CORE--STEM CELL

干细胞核心将为大规模分离小鼠和人类来源的造血干细胞群体提供一个中央设施。来源将包括正常小鼠骨髓(Balb/c)、正常捐赠者的人骨髓、某些人群(地中海贫血、G6PD缺乏症)的骨髓、正常捐赠者动员的G-CSF外周血和脐带血。通过FACS分析，通过阳性免疫磁选获得的标准人类制剂将是90-95%纯度的CD34+人群。用于更专门研究的第二代产品，例如CD34+CD38-或CD34+/Thy1+，将通过使用免疫磁性微球的无菌FAC分选和/或阳性选择从初选的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，单独的“谱系阴性”细胞(LIN-)将与一组针对各种分化抗原的抗体(LIN-CD34-)一起用于免疫珠子去除，无论是否有CD34+去除。获得浓缩干细胞的替代程序将包括对来自小鼠和人骨髓或血液的Hoechst染料SP组分、根据乙醛脱氢酶表达或表达多药外排MDR-1基因的细胞(罗丹明染料除外)进行FACS分选。小鼠干细胞将通过SCA1+、Lin-、c-Kit+、Thy-1 Low和罗丹明Low表型进行选择。这些制剂将通过标准的体外干细胞和祖细胞含量测定，在某些情况下通过NOD-SCID植入试验、SDF-1趋化试验、TRAP试验和端粒长度进行评估。核心还将为项目4提供来自细胞因子刺激的CD34+细胞或从正常黄褐色涂层制剂中获得的单核细胞的人树突状细胞群体。流式细胞仪的特性和在MLC中的刺激活性将在每种制剂中确定。脐带血CD34+细胞的体外培养将产生相当于扁桃体幼稚B细胞群的体外产生的人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.