Flt3 Ligand Enhances the Yield of Primitive Cells After Ex Vivo Cultivation of CD34+ CD38dim Cells and CD34+ CD38dim CD33dim HLA-DR+ Cells

Abstract

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.

Endurance Exercise Improves Molecular Pathways of Aerobic Metabolism in Patients With Myositis

“…In the current study the observed increase in capillary density in the exercise group is consistent with an increased expression of genes related to capillary growth (i.e., FLT3L), which was not found in the control group . Also, we observed an up‐regulation of genes that promote vascular endothelial growth factor–mediated angiogenesis, such as the gene for insulin‐like growth factor 1 receptor (IGF‐1R) .…”

Selective in vitro expansion and efficient retroviral transduction of human CD34+ CD38– haematopoietic stem cells

“…In contrast, with the combination of FL, TPO and SCF, we observed strong proliferation of both CD34 + and CD34 + CD38 – cells. As it has been reported (Shah et al , 1996; Dooley et al , 1997) that FL combined with IL‐3, IL‐6 and SCF acts as a potent growth factor mixture for HSC, we systematically compared the expansion of CD34 + CD38 – cells from CD34 + enriched BM, CB and PBSC, using the combinations FL/TPO/SCF and FL/IL‐3/IL‐6/SCF. Stimulation of purified CD34 + BM cells, containing approximately 1% to 2% CD34 + CD38 – cells, with both cytokine mixtures resulted in the differentiation of a substantial proportion of cells as evidenced by the progressive loss of CD34 expression.…”

“…While our studies were in progress, several groups reported ex vivo expansion of CD34 + or CD34 + CD38 – cells using different combinations of interleukin 3 (IL‐3), IL‐6, FL, TPO, stem cell factor (SCF) and leukaemia inhibitory factor (LIF) (Petzer et al , 1996; Shah et al , 1996; Ramsfjell et al , 1997; Luens et al , 1998). It was demonstrated that FL is a potent growth factor for CD34 + CD38 – cells (Dooley et al , 1997) and CD34 + /Thy‐1 + bone marrow (BM) or peripheral blood stem cells (PBSC), a population with a high degree of overlap with the CD34 + CD38 – population (Craig et al , 1993). Purified PBSC CD34 + /Thy‐1 + cells that are stimulated with FL/TPO/SCF retain primitive stem cell activity as measured by their capacity to repopulate SCID mice with haematopoietic cells and to form distinct colonies in the CAFC assay (Murray et al , 1999; Young et al , 1999).…”

Ex vivo expansion of long‐term culture initiating marrow cells by IL‐10, SCF, and IL‐3

“…Maintenance or even expansion of LTC-ICs may be achieved by cultivation of marrow progenitor cells on preformed stroma layers of marrow cells or on engineered murine stroma layers by enrichment from highly purified primitive hematopoietic subpopulations supplemented with early acting stimulative cytokines or by frequent media exchange in bioreactors. 11,[33][34][35] However, cell loss during purification of progenitor cells might reduce the net expansion of progenitor cells in this timeconsuming and extensive procedure, and accessory mar-row cells lost in this procedure may play an important role in expansion of LTC-ICs. 16 The exact mechanism by which IL-10 sustains LTC-ICs and expands these cells in combination with IL-3 and SCF, respectively, remains unclear.…”