Multiparameter analysis of murine bone marrow side population cells

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Abstract
We describe the multiparameter flow cytometric analysis of the relationship between side population (SP) formation and well-characterized, antigen-defined stem cell subsets. We also compared the competitive repopulation ability of subsets defined by the SP profile to those identified by antigenic markers. The vast majority of SP cells possessed a primitive cell phenotype (c-kit+, SCA-1+, Thy-1+, CD31+, CD135neg, lineageneg), but only a minority of antigen-defined subsets were SP cells. Hence, although SP cells are identified independently of antigenic markers, they are not distinct from established stem cell phenotypes, but are a small subset of them. Approximately half of SP cells expressed CD34 at readily detectable levels, and one-third of SP cells possessed the primitive c-kit+, SCA-1+, lineageneg, CD34neg cell phenotype. Since most SP cells are a subset of c-kit+, Thy-1+, lineageneg, SCA-1+ cells (KTLS), we determined whether the SP cell subset represents a further enrichment in long-term repopulating cell content. The SP+ subset of KTLS+ cells was more enriched for competitive repopulation units than the SP- fraction of KTLS+ cells. Hence, the SP cell fraction highlights a subset of the long-term repopulating cells found within the already highly purified KTLS fraction.