Mitochondria recipient AML cells and LICs display resistance to cytarabine-induced apoptosis and an enhanced replating potential. (A-B) FACS analysis of AML sample #5957 cells and AML cell lines cocultured for 72 hours with MitoTracker Green–loaded MS-5 with or without 3 µM cytarabine (ARA). (A) (Upper) FACS histogram analysis of MT uptake by AML #5957 cells (FSC-low, huCD45⁺CD34⁺DAPI⁻). The gray dashed line represents the MT fluorescence intensity of MS-5 cells (FSC-high, huCD45⁻). AML cells were gated for negative-low MT (MT low) and medium-high MT (MT high) populations as defined by the vertical dashed black line. (Lower) MMP using TMRE for MT low or MT high populations in the untreated (CT; lower left panel) and cytarabine conditions (ARA; lower right panel). Dashed black lines define TMRE fluorescence limit for AML cells with polarized (intact ΔΨm) and depolarized (lost ΔΨm) MMP. (B) Data shown represent the mean ± SEM percentage values of AML #5957 cells (left histogram) or for AML cell lines HL60, Kasumi-1, KG-1, THP-1, and U937 (right histogram) with polarized and depolarized MMP in the MT low and the MT high populations in CT and ARA conditions. ^NSP > .05, **P < .01, ***P < .001 by ANOVA. (C) HL-60, Kasumi-1, Molm-14, and U937 cells were cocultured for 72 hours with MS-5 OMI/HTRA2-mCherry with or without cytarabine 3 µM. Histogram shown represents the mean percentage of Annexin-V⁻ and DAPI⁻ in the mCherry⁻ or mCherry⁺ cells of the GFP⁺ AML population. (D) Functional mitochondria in stromal cells is required to maintain regrowing potential after ARA treatment. The specified AML cells were cocultured with MS-5 or p0 MS-5 with ARA for 1 week. Then, cocultures were washed, medium was changed, and leukemic regrowth was monitored per well over 5 weeks. (E) T₀-sorted LICs and non-LICs of AML samples #C3-1 and #3315 were cocultured with MT-loaded MS-5 for 72 hours with or without ARA 3 µM. Histograms represent the percentage values of Annexin-V⁻ and DAPI⁻ cells in the MT low or the MT high population of LIC and non-LIC in CT or ARA conditions. Refer to supplemental Figure 8A-B and to “Materials and methods” for LICs and non-LICs population definition. (F) Mitochondria recipient AML cells have a selective advantage for replating potential. AML #C3-1, #C3-2, #C3-4, and #5957 cells were cocultured for 72 hours with MT-loaded MS-5 with or without 3 µM cytarabine (ARA). MT low or MT high populations were sorted at 72 hours from CT or ARA conditions and replated in limiting dilution. Data represent leukemic long-term culture initiating cells frequencies determined at week 5 after replating. * P < .05 by paired t test.