title: Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa.
abstract: We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.
authors.affiliation: Pharmaceutical Research Laboratory, Kirin Brewery Company, Limited, Gunma, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Miyazaki
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: M
authors.initials: M
authors.lastname: Yanagida
authors.forename: K
authors.initials: K
authors.lastname: Horie
authors.forename: T
authors.initials: T
authors.lastname: Mikayama
authors.forename: H
authors.initials: H
authors.lastname: Ohashi
authors.forename: M
authors.initials: M
authors.lastname: Nishikawa
authors.forename: T
authors.initials: T
authors.lastname: Suzuki
authors.forename: T
authors.initials: T
authors.lastname: Sudo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002469:Cell Separation / Q000379:methods*; D003208:Concanavalin A / Q000494:pharmacology; D003470:Culture Media; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000276:immunology*; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000494:pharmacology; D013154:Spleen / Q000166:cytology; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D016298:Hematopoietic Cell Growth Factors; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D003208:Concanavalin A