Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL- 1/IL-3. This indicates that an earlier cell type was affected by IL- 3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL- 6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

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