5-FLUOROURACIL PERMITS ACCESS TO A PRIMITIVE SUBPOPULATION OF PERIPHERAL-BLOOD STEM-CELLS

Peripheral blood stem cells (PBSC) contain a mixture of mature and imm ature hematopoietic progenitors. Resistance to 5-Fluorouracil (5-FU) h as been used to identify and characterize primitive quiescent stem cel ls among bone marrow (BM) cells. To see if the same technique could be used to isolate a similar population of cells among PBSC, low-density peripheral blood mononuclear cells (PBMNC) were collected by cytapher esis in the regenerative phase after high-dose chemotherapy from patie nts with hematological malignancies. These PBMNC were incubated with i ncreasing concentrations of 5-FU for 24 h. The viable 5-FU resistant c ells were then cultured in semi-solid media in the presence of either single cytokines: TCM 5637, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), or a combination of cytokines: interleukin 1 (IL-1) I L-1+IL-3+5637, 3+Stem Cell Factor (SCF). Low concentrations (5-10 pg/m l 5-FU) eliminated mature day 7 Colony Forming Units-Granulocyte Macro phage (CFU-GM) and spared day 7 clusters while enriching for day 14 CF U-GM, irrespective of the growth factors used. Higher concentrations o f 5-FU (15, 20, 25 mug/ml) selected for later forming clonogenic eleme nts. A combination of synergistic growth factors was required for the development of morphologically identifiable clonogenic elements resist ant to 25 mug/ml 5-FU at day 21 of culture. Further experimentation de monstrated that SCF could effectively replace TCM 5637 in the cytokine combination for the detection of primitive late forming clonogenic el ements. The presence of SCF potentiated colony formation by 5-FU resis tant PBMNC. It was confirmed that GM-CSF alone was unable to support c olony formation by PBMNC resistant to 25 mug/ml. These observations de monstrate that PBSC contain a heterogenous mixture of hematopoietic pr ogenitors and that incubation with 25 mug/ml 5-FU permits access to a quiescent primitive stem cell population that requires a combination o f svnergistic growth factors for the development of morphologically id entifiable clonogenic elements at day 21. Taken together, these result s suggest that PBSC have similar characteristics to BM derived stem ce lls.