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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 509-518
Neutrophilic Cell Production by Combination of Stem Cell Factor and
Thrombopoietin From CD34+ Cord Blood Cells in
Long-Term Serum-Deprived Liquid Culture
Nobukuni Sawai,
Kenichi Koike,
Susumu Ito,
Hadija Hemed Mwamtemi,
Yumi Kurokawa,
Tatsuya Kinoshita,
Kazuo Sakashita,
Tsukasa Higuchi,
Kouichi Takeuchi,
Masaaki Shiohara,
Hiroshi Miyazaki,
Takashi Kato, and
Atsushi Komiyama
From the Department of Pediatrics and the Blood Transfusion Service,
Shinshu University School of Medicine, Matsumoto, Japan; and the
Pharmaceutical Research Laboratory, Kirin Brewery Co, Ltd, Takasaki,
Japan.
In the present study, we investigated the effects of stem cell
factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34+ cells using a serum-deprived liquid
culture system. Although SCF alone supported a modest production of
neutrophilic cells and a remarkable generation of mast cells, the
addition of TPO to the culture containing SCF caused an apparent
generation of neutrophilic cells, identified by immunocytochemical
staining and flow cytometric analysis. The significant production of
neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or
granulocyte-macrophage colony-stimulating factor (GM-CSF). The addition
of neutralizing antibody against G-CSF or GM-CSF did not influence the
SCF + TPO-dependent neutrophilic cell production. A single-cell
culture study showed that not only
CD34+CD38+ c-kit+ cells but
also CD34+CD38 c-kit+ cells
were responsible for the neutrophilic cell generation. In clonal cell
cultures, GM progenitors as well as erythroid progenitors and
multipotential progenitors expanded in the cultures supplemented with
SCF and TPO. The neutrophilic cells grown by SCF + TPO were at
myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the
CD34c-kit/low CD15+ cells
grown by SCF + TPO into a culture containing SCF + G-CSF permitted
both the terminal maturation into segmented cells and the appearance of
ALP and CD35. These results indicate the existence of a
G-CSF/GM-CSF-independent system of neutrophilic cell production.
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