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

Publisher Website
Google Scholar
Abstract
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 My 10 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 My 10 and low densities of HLA‐DR (My 10+++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‐lα nor G‐CSF alone augmented MK colony formation above baseline (2.5 ± 0.8/5 × 103 My 10+++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 of IL‐3 and 25 units/ml of GM‐CSF. At maximal concentration, IL‐3 had a greater ability to promote megakaryocyte 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 My 10+++DR+ cells are largely devoid of these accessory cells.