Isolation of human megakaryocytes by immunomagnetic beads

Abstract

A simple method was developed to purify human megakaryocytes to homogeneity from normal bone marrow aspirates. An initial separation of marrow between 1.020 and 1.050 g/ml. Percoll density cut was used to enrich megakaryocytes. After washing, the cells were suspended with immunomagnetic beads which were coated with sheep anti-mouse IgG antibody and treated with anti-human glycoprotein (GP) IIb/IIIa monoclonal antibody, or the cells were treated with human platelet GP IIb/IIIa monoclonal antibody and suspended with the immunomagnetic beads which were coated with sheep anti-mouse IgG antibody. Megakaryocytes were selectively separated using a magnet. All of the isolated cells were morphologically recognizable megakaryocytes. 1.5-3.1 x 10(4) megakaryocytes were obtained from 1.7-4.5 x 10(8) bone marrow nuleated cells. These cells were all positive in immunoenzymatic staining for GP IIb/IIIa. Megakaryocytes obtained by this method responded to recombinant human GM-CSF (rhGM-CSF) showing an increased 3H-thymidine (3H-dT) incorporation. These data show that this method is useful for obtaining pure megakaryocyte populations which can be submitted to comprehensive biological studies.