EFFECT OF FLT3 LIGAND ON IN-VITRO GROWTH AND EXPANSION OF COLONY-FORMING BONE-MARROW CELLS FROM PATIENTS WITH APLASTIC-ANEMIA

To determine the value of flt3 ligand (flt3L) in stimulating hematopoi esis in human hypoproliferative bone marrow disorders, we examined its in vitro effect on bone marrow cells from patients with aplastic anem ia (AA). Growth response to flt3L, alone and in combination with other hematopoietic growth factors, was investigated in clonogenic methylce llulose assays, in long-term liquid and stroma cultures. Bone marrow c ells were derived from 13 AA patients with persisting in vitro growth defect after immunosuppressive treatment and from nine normal bone mar row donors. In methylcellulose cultures, flt3L stimulated formation of hematopoietic colonies only weakly, whereas it had an additive effect when combined with erythropoietin (Epo), stem cell factor (SCF), inte rleukin-3 (IL-3), interleukin-11 (IL-11), or granulocyte colony-stimul ating factor (G-CSF). Flt3L was less effective than SCF and did not fu rther enhance the number of hematopoietic colonies formed in response to SCF-containing combinations of multiple cytokines. In long-term liq uid suspension cultures, flt3L was less mitogenic than SCF but its eff ect on the maintenance of progenitors was superior that of SCF and of IL-3, IL-11, and G-CSF. The total number of clonogenic AA cells increa sed as much as four-fold during the first culture week and FACS analys is demonstrated expansion of the CD34(+)CD38(+) progenitor cell subset . Despite this enhancement, survival of AA cells remained significantl y poorer than that of normal cells, in which the primitive subset of C D34(+)CD38(-) cells was maintained up to 4 weeks when flt3L was used a s a single factor. Both in normal and AA cultures, flt3L promoted diff erentiation of cells of the myeloid lineages. In cultures of bone marr ow stroma, flt3L had almost no effect on growth and survival of AA pro genitors, while in cultures of normal cells the number of colony-formi ng cells increased up to 10-fold. Although flt3L does not overcome the proliferative defect of AA precursors, we conclude that the ligand is capable of in vitro stimulation and expansion of the reduced progenit or cell pool in AA, when used in appropriate culture conditions. The i n vitro effects of flt3L on AA cells differ in many aspects from those of the structurally related cytokine SCF, suggesting a benefit in use of a combination of these two early-acting growth factors.