isolation of hematopoietic stem cell populations from both murine and human sources. The source will include normal murine bone marrow (Balb/c), human marrow from normal donors, marrow from certain populations (thalassemia, G6PD deficiency), G-CSF mobilized peripheral blood from normal donors, and umbilical cord blood. The standard human preparation will be a CD34+ population 90-95% pure by FACS analysis and obtained by positive immunomagnetic selection. Second generation products for more specialized studies, for example CD34+CD38 - or CD34+/Thy1+, will be obtained from the primary selected CD34+ cells by sterile FACS sorting and/or positive selection using immunomagnetic microbeads. Examples of these products would be AC133+/CD34+, KDR++CD34+, KDR+/AC133+, CD34+CD38-, CD34+Thy1+. For some studies "lineage negative" cells (Lin-) alone will be obtained for immunobead depletion with a panels of antibodies to various differentiation antigens, with or without CD34+ depletion (Lin-CD34-). "Alternative" procedures for obtaining enriched stem cells will include FACS sorting of Hoechst dye SP fractions from murine and human marrow or blood, cells selected on the basis of aldehyde dehydrogenase expression or an expression of the multi-drug efflux MDR-1 gene (Rhodamine dye exclusion. Murine stem cells will be selected by SCA1+, Lin-, c-Kit+, Thy-1 low and Rhodamine low phenotype. The preparations will be evaluated for stem cell and progenitor content by standard in vitro assays, in some case by NOD-SCID engraftment assays, for SDF-1 chemotaxis, TRAP assay, and telomere length. The Core will also provide project 4 with populations of human dendritic cells derived from either cytokine-stimulated CD34+ cells or blood monocytes obtained from normal buffy coat preparations. FACS characterization and stimulatory activity in MLC will be determined on each preparation. In vitro generated human B cells equivalent to the tonsillar naive B cell population will be generated by in vitro culture of cord blood CD34+ cells.