Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.
The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.
Nishi
N
N
Pharmaceutical Research Laboratory of Kirin Brewery Co., Ltd., Gunma, Japan.
Ishikawa
R
R
Inoue
H
H
Nishikawa
M
M
Kakeda
M
M
Yoneya
T
T
Tsumura
H
H
Ohashi
H
H
Yamaguchi
Y
Y
Motoki
K
K
Sudo
T
T
Mori
K J
KJ
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D002454:Cell Differentiation / Q000187:drug effects; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004357:Drug Synergism; D016179:Granulocyte Colony-Stimulating Factor / Q000302:isolation & purification / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008562:Membrane Glycoproteins; D051379:Mice; D009699:N-Glycosyl Hydrolases; D019089:Stem Cell Factor / Q000302:isolation & purification / Q000494:pharmacology*; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D017077:Culture Media, Conditioned; D008562:Membrane Glycoproteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1