THE MEMBRANE-BOUND ISOFORM OF STEM-CELL FACTOR SYNERGIZES WITH SOLUBLE FLT3 LIGAND IN SUPPORTING EARLY HEMATOPOIETIC-CELLS IN LONG-TERM CULTURES OF NORMAL AND APLASTIC-ANEMIA BONE-MARROW

The hematopoietic growth factors stem cell factor (SCF) and flt3 ligan d (flt3L) are produced within the hematopoietic microenvironment in a membrane-bound and soluble isoform. To elucidate the relevance of the two isoforms in the network of early-acting cytokines, we examined the interaction of membrane-bound SCF with the soluble forms of SCF and f lt3L in long-term cultures of human bone marrow cells. Feeder layers o f the murine SCF-deficient Steel stromal cell line transfected with hu man cDNA stably expressing SCF as a transmembrane molecule were used t o support growth of mononuclear cells and CD34(+) progenitors derived from normal human bone marrow or from hypoplastic marrow of patients w ith aplastic anemia (AA). The output of nonadherent progenitor cells r epresenting colony-forming units (CFU) and high-proliferative potentia l colony-forming cells (HPP-CFC) was scored weekly in secondary methyl cellulose cultures; the number of colonies derived from long-term cult ure-initiating cells (LTC-IC) was determined in nonadherent and adhere nt cells at 5 weeks. Membrane-bound SCF expressed in the stromal layer was more effective than soluble SCF and soluble flt3L in maintaining clonogenic progenitors. Furthermore, the transmembrane form of SCF eff ectively synergized with both exogenously supplied factors, although t he effect of flt3L was superior to the effect of soluble SCF. In cultu res of normal bone marow cells, addition of flt3L enhanced the total n umber of CFU and HPP-CFC-type progenitors, primarily of the granulocyt e/macrophage lineage, by six-to ninefold after 3 weeks and of LTC-IC-d erived colonies by 13-fold after 5 weeks of culture. In cultures of AA cells, both the number and the survival rate of clonogenic precursors were severely impaired even in the presence of flt3L, which, however, yielded a two-to sixfold enhancement of CFU and HPP-CFC numbers at 1 to 2 weeks. In comparison with the hematopoietic function of human Dex ter-type stroma cultures, murine feeders expressing high levels of mem brane-associated human SCF were equivalent in supporting hematopoiesis during the initial 3 to 4 culture weeks when supplemented with flt3L. These results demonstrate that soluble flt3L interacts with membrane- bound SCF in supporting the long-term growth of bone marrow progenitor cells. The hypothesis that SCF and flt3L function synergistically dur ing the very early stages of human hematopoiesis is thereby reinforced .