Cell cycle analysis of fresh, ex vivo–cultured and BM- and spleen-homed MPB and BM CD34⁺ cells.

CD34⁺ cells from fresh MPB (light bars) or BM (dark bars) were enriched by immunomagnetic selection and stained with CFSE as described in “Materials and methods.” A sample was retained for cell cycle analysis at time zero and for initiating short-term ex vivo cultures. Cells in culture were maintained with a 6-cytokine cocktail containing human SCF at 100 ng/mL, Flt-3 ligand at 50 ng/mL, MGDF at 50 ng/mL, IL-3 at 100 ng/mL, IL-6 at 100 ng/mL, and GM-CSF at 20 ng/mL. Remaining cells were transplanted into conditioned NOD/SCID mice, and 40 hours AT bone marrow (AT/BM) and spleen (AT/Spl) cells were recovered from individual mice and human CFSE⁺ cells were isolated by flow cytometric cell sorting. Sorted cells and cells maintained in culture were stained with PI and analyzed for cell cycle status. Each bar represents the mean ± SD of 2 to 6 measurements for MPB samples (from 3 independent experiments) and 4 to 5 measurements for BM samples (from 5 independent experiments). Differences between fresh and cultured BM and MPB cells,P < .01; differences between ex vivo–cultured BM and MPB and BM- or spleen-homed cells, P < .01; differences between fresh and BM- or spleen-homed BM and MPB cells,P > .01.