The Stem Cell Core will support all projects by providing expertise in the purification and characterization of primitive hematopoietic cell populations as well as the tracking of genetically modified cells in bone marrow and peripheral blood following reconstitution. Expertise has been established in the preparation of primitive hematopoietie cell populations from three species: humans, rhesus macaques and mice. Human peripheral blood cells obtained from normal volunteers following administration of G-CSF are purified using the Clinimacs and/or the Aria cell sorter. The shared resource is equipped with Miltenyi MACS system as well as a state-of-the-art Becton-Dickinson Aria cell sorter. The Clinimacs is also used to recover CD34 + cells from peripheral blood or bone marrow of rhesus macaques following administration of G-CSF and SCF. Methodologies depending on depletion of cells expressing lineage markers are being developed using the AutoMACS device with enrichment in primitive cells monitored by quantitating the side population (SP) cells defined by dye efflux. Two methodologies are available within the Core for preparation of cell populations highly enriched in murine repopulating stem cells. The first involves the use of dye efflux to isolate the SP population. The expertise in purifying functional SP cells will be adapted for use in the rhesus model. The methodology is sufficiently developed to allow reconstitution of mice with as few as 250 transplanted SP calls. The second strategy involves selection of lineage -ckit+, Sca1+ cell by FACS. Overall, the Core provides essential expertise, instrumentation and experience in complex methodologies necessary to purify, eharacterize and analyze primitive hematopoietic cell populations.
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