Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay.
In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine-RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by 125I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34+CD38 cells (approximately 1600 sites/cell), and decreased in the following order: CD34+CD38- cells > CD34+CD38+ cells > CD34-CD38+ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34+ fraction expressed more EpoR than their CD34- fraction. However, the expression levels of EpoR of these fractions were lower than CD34+CD38- cells. These results indicated that EpoR was highly expressed on CD34+ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.
Shinjo
K
K
Department of Medicine III, Hamamatsu University School of Medicine, Japan.
Takeshita
A
A
Higuchi
M
M
Ohnishi
K
K
Ohno
R
R
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002454:Cell Differentiation; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006021:Glycophorins / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D008214:Lymphocytes / Q000378:metabolism; D008562:Membrane Glycoproteins; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D006021:Glycophorins; D008562:Membrane Glycoproteins; D017467:Receptors, Erythropoietin; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1