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.
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