CORE--STEM CELL

干细胞中心将为大规模分离小鼠和人造血干细胞群提供中心设施。来源将包括正常小鼠骨髓（Balb/c）、正常供者的人骨髓、某些人群（地中海贫血、G6PD缺乏症）的骨髓、正常供者动员的G-CSF外周血和脐带血。标准人制剂将是CD34+群体，经FACS分析纯度为90-95%，并通过阳性免疫磁选择获得。用于更专业研究的第二代产品，例如CD34+CD38 -或CD34+/Thy1+，将通过无菌FACS分选和/或免疫磁微珠阳性选择从原代选择的CD34+细胞中获得。这些产物的例子是AC133+/CD34+， KDR++CD34+, KDR+/AC133+, CD34+CD38-, CD34+Thy1+。在一些研究中，将单独获得“谱系阴性”细胞（Lin-），用各种分化抗原的抗体面板进行免疫珠消耗，有或没有CD34+消耗（Lin-CD34-）。获得富集干细胞的“替代”程序将包括从小鼠和人类骨髓或血液中对Hoechst染料SP组分进行FACS分选，根据醛脱氢酶表达或多药外排MDR-1基因的表达选择细胞（罗丹明染料排除）。小鼠干细胞将通过SCA1+， Lin-, c-Kit+， Thy-1低和罗丹明低表型选择。这些制剂将通过标准的体外测定来评估干细胞和祖细胞含量，在某些情况下通过NOD-SCID植入测定来评估SDF-1趋化性、TRAP测定和端粒长度。Core还将为项目4提供从细胞因子刺激的CD34+细胞或从正常的灰褐色涂层制备的血液单核细胞中获得的人类树突状细胞群。每种制剂将确定MLC中的FACS表征和刺激活性。脐带血CD34+细胞的体外培养将产生与扁桃体初始B细胞群相当的体外生成的人B细胞。

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.