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Granulocyte colony-stimulating factor induction of normal human bone marrow
progenitors results in neutrophil-specific gene expression
N Berliner, A Hsing, T Graubert, F Sigurdsson, M Zain, E Bruno and R Hoffman
Department of Internal Medicine, Yale University School of Medicine, New
Haven, CT 06510.
We have used a combination of hematopoietic growth factors to induce in
vitro granulocytic maturation. A fraction of marrow cells enriched for
hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from normal
human bone marrow by monoclonal antibody staining and
fluorescence-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 myeloid
gene expression. Maturation was observed to progress to the late
metamyelocyte and band stage over a period of 10 to 12 days.
Neutrophil-specific gene expression was assayed by reverse
transcription-polymerase chain reaction (RT-PCR). Induction with G-CSF
resulted in sequential expression of primary and secondary granule
proteins, with asynchronous expression of primary granule proteins starting
from days 1 to 5, and synchronous expression of lactoferrin and
transcobalamin I (secondary granule proteins) from days 7 to 8.
Interestingly, myeloperoxidase (MPO) mRNA expression was easily detected in
both the freshly isolated CD34+, HLA-DR+ cells and cells at all subsequent
stages of induction. This suggests that MPO mRNA is expressed very early
during neutrophil development, perhaps before the development of
significant numbers of phenotypically recognizable granules. This
recapitulation of a program of sequential expression of primary and
secondary granule protein genes suggests that in vitro marrow culture
suspensions to which appropriate growth factors are added can mimic normal
granulocytic maturation. This system should provide an important model for
the study of neutrophil-specific gene expression.
Volume 85,
Issue 3,
pp. 799-803,
02/01/1995
Copyright © 1995 by The American Society of Hematology
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