Flt3 ligand enhances the yield of primitive cells after ex vivo cultivation of CD34(+) CD38(dim) cells and CD34(+) CD38(dim) CD33(dim) HLA-DR+ cells

Flt3 ligand (FL) has been proposed as a possible modulator of early hematopoietic cell growth. The purpose of this study was to analyze the impact of FL on ex vivo expansion of hematopoietic cells obtained from adult donors. We sought to precisely identify hematopoietic populations responsive to FL and to quantitate the ability of FL to enhance the survival and/or proliferation of early hematopoietic precursors in a stroma free culture system. Towards that end, four CD34(+) subsets were isolated and their response to FL was characterized. In methylcellulose, FL significantly increased colony formation by CD34(+) CD38(dim) cells but not CD34(+) CD38(+) cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34(+) CD38(dim) fraction than the CD34(+) CD38(+) fraction. FL stimulated the generation of colony-forming unit-granulocyte-macrophage (CFU-GM) from the CD34(+)CD38(dim) fraction by 14.5- +/- 5.6-fold. To determine if CD34(+) CD38(dim) cells responded uniformly to FL, the population was subdivided into a CD34(+) CD38(dim) CD33(dim) HLA-DR+ (HLA-DR+) fraction and a CD34(+) CD38(dim) CD33(dim) HLA-DRdim (HLA-DRdim) fraction. FL was far more effective at stimulating cell and progenitor growth from the HLA-DR+ fraction. To determine if FL enhanced or depleted the number of precommitted cells in expansion culture, CD34(+) CD38(dim) and HLA-DR+ fractions were incubated ist liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34(+) CD33(dim) cells and CD34(+) HLA-DRdim cells after 5 to '12 days of cultivation. To confirm immunophenotypic data, the effect of FL on long-term culture-initiating cells (LTCIC) was determined. After 2 weeks of incubation of CD34(+) CD38(dim) or HLA-DR+ cultures, LTCIC recoveries were significantly higher with FL in 5 of 6 trials (P <.05). For HLA-DR+ cells, LTCIC recoveries averaged 214% +/- 87% of input with FL and 24% +/- 16% without FL. in contrast, HLA-DRdim LTCIC could not be maintained in stroma-free culture. We conclude that less than 10% of CD34(+) cells respond vigorously to FL and that those cells are contained within the HLA-DR+ fraction. FL stimulates the expansion of total cells, CD34(+) cells, and CFU-GM and enhances the pool of early CD34(+) CD33(dim) cells, CD34(+) HLA-DRdim cells, and LTCIC. These data indicate that it is possible to expand hematopoietic progenitors from adult donors without losing precursors from the precommitted cell pool. (C) 1997 by The American Society of Hematology.