Primary human AML cells resulted in BM stromal cell alterations in NSG-SGM3 mice. Primary patient AML cells with or without MLL-AF9 mutations were transplanted by intrafemoral injection into sublethally irradiated NSG-SGM3 mice. Femurs and tibias were collected to analyze AML cell engraftment and BM stromal cell phenotype 6 to 20 weeks after transplantation. Engraftment levels (3.8% to 94.6% of total BM cells) in NSG-SGM3 mice were included in the analysis. (A) Representative FACS profile showing the gating strategy used for phenotypic analysis of BM stromal cells, including ECs, MSCs, and MPCs, and human CD45 (hCD45)⁺ cells in NSG-SGM3 mice transplanted with phosphate-buffered saline (control) or normal BM CD34⁺ cells or patient AML mononuclear cells (AML). CD44⁻ cells were first gated within CD45⁻TER119⁻CD31⁻ cells and then subdivided into SCA1⁺CD51⁺ MSCs and SCA1⁻CD51⁺ MPCs, as indicated. CD31⁺ ECs were gated within CD45⁻TER119⁻ cells. Human CD45⁺ cells were gated within live (propidium iodide negative [PI⁻]) mononuclear cells. (B) Engraftment of primary AML cells from patient samples and CD34⁺ cells from healthy donors in NSG-SGM3 mice. (C) Increased frequencies of BM SCA1⁺CD51⁺ MSCs, SCA1⁻CD51⁺ MPCs, CD45⁻TER119⁻CD31⁺ ECs, and CD45⁻TER119⁻CD31⁺SCA1⁺ arteriolar ECs in NSG-SGM3 mice after transplantation. (D) Total BM cell counts in xenografted NSG-SGM3 recipient mice. (E) Absolute counts of ECs (CD45⁻TER119⁻CD31⁺), SCA1⁺CD51⁺ MSCs, and SCA1⁻CD51⁺ MPCs in NSG-SGM3 recipient mice. The green dots in panels B-E represent the cell counts in recipient mice 8 weeks after transplantation of healthy CD34⁺ BM cells. The blue dot represents cell count in the NSG mouse with 94.6% AML engraftment. Black dots indicate data from adult AML cells transplantation; red dots, pediatric AML donor; orange dots, pediatric AML donor with MLL-AF9 mutation. The horizontal bars in panels B-C,E show median values. Data are from 4 independent experiments. Differences were determined using an unpaired Student t test or Mann-Whitney U test.