Murine stromal cells counteract the loss of long-term culture-initiating cell potential induced by cytokines in CD34(+)CD38(low/neg) human bone marrow cells

Evidence has been provided recently that shows that high concentrations of cytokines can fulfill functions previously attributed to stromal cells, suc h as promote the survival of, and led to a net increase in human primitive progenitors initiating long-term cultures in vitro (LTC-IC) or engrafting N OD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in v ivo, These data prompted us to reevaluate whether stromal cells will furthe r alter the properties of primitive progenitor cells exposed to cytokines. Single CD34(+)CD38(low) and CD38(neg) cells were incubated 10 days in serum -containing or serum-free medium in the presence or in the absence of murin e marrow-derived stromal cells (MS-5). Recombinant human cytokines stem cel l factor (SCF), pegylated-megakaryocyte growth and differentiation factor ( PEG-MGDF), FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage col ony-stimulating factor (GMCSF) were systematically added at various concent rations (10 to 300 ng/mL). Cell proliferation and LTC-IC potential were eva luated in each clone after 10 days. A striking and consistent observation w as the retention of a high LTC-IC potential in clones exposed to cytokines in the presence of stromal feeders, whereas clones exposed to cytokines alo ne in the absence of stromal feeders rapidly lost their LTC-IC potential as they proliferated. This was reflected both by the higher proportion of wel ls containing LTC-IC and by the high numbers of CFC produced after 5 weeks in clones grown with MS-5 during the first 10 days, We further showed by an alyzing multiple replicates of a single clone at day 10 that MS-5 cells pro moted a net increase in the LTC-IC compartment through self-renewal divisio ns. Interestingly, these primitive LTC-IC were equally distributed among sm all and large clones, as counted at day 10, indicating that active prolifer ation and loss of LTC-IC potential could be dissociated. These observations show that, in primitive cells, stromal cells counteract differentiation ev ents triggered by cytokines and promoted self-renewal divisions. Furthermor e, the almost identical distribution of the size of the clones with or with out MS-5 suggests that proliferation and function of human primitive cells may be independently regulated by external signals, and that the former is primarily under the control of cytokines. (C) 1999 by The American Society of Hematology.