Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34+ murine bone marrow cells

Objective. The aim of this study was to determine whether the combination of a sizable generation of colony-forming cells (CFC) with the maintenance of their progenitors (pre-CFC) ensured by incubation in hypoxia is associated with a certain degree of cell cycling, ultimately responsible for "self- renewal' I of pre-CFC. The effects of interleukin-3 (IL-3) on the cycling and CFC-generation potential of pre-CFC also was investigated. Materials and Methods. In severely hypoxic (0.9 % O-2) murine bone marrow cell cultures containing stem cell factor, interleukin-6, and granulocyte colony-stimulating factor, pre-CFC maintenance was characterized by the culture-repopulating ability assay, an in vitro analogue of the marrow-repopulating ability assay. The proliferative history of CD34(+) cells in primary cultures was determined by PKH2 staining and related to their CFC-generation potential. In some experiments, subsets of CD34(+) cells sorted on the basis of the number of cell divisions (0, 1, >1) were independently characterized. Results. In hypoxia, the numbers of CFC and CD34(+) cells were markedly reduced, whereas pre-CFC were maintained better than in air. Addition of 5-fluorouracil to hypoxic cultures for 2 days suppressed their CITC-generation potential. The CFC-generation potential of divided CD34(+) cells was maintained or increased with respect to that of undivided cells in hypoxia but not in air. IL-3 decreased CFC-generation potential at both oxygen concentrations. IL-3 also increased the number of CD34(+) cells that divided more than once in hypoxia, decreasing their CITC-generation potential. Conclusions. Maintenance of CFC-generation potential in hypoxia occurs mainly in a subset of cycling progenitors, despite their proliferation (self-renewal). IL-3 decreased CFC-generation potential by increasing the rate of pre-CFC proliferation beyond the first cycle, which probably results in their differentiation and loss of CFC-generation potential. (C) 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.