Effects of GST-hDll1^NDSL in myelopoiesis.

Mouse Lin⁻ bone marrow cells were cultured for 7 days in the serum-free medium with IL-3, GM-CSF, and IL-1. GST-hDll1^NDSL or control GST was added to the cultures at 0.1 μmol/L every 3 days. The cultures were initiated with 3 × 10⁴ Lin⁻cells in 3 mL serum-free medium. All values represent mean ± SE from five independent experiments. (A) Differentiation analysis. Lin⁻ cells cultured for 7 days with GST or GST-hDll1^NDSL were washed and labeled with FITC- and phycoerythrin (PE)-conjugated mAbs directed to Mac-1 or Gr-1. The top panel of fluorescence histograms shows the cells stained with control isotype antibodies (IgG_2b-FITC and IgG_2b-PE). The middle panel shows the cells cultured with GST and stained with anti-Mac-1 or anti-Gr-1 antibodies. The bottom panel shows the cells cultured with GST-hDll1^NDSL and stained with the above two antibodies. The x-axis represents log fluorescence intensity, and the y-axis represents cell numbers. Percentages of Mac-1- or Gr-1-positive cells are indicated in parentheses, and the MIF is shown for the corresponding M1 population of cells. (B) Progenitor cell assay. Lin⁻ cells before and after culture for 7 days with GST and GST-hDll1^NDSL were plated in triplicate in methylcellulose supplemented with IL-3, GM-CSF, KL, and Epo. Colonies were scored under microscope, including the CFU mix consisting of multiple lineages of at least granulocyte/macrophage and erythroid clusters, CFU-GM, burst-forming unit–erythrocyte (early BFU-E consisting of multiple erythroid clusters and late BFU-E as a single compact erythroid colony at day 7 of culture), and high-proliferation potential–CFU colonies (larger than 0.5 mm in diameter). Columns represent fold expansion of colonies formed by the progenitors. The fold expansion was calculated as the number of colonies from the day 7 cultured cells divided by the colonies from the cells before culture. Differences between the GST and GST-hDll1^NDSL groups are statistically significant for CFU mix and early BFU-E (P = .02) and very significant for CFU-GM (P = .02), late BFU-E (P = .01), and total CFU (P = .001).