Stromal cells expressing Notch ligand block myelopoiesis in vitro. (A) Schematic representation of the in vitro coculture of CMP cells with OP9 cells expressing Notch ligands with cytokine cocktails. (B) Day 14 culture of bone marrow–derived CMP cells, cocultured with OP9-control cells (no ligand) or OP9 cells expressing Notch ligands, were analyzed for Gr-1 expression. Data are mean values of at least 4 determinations. The bar graph represents the percentage of Gr-1⁺ and Gr-1⁻ cells among total viable cells. (C) May-Grünwald-Giemsa–stained cytospins of day 14 cultured cells with OP9-control, OP9-Jagged1, and OP9-Dll1. (D) Short-term culture (60 hours) of CMP cells with SCF was analyzed for the percentages of GMP and MEP generated from CMP (numbers shown to right of graphs). Data are representative of at least 3 independent experiments. (E) Myelopoiesis transcription factors (PU.1 and C/EBPα) were differentially expressed in OP9-Dll1 versus OP9-control cultures. Results (means ± SD; n = 4) have been standardized for GAPDH levels and were expressed as percentage of expression relative to the levels detected in OP9-control (set at 1). ^aTotal number of cells present at the end of culture with 500 cells input grown on OP9 stromal cells with SCF (50 ng/mL), IL-3 (5 ng/mL), Flt3L (10 ng/mL), and GM-CSF (2.5 ng/mL). Data are means plus or minus SD of 6 independent experiments (*P < .05, #P < .01).