GRANULOCYTE-COLONY-STIMULATING FACTOR INDUCTION OF NORMAL HUMAN BONE-MARROW PROGENITORS RESULTS IN NEUTROPHIL-SPECIFIC GENE-EXPRESSION

We have used a combination of hematopoietic growth factors to induce i n vitro granulocytic maturation. A fraction of marrow cells enriched f or hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from n ormal human bone marrow by monoclonal antibody staining and fluorescen ce-activated cell sorting. Cells were cultured in a suspension system for 3 days in the presence of stem cell factor and interleukin-3 (IL-3 ), after which granulocyte colony-stimulating factor (G-CSF) was added . Cells were harvested daily and analyzed for phenotypic maturation by morphologic criteria, and total RNA was obtained for analysis of myel oid gene expression. Maturation was observed to progress to the tate m etamyelocyte and band stage over a period of 10 to 12 days. Neutrophil -specific gene expression was assayed by reverse transcription-polymer ase chain reaction (RT-PCR). Induction with G-CSF resulted in sequenti al expression of primary and secondary granule proteins, with asynchro nous expression of primary granule proteins starting from days 1 to 5, and synchronous expression of lactoferrin and transcobalamin I (secon dary granule proteins) from days 7 to 8. Interestingly, myeloperoxidas e (MPO) mRNA expression was easily detected in both the freshly isolat ed CD34+, HLA-DR+ cells and cells at all subsequent stages of inductio n. This suggests that MPO mRNA is expressed very early during neutroph il development, perhaps before the development of significant numbers of phenotypically recognizable granules. This recapitulation of a prog ram of sequential expression of primary and secondary granule protein genes suggests that in vitro marrow culture suspensions to which appro priate growth factors are added can mimic normal granulocytic maturati on. This system should provide an important model for the study of neu trophil-specific gene expression. (C) 1995 by The American Society of Hematology.