VPA treatment generates HSCs with a remodeled mitochondrial network. (A) Mitochondrial mass, membrane potential, and superoxide anion levels (ROS) in different subsets of CD34⁺ cells expanded for 4 days with VPA, as determined by staining with MitoTrackerGreen, tetramethylrhodaminemethyl ester, or MitoSOXRed. Bars represent the fold change of median fluorescence intensity (MFI) relative to the corresponding CD34⁺ subsets cultured with cytokines alone (dotted line) (n = 11). (B-C) Flow cytometer histograms indicating mitochondrial ROS levels (B) and mitochondrial mass (C) in CD34^highCD90^high (gray) and CD34^lowCD90^low cells (red) expanded for 4 days with VPA (n = 13). Similar numbers of CD34^highCD90^high and CD34^lowCD90^low cells were analyzed in panels B and C. Numbers in panel B represent the average fold change of ROS levels. (D) Mitochondrial to nuclear DNA ratio in the isolated CD34⁺ cells. Graphs represent the relative fold change of mtDNA/nDNA ratio of CD34⁺ cells treated with VPA normalized to that of CD34⁺ cell treated with cytokines alone within each experiment (n = 8). (E-F) Electromicrographs of CD34⁺ cells isolated after 4 days of expansion. Images were captured: (a) magnification ×1000; scale bar, 10 μm; (b) magnification ×2500; scale bar, 5 μm; (c) magnification ×10 000; scale bar, 1 μm. Numbers represent the average mitochondrial number per each cell (n = 16). (G) Confocal microscopic images of CD34⁺ cells isolated after 4 days of expansion and stained with 4′,6-diamidino-2-phenylindole (blue-nuclei) and MitoTrackerOrange (red; scale bar, 10 μm) or stained for mitochondrial membrane protein Tomm20 (green; scale bar, 7.5 μm). (H-I) Oxygen consumption rate (H), basal and maximal oxygen respiration and ATP production (I) measured by seahorse analyzer in CD34⁺ cells expanded for 4 days (n = 7). (J) Enrichment of glycolysis pathway analyzed by GSEA on the upregulated genes identified by the bulk RNA-seq in CD34⁺ cells cultured for 4 days with cytokines with VPA relative of CD34⁺ cells cultured with cytokines alone. (K) Fold change of the metabolic potential represented as glycolytic (extracellular acidification rate [ECAR]) to aerobic (oxygen consumption rate [OCR]) metabolism ratio of CD34⁺ cells expanded for 4 days with VPA relative to CD34⁺ cells treated with cytokines alone.³⁹  Metabolic potential represents the percentage of stressed OCR and ECAR values measured on stimulation with oligomycin and carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) over basal OCR and ECAR levels, respectively (n = 3). Experiments in H, I, and K were performed in triplicates, and values in I and K were normalized within each experiment. “n” is the number of biological replicates. Error bars with SEM, *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001 were determined by 2-way ANOVA for panel A and unpaired Student t test for panels B, C, F, I, and K.