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Institute of Hematology, Erasmus University Rotterdam, Rotterdam, The Netherlands
Key Words. SCF • EPO • IL-6 • GM-CSF • Receptor • Hemopoiesis • Flow cytometry • Biotin • Digoxigenin
Dr. Gerard Wagemaker, Institute of Hematology, Room Ee1314B, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
The detection of functional growth factor (GF) receptors on subpopulations of hemopoietic cells may provide a further dissection of immature cell subsets. Since little information is available about coexpression of different GF receptors at the level of single hemopoietic cells, we studied the feasibility of simultaneous cell staining with a combination of biotin- and digoxigenin-labeled GFs for flow cytometric detection of functional receptors. Using this methodology, coexpression of Kit and receptors for erythropoietin (EPO), interleukin 6 (IL-6), and GM-CSF on hemopoietic cells was studied by triple-staining of rhesus monkey bone marrow (BM) cells with labeled GFs and antibodies against other cell surface markers. Most of the immature, CD34++ cells were Kit+ but did not display detectable levels of EPO-receptors (EPO-Rs) or GM-CSF-R. Approximately 60% of these CD34++/Kit+ cells coexpressed the IL-6-R, demonstrating that immature cells are heterogeneous with respect to IL-6-R expression. Maturation of monomyeloid progenitors, as demonstrated by decreasing CD34 and increasing CD11b expression, is accompanied by a decline of Kit and an increase in GM-CSF-R expression in such a way that Kit+/GM-CSF-R+ cells are hardly detectable. IL-6-R expression is maintained or even increased during monomyeloid differentiation. IL-6-R and GM-CSF-R were not identified on most CD71++ cells, which indicated that these receptors are probably not expressed during erythroid differentiation. Together with previous results, our data show that both Kit and CD71 are upregulated with erythroid commitment of immature progenitors. Upon further differentiation, Kit+/EPO-R– cells lose CD34 and acquire EPO-R. Maturing erythroid cells eventually lose CD71 and Kit expression but retain the EPO-R. In conclusion, this approach enables further characterization of the specificity of GFs for different bone marrow subpopulations. Apart from insight into the differentiation stages on which individual GFs may act, information about receptor coexpression may be used to identify individual cells that can respond to multiple GFs, and allows for further characterization of the regulation of lineage-specific differentiation.
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