In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.
When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect 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
Yoneya
T
T
Kakeda
M
M
Tsumura
H
H
Ohashi
H
H
Mori
K J
KJ
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D001854:Bone Marrow Cells; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D004247:DNA / Q000096:biosynthesis; D004357:Drug Synergism; D005312:Fetal Blood; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins; D051379:Mice; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D004247:DNA; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1; D013936:Thymidine