Evidence for a qualitative hierarchy within the Hoechst-33342 'side population' (SP) of murine bone marrow cells

In vitro cobblestone area ( CA)- forming cell ( CAFC) and in vivo ( short- term and competitive repopulation) assays demonstrate that a qualitative hierarchy exists within the Hoechst- 33342- defined side population ( SP) in murine bone marrow ( BM). Consistent with and extending previous studies, we demonstrate that ( i) hematopoietic activity found in whole BM ( WBM) is concentrated within the SP, rather than the non- SP ( NSP); and ( ii) within the SP, those cells that more strongly efflux the dye ( lower SP, LSP) are qualitatively different from those that less strongly efflux the dye ( upper SP, USP). Qualitative differences are highlighted by evidence that ( i) CA derived from LSP CAFC persist in culture significantly longer than CA derived from USP CAFC; ( ii) short- term, multilineage repopulation of lethally irradiated mice by LSP cells is more rapid than that in mice receiving USP, NSP, whole SP ( WSP), or WBM cells and ( iii) LSP cells out- compete USP cells in the multilineage hematopoietic repopulation of lethally irradiated recipients. These data suggest that LSP cells are of higher quality than USP cells and potentially provide a means by which qualitative changes in primitive hematopoietic progenitors occurring naturally with aging, or clinically as a consequence of therapeutic manipulation, can be assessed.