Clonogenicity, gene expression and phenotype during neutrophil versus erythroid differentiation of cytokine‐stimulated CD34+human marrow cellsin vitro

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Abstract
Summary: With the objective to correlate clonogenicity, gene expression and phenotype during differentiation, human bone marrow CD34+cells were culturedin vitroto stimulate erythroid or neutrophil development, and sorted into five subpopulations according to their surface expression of CD15/CD33 and blood group antigen A/CD117 respectively. Sorted cells were cultured in methylcellulose and analysed by real‐time reverse transcription polymerase chain reaction for expression of neutrophil and erythroid marker genes. Surface expression of CD15 coincided with restriction to neutrophil/monocyte differentiation and A antigen with restriction to erythroid differentiation. GATA‐2 mRNA was down‐regulated during both neutrophil and erythroid maturation, whereas GATA‐1, SCL, ABO, erythropoietin receptor, Kell, glycophorin A,β‐globin andα‐haemoglobin stabilizing protein were up‐regulated during erythroid differentiation and silenced during neutrophil differentiation. CCAAT/enhancer‐binding protein (C/EBP)‐α, PU.1, granulocyte colony‐stimulating factor receptor, PR3, C/EBP‐ɛand lactoferrin were sequentially expressed during neutrophil differentiation but rapidly down‐regulated during the early erythroid stages. Nuclear factor erythroid‐derived 2 (NF‐E2) and glycophorin C were expressed both during neutrophil and erythroid differentiation. Our data support the notion of early expression of several lineage‐associated genes prior to actual lineage commitment, defined by surface expression of CD15 and A antigen as markers for definitive neutrophil/monocyte and erythroid differentiation respectively. Previous findings, primarily from cell lines and mouse models, have been extended to adult human haematopoiesis.