Effect of FLT3 ligand on in vitro expansion of colony-forming bone marrow cells from patients with aplastic anemia

To determine the value of flt3 ligand (flt3L) in stimulating hematopoiesis in hypoproliferative bone marrow disorders, we examined its in vitro effect on bone marrow cells from patients with aplastic anemia (AA). In methylcellulose cultures, fli3L stimulated formation of hematopoietic colonies only weakly; it had an additive effect when combined with erythropoietin, stem cell factor (SCF), interleukin (IL)-3 and IL-11 or granulocyte colony-stimulating factor (G-CSF). Flt3L was less effective than SCF and did not further enhance the number of colonies formed in response to SCF-containing combinations of multiple cytokines. In long-term liquid suspension cultures, flt3L was less mitogenic than SCF but its effect on the maintenance of progenitors was superior to the effect of SCF and of IL-3, IL-11 and G-CSF. The tola! number of clonogenic cells increased up to 4 fold and FACS analysis demonstrated an expansion of the CD34 ⁺ CD38 ⁺ progenitor cell subset during the first culture week. Survival of AA cells was significantly poorer than of normal cells of which the primitive subset of CD34 ⁺ CD38 ⁺ cells was maintained for up to 4 weeks with flt3L used as a single factor. In cultures of bone marrow stroma, flt3L had almost no effect on growth and survival of AA progenitors, while it enhanced the number of normal colonyforming cells up to 10 fold. Flt3L was also tested in cultures supported by feeder layers of murine Steel stromal cell line S/AS/ ^h220 transfected with human cDNA expressing SCF as a membrane-bound molecule. In cultures of normal marrow cells, flt3L enhanced the output of progenitor cells representing colony-forming units (CPU) and high-proliferative potential colony-forming (HPP-CFC)-. type progenitors by 6-9 fold after 3 weeks, and of long-term culture-initiating cells (LTC-lC)-derived colonies by 13-fold after 5 weeks of culture. In Sl/Sl ^h220 cultures of AA cells, the presence of flt3L yielded a 2-6 fold enhancement of CFU and HPPCFC at 1-2 weeks. Both in normal and AA cultures, fH3L promoted primarily differentiation of the granulocyte/macrophage lineage. Although in none of the culture systems employed, flt3L could overcome the proliferative defect of AA precursors, we conclude that the ligand is capable of in vitro stimulation and expansion of the reduced progenitor cell pool in AA, when used in appropriate culture conditions. The in vitro effects of flt3L on AA cells differ in many aspects from those of the structurally related cytokine, SCF.