Separation of myeloid and erythroid progenitors based on expression of CD34 and c-kit

Abstract

In this report, a novel approach is described to physically separate erythroid progenitors from monocyte and granulocyte progenitors, based on the expression of CD34 and Kit. Using biotin-labeled human Kit ligand (KL) and flow cytometry, Kit was detectable on 2% to 3% of the nucleated cells in rhesus monkey bone marrow. Combination of biotin-KL with CD34 monoclonal antibodies (MoAb) showed that Kit was expressed on subsets of CD34(low) and CD34(pos) cells. Our data clearly demonstrate that CD34(pos) cells are more heterogeneous with respect to Kit expression than observed in studies using Kit MoAb. A small cluster, approximately 7% of the CD34(pos) cells, expressed CD34 at submaximal levels and stained brightly with biotinylated KL This CD34(pos)/kit(hi) fraction contained predominantly erythroid progenitors (burst-forming units-erythroid; BFU-E). The majority of the granulocytic and monocytic progenitors (colony-forming units-granulocyte/macrophage; CFU-GM) were CD34(pos)/kit(med). Some BFU-E were also detected in the CD34(pos)/kit(med) and CD34(low)/kit(pos) fractions at low frequency. In the latter subset, most erythroid colony-forming units (CFU-E) were recovered. Using three-color flow cytometry, we analyzed expression of Kit in relation to that of CD34 and the class II major histocompatibility antigen, RhLA-DR. The most immature bone marrow cells that can be identified in vitro, ie, CD34(pos)/RhLA-DR(low) cells, were kit(med). The CD34(pos)/kit(hi) and CD34(pos)/kit(neg) subsets predominantly contained the more mature RhLA- DR(bright) cells. Our results demonstrate that erythroid precursors express c-kit at much higher levels than monomyeloid precursors and pluripotent progenitors. The difference in expression levels of CD34 and c-kit can be exploited to isolate BFU-E populations that are virtually devoid of nonerythroid cells.