In vitro expansion of human AML cells. (A) Growth curves (top panels) and morphology (bottom panels) of primary AML cells plated on different stromal cells for 3 weeks. All cells were grown in the presence of human IL3, IL6, SCF, TPO, and FLT3 ligand. (B) Metaphase fluorescence in situ hybridization (FISH) for MLL rearrangement on UPN 410324 after 1 week of stromal coculture with a dual-color break apart probe, showing 1 normal 11q23 locus (yellow, arrow) and a typical rearrangement (separate red and green), as well as an extra 3′ MLL signal (red). This pattern was seen in 100 of 100 cells, and the identical rearrangement pattern was seen at the time of diagnosis. (C) Identification of AML-specific mutations in 2 primary AML samples. Shown are the variant allele frequencies at day 0 (black) and after 7 days of culture on HS27 stroma (red). (D) Engraftment of UPN 476081 at 16 weeks in the bone marrow of NSG mice after 2 weeks of growth on HS27 (right), compared with 476081 after overnight culture (left).