Purification of human marrow progenitor cells and demonstration of the direct action of macrophage colony-stimulating factor on colony-forming unit-macrophage.
To facilitate the investigation of the direct interaction between hematopoietic progenitors and colony-stimulating factors, we have developed a method to purify human marrow progenitor cells. Using density centrifugation, negative panning with concanavalin A coated plates, positive selection of CD34-positive cells with immunomagnetic microspheres, overnight adherence to a plastic dish, negative selection with a panel of monoclonal antibodies, and density centrifugation, human marrow progenitor cells were purified from 1.5% to 53.2%, a 42-fold purification, with a 4.8% yield. The purified cells consisted of 38% erythroid, 9% colony forming unit-granulocyte (CFU-G), 29% CFU-macrophage (CFU-M), 12% CFU-eosinophil/basophil (CFU-Eo/Ba), and 4% CFU-mix. The purified cells cultured in serum-free fibrin clots with recombinant human macrophage colony-stimulating factor (rM-CSF) for 14 days developed a pure population of CFU-M colonies. An appearance of CFU-M colonies was present after the addition of 1 U/mL of rM-CSF and the maximum stimulation was found at 100 U/mL. When the purified cells were cultured in serum-free medium with rM-CSF in a limiting dilution assay and the percentage of nonresponder wells for CFU-M colonies was plotted against cell concentration, serum-free cultures yielded a straight line through the origin, indicating that CFU-M development did not depend on accessory cells and that rM-CSF acted directly on the CFU-M.
Sato
N
N
Second Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo.
Sawada
K
K
Kannonji
M
M
Tarumi
T
T
Sakai
N
N
Ieko
M
M
Sakurama
S
S
Nakagawa
S
S
Yasukouchi
T
T
Krantz
S B
SB
D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D001854:Bone Marrow Cells; D002455:Cell Division; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A; D003470:Culture Media; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology*; D008280:Magnetics; D008863:Microspheres; D011994:Recombinant Proteins / Q000494:pharmacology
D015703:Antigens, CD; D018952:Antigens, CD34; D003470:Culture Media; D011994:Recombinant Proteins; D003208:Concanavalin A; D016173:Macrophage Colony-Stimulating Factor