title: An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow.
abstract: An improved negative immunomagnetic selection strategy has been devised for the enrichment of primitive hemopoietic cells using the high proliferative potential colony-forming cell (HPP-CFC) assay as an index of stem cell purification. Immunomagnetic selection was carried out using goat anti-rat conjugated M-450 Dynabeads and a cocktail of rat monoclonal antibodies directed against lineage antigens expressed on B-lymphocytes (B220), neutrophils and activated macrophages (7/4), differentiating erythroid cells (YW 25.12.7), and T-lymphocyte subsets (Lyt-2 and L3T4). This negative selection strategy results in the highly reproducible enrichment of HPP-CFC with negligible loss of HPP-CFC at the immunomagnetic selection step. A 30-fold enrichment of HPP-CFC stimulated by interleukin 3 (IL-3) plus colony-stimulating factor (CSF-1), or interleukin 1 alpha (IL-1 alpha) plus IL-3 plus CSF-1, is obtained with simultaneous resolution of HPP-CFC from progenitor cells of low proliferative potential responsive to CSF-1 alone (LPP-CFC). Flow cytometric analysis of these lineage-negative cells reveals that they almost exclusively exhibit the light-scattering characteristics of blast cells and the morphology of a candidate hemopoietic stem cell. Positive fluorescence-activated cell sorting of immunomagnetically pre-enriched normal bone marrow cells using wheat germ agglutinin yields cell preparations with a cloning efficiency of up to 45% and a HPP-CFC content of 20%.
authors.affiliation: Cell Biology Group, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
authors.forename: I
authors.initials: I
authors.lastname: Bertoncello
authors.forename: T R
authors.initials: TR
authors.lastname: Bradley
authors.forename: S M
authors.initials: SM
authors.lastname: Watt
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001853:Bone Marrow / Q000187:drug effects / Q000276:immunology; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D007158:Immunologic Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D008280:Magnetics; D051379:Mice; D008815:Mice, Inbred Strains
chemical_list: D000911:Antibodies, Monoclonal; D003115:Colony-Stimulating Factors; D007375:Interleukin-1; D007377:Interleukin-3