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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 529-538
Murine Stromal Cells Counteract the Loss of Long-Term
Culture-Initiating Cell Potential Induced by Cytokines in
CD34+CD38low/neg Human Bone Marrow Cells
Annelise Bennaceur-Griscelli,
Cristina Tourino,
Brigitte Izac,
William Vainchenker, and
Laure Coulombel
From INSERM U 362, Institut Gustave Roussy, Villejuif, France.
Evidence has been provided recently that shows that high
concentrations of cytokines can fulfill functions previously attributed to stromal cells, such 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 NOD-SCID (nonobese diabetic severe-combined immunodeficient) recipients in vivo. These data prompted us to re-evaluate whether stromal cells will further alter the
properties of primitive progenitor cells exposed to cytokines. Single
CD34+CD38low and CD38neg cells
were incubated 10 days in serum-containing or serum-free medium in the
presence or in the absence of murine marrow-derived stromal cells
(MS-5). Recombinant human cytokines stem cell factor (SCF),
pegylated-megakaryocyte growth and differentiation factor (PEG-MGDF),
FLT3-L, Interleukin (IL)-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were systematically added at various concentrations (10 to 300 ng/mL). Cell proliferation and LTC-IC
potential were evaluated in each clone after 10 days. A striking and
consistent observation was the retention of a high LTC-IC potential in
clones exposed to cytokines in the presence of stromal feeders, whereas
clones exposed to cytokines alone in the absence of stromal feeders
rapidly lost their LTC-IC potential as they proliferated. This was
reflected both by the higher proportion of wells 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 analyzing multiple
replicates of a single clone at day 10 that MS-5 cells promoted a net
increase in the LTC-IC compartment through self-renewal divisions.
Interestingly, these primitive LTC-IC were equally distributed among
small and large clones, as counted at day 10, indicating that active
proliferation and loss of LTC-IC potential could be dissociated. These
observations show that, in primitive cells, stromal cells counteract
differentiation events triggered by cytokines and promoted self-renewal
divisions. Furthermore, the almost identical distribution of the size
of the clones with or without 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.
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