BM cellular niche alterations in MLL-AF9 AML mice. (A) Strategy for establishing the MLL-AF9 mouse–induced AML mouse model. Normal and MLL-AF9–expressing BM KIT⁺ cells were transplanted to nonirradiated wild-type C57BL/6 mice. BM stromal cells from control and AML mice were collected and sorted by FACS for subsequent analysis when the mice receiving MLL-AF9–expressing cells developed AML. (B) Platelet numbers in the blood of the AML mice. (C) Increased spleen weight of AML mice. (D) Representative FACS profile showing the gating strategy used for analysis and sorting of BM MSCs and MPCs from control and AML mice. SCA1⁺CD51⁺ MSCs and SCA1⁻CD51⁺ MPCs were first gated within CD45⁻TER119⁻CD31⁻CD44⁻ cells. CD31⁺ ECs were gated within CD45⁻TER119⁻ cells. PI, propidium iodide. (E) Frequencies of CD45⁻TER119⁻ cells and CD31⁺ ECs in AML mouse BM. (F) Frequency and absolute number of SCA1⁺CD31⁺ arteriolar ECs and SCA1⁻CD31⁺ sinusoidal ECs in AML mice. (G) FACS histogram of SCA1⁺ arteriolar ECs in AML mice. Cells were gated from CD45⁻TER119⁻CD31⁺ cells. (H) Frequencies of BM MSCs and MPCs in AML mice. (I) Absolute numbers of BM MSCs and MPCs in AML mice. (J) Increased frequency and absolute number of CFU-Fs in AML mice. (K) CFU-F activities in freshly sorted MSCs from control mice and AML mice. (L) Enhanced adipocyte and osteoblast differentiation potential of AML MSCs in vitro. The adipogenic, osteogenic, and chondrogenic differentiations were stained with oil red, alizarin red, and toluidine blue, respectively. Scale bars represent 250 μm (left), 500 μm (middle), and 100 μm (right). The inserts are the control (Ctr) images of the nondifferentiated culture. P0, postnatal day 0. (M) An overview image of alizarin red staining of the induced osteoblast differentiation of control and AML MSCs. Data were generated from the same experiment as shown in panel L. Scale bar, 500 μm. Statistical analysis was performed using an unpaired 1-tailed (B-C) or 2-tailed (E-K) Student t test or Mann-Whitney U test. Each dot represents data from an individual mouse. The horizontal bars show mean values. Data are from 2 independent experiments.