Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T-lymphocyte depleted (NALT^-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 ± 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10⁺⁺⁺DR⁺). This procedure resulted in an 18-fold increase in CE over NALT^- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1α (IL-1α), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin-3 (IL-3) on MK colony formation by My10⁺⁺⁺DR⁺ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1α nor G-CSF alone augmented MK colony formation above baseline (2.5 ± 0.8/5 x 10³ My10⁺⁺⁺DR⁺ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml or IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote magakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10⁺⁺⁺DR⁺ cells are largely devoid to these accessory cells.