Maturity-dependent fractionation of neutrophil progenitors: A new method to examine in vivo expression profiles of differentiation-regulating genes

Abstract

To investigate differentiation-dependent gene expression during granulopoiesis, we established a new method to isolate six sequential differentiation stages of neutrophil progenitors from bone marrow. Neutrophil progenitors were divided into three populations by density centrifugation, followed by depletion of other lineages, and further separated by fluorescence-activated cell sorting based on the expressions of CD34, CD11b, and CD16: CD34(+) fraction from a low-density population (F1), CD11b(-)/CD16(-) (F2), CD11b(+)/CD16(-) (F3), and CD11b(+)/CD16(low) (F4) fractions with intermediate density, and CD11b(+)/CD16(int) (F5) and CD11b(+)/CD16(high) (F6) fractions from a high-density population. To examine whether this fractionation was applicable to the study of in vivo gene expression profiles during granulopoiesis, we analyzed messenger RNA levels of AML-1 and CCAAT/enhancer binding protein (EBP)-ε and two target genes of C/EBP-ε, granulocyte-macrophage colony-stimulating factor receptor common β subunit and lactoferrin, in the six fractions and peripheral blood-derived neutrophils (F7). Expression of AML-1 and C/EBP-ε peaked at F1 and F4, respectively, followed by a gradual decrease. Although granulocyte-macrophage colony-stimulating factor receptor common β subunit messenger RNA levels remained low from F1 through F6 and elevated at F7, lactoferrin messenger RNA showed a drastic increase at F3 and dropped at F5. The difference in the expression profiles of the two C/EBP-ε target genes suggests the involvement of regulators other than C/EBP-ε in the induction of the two genes. The new fractionation method is able to provide new information on maturation-dependent gene expression during granulopoiesis.