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+ CD38dim cells but not CD34+ CD38+ cells. In suspension culture, the enhancement of cell expansion by FL was 10 times greater with the CD34+ CD38dim fraction than the CD34+ CD38+ fraction. FL stimulated the generation of colony-forming unit–granulocyte-macrophage (CFU-GM) from the CD34+CD38dim fraction by 14.5- ± 5.6-fold. To determine if CD34+ CD38dim cells responded uniformly to FL, the population was subdivided into a CD34+ CD38dim CD33dim HLA-DR+ (HLA-DR+) fraction and a CD34+ CD38dim CD33dim 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+ CD38dim and HLA-DR+ fractions were incubated in liquid culture and analyzed by flow cytometry. Inclusion of FL enhanced the absolute number of primitive CD34+ CD33dim 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+ CD38dim 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+ CD33dim 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.
Myeloid engraftment after bone marrow transplantation (BMT) is influenced by a number of variables, including cytoreductive chemoradiotherapy, genetic disparity, number of reinfused committed myeloid progenitor cells, healthy microenvironment, and the presence of hematopoietic growth factors. Granulocyte colony-stimulating factor (G- CSF) stimulates proliferation of myeloid progenitor cells and enhances myeloid engraftment after BMT. We investigated the temporal relationship between endogenous G-CSF production and myeloid engraftment in both children and adults after allogeneic (ALLO) and autologous (AUTO) BMT. Circulating endogenous G-CSF levels ranged between 0 and 2552 pg/mL. The correlation coefficient between circulating serum G-CSF levels and the peripheral absolute neutrophil count (ANC) was r = -.875 (P less than .001). The endogenous serum G- CSF level was highest during the first week after BMT, when the ANC was less than or equal to 200/microL (699 +/- 82.3 pg/mL) (P less than .001). Both children and adults demonstrated a similar inverse relationship between circulating G-CSF level and degree of neutropenia. One patient failed to engraft after AUTO BMT and also failed to generate any endogenous G-CSF production. Lastly, once the serum G-CSF level decreased to less than 200 pg/mL, a mean of 6.1 +/- 0.9 days elapsed before the ANC was greater than or equal to 500/microL for 2 consecutive days. This study demonstrates that endogenous G-CSF production is associated with myeloid engraftment in both children and adults after AUTO and ALLO BMT and that the rate of increase and decrease in endogenous G-CSF may be predictive of either failure to engraft or duration of neutropenia.
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