Mitochondria uptake is an enhanced phenomenon for primary AML sample compared with normal CB CD34⁺ cells and is increased in vitro and in vivo under cytarabine treatment. (A-C) Exogenous mitochondrial incorporation occurs to a greater extent for primary AML blasts compared with normal lymphoid or CB cells. (A) AML cell lines (green bars, n = 4) or AML primary (red bars, n = 4) or CB mononuclear samples (blue bars, n = 3) were cocultured with MS-5 control or MS-5 OMI/HTRA2-mCherry for 72 hours. Data show the absolute percent of mCherry-positive human cells determined by FCM. (B) AML primary or CB mononuclear samples were cultured for 72 hours with MitoTracker Green loaded MS-5. (Upper) Gating strategy for monitoring the MitoTracker green fluorescence intensity for 1 AML sample in the human live (hCD45⁺SCA-1⁻Annexin-V⁻DAPI⁻) myeloblasts (hCD34⁺hCD3⁻; red, blasts) and lymphoid (hCD34⁻hCD3⁺; green, lympho) populations. (Lower) Representative histograms plots for 1 AML patient and 1 CB. Gray dashed line represents the MitoTracker fluorescence intensity of MS-5 cells (FSC-high, SCA-1⁺). (C) MitoTracker MFI in the human live blasts and lymphoid populations determined for 14 AML and 9 CB primary samples. Paired t test was applied for blasts vs lympho compartments for intrasample comparison. A Mann-Whitney t test was applied for AML vs CB comparison. (D) ARA treatment increases AML uptake of functional mitochondria. AML#5957 cells (left) or AML#C3-1 cells (right) were cocultured with MitoTracker Green (MT)–loaded MS-5 or MS-5 OMI/HTRA2-mCherry cells in triplicate wells for 72 hours with the indicated dose of ARA. For each sample, data show the mean percentage (±standard deviation [SD]) of human live cell count and the mean ± SD MitroTracker MFI or the mean percentage ± SD of mCherry⁺ normalized to control condition for the hCD45⁺SCA-1⁻hCD34⁺Annexin-V⁻DAPI⁻ AML cells. (E) Primary AML samples or MOLM-14 cells were injected in NSG mice. Recipient mice for MOLM-14 were treated at week 2 after injection with ARA 60 mg/kg per day over 5 days. Representative FACS analysis of hCD45 and CD33 expression before and after cell sorting of mice bone marrow at week 12 after injection of AML#C3-1. (F) Gels showing amplified DNA sequences from 4 primary AML isolated or sorted after in vivo engraftment and from isolated NSG mice bone marrow cells; hu GAPDH, human glyceraldehyde-3-phosphate dehydrogenase; mu Rplp0, mouse ribosomal protein, large, P0; mu mt-Co2, mouse mitochondrially encoded cytochrome c oxidase II. (G) Murine and human nuclear and mitochondrial gene expression profiles determined by quantitative real-time PCR from isolated NSG mice bone marrow cells, MOLM-14 isolated or sorted after in vivo engraftment from untreated (untr) or ARA-treated mice (n = 3 mice per group). 36b4, mouse nuclear encoded 60S acidic ribosomal protein P0; murine or human mt-CO2, mitochondrial encoded cytochrome c oxidase II. ΔCt corresponds to the difference between Ct of the tested specimens and the Ct of isolated human or murine cells. ^NSP > .05, *P < .05, ***P < .001 determined by ANOVA.