DYE EFFLUX STUDIES SUGGEST THAT HEMATOPOIETIC STEM-CELLS EXPRESSING LOW OR UNDETECTABLE LEVELS OF CD34 ANTIGEN EXIST IN MULTIPLE SPECIES

We previously described a method for isolating murine hematopoietic st em cells capable of reconstituting lethally irradiated recipients, whi ch depends solely on dual-wavelength flow cytometric analysis of murin e bone marrow cells stained with the fluorescent DNA-binding dye Hoech st 33342. This method, which appears to rely on the differential abili ty of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here t hat dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells . Like the murine SP cells, both human and rhesus SP cells are primari ly CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term c ulture-initiating cells (LTC-ICs), an indicator of primitive hematopoi etic cells, and have the capacity for differentiation into T cells. Al though rhesus SP cells do not initially possess any hematopoietic colo ny-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conver sion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells tha t lack the CD34 surface marker classically associated with primitive h ematopoietic cells.