Donor hematopoietic cells transfer functional mitochondria to the irradiated host BM MSC following total body irradiation. (A-C) Schematic illustration of transplantation protocol. Lethally irradiated congenic WT mice transplanted with CD45⁺ BM cells obtained from Dendra2-mito WT mice and analyzed 2 weeks and 1 month posttransplantation (A). Representative histograms after 1 month posttransplantation (B) and quantified analyses (C) show the levels of Dendra2⁺ mitochondria transfer from donor HSPC to host BM-MSC (CD45⁻/PDGFRα⁺/Sca-1⁻). Data are the average of 3 to 5 mice per group. (D-F) Lethally irradiated WT Dendra2-mito mice transplanted with CD45⁺ BM cells obtained from congenic WT mice and analyzed 2 weeks and 1 month posttransplantation (D). Representative histograms after 1 month posttransplantation (E) and quantified summary (F) show the levels of Dendra2⁺ mitochondria transfer from host Dendra2-mito stromal cells to WT donor HSC (CD34⁻/Lin⁻/Sca-1⁺/c-Kit⁺). Data are the average of 3 to 6 mice per group. (G-I) BM MSC were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice for 16 hours, and the transfer of mitochondria from Dendra2-mito CD45⁺ cells to MSC was analyzed. Histograms (G) and bar diagram (H) representing the percentage of MSC containing donor-derived Dendra2⁺ mitochondria. (I) Relative quantification of mitochondrial content in stromal cells cocultured with or without Dendra2-mito CD45⁺ cells was analyzed by real-time polymerase chain reaction using ND1 gene belonging to mitochondrial DNA (mND1) and nuclear hexokinase 2 (nHK2) gene. n = 4-6 independent experiments. (J) Representative example of mitochondrial transfer kinetics followed for up to 225 min. Confocal spinning disk microscopy of hematopoietic Dendra2-mito cells cocultured with WT stromal precursors. Heat map shows the Dendra2 signal intensity. Images taken at indicated time points show the transfer of mitochondria from 1 hematopoietic cell (H) toward a neighboring stromal cell (S) after a long, thin hematopoietic cell extension. White arrows depict mitochondria moving away from H to S and red arrows depict mitochondria already transferred to S. (K) TEM images of mitochondria transfer in an in vivo setting. WT Dendra2-mito CD45⁺ cells were transplanted in lethally irradiated WT congenic mice and the transfer of Dendra2⁺ mitochondria from HSPC to MSC was analyzed 4 months posttransplantation. (a) Representative TEM images showing donor (D) and recipient (R) cells, and mitochondrial cristae. (b) Overlay of TEM image with identical fluorescent micrograph (correlative light electron microscopy, CLEM). (c) Fluorescent microscopy image showing donor CD45⁺ hematopoietic cells (red) and Dendra2⁺ mitochondria (green). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) . The short white arrows in panels a-c show Dendra2⁺ mitochondria in donor CD45⁺ cells. The short red arrows in panel a-c represent donor Dendra2⁺ mitochondria in recipient stromal cells. The boxed areas (d, e, and f) in panel a are magnified in TEM image panels d, e and f, respectively. TEM magnification of mitochondria indicated by a long magenta arrow in panel d is presented in TEM micrograph (g). TEM magnification of mitochondria indicated by a long yellow arrow and a green arrow in panel e are presented in TEM micrographs panels h and i, respectively. TEM magnification of mitochondria indicated by a long red arrow in panel f is presented in TEM micrograph (j). All these mitochondrial images represent donor-derived Dendra2⁺ mitochondria which either persist in the hematopoietic (CD45⁺, red fluorescent) donor cell (d, g) or have been transferred to a recipient BM stromal cell (CD45⁻, with no red fluorescence; in e, f, h, i, and j). TEM micrographs in panels g, h, i, and j provide morphological detail on mitochondrial cristae and membranes. Scale bar, 2 µm, 500 nm, and 200 nm. (L-M) Mitochondrial ROS levels (L) and (M) membrane potential in host MSC containing (Dendra2-mito⁺) or not (Dendra2-mito⁻) donor-derived Dendra2⁺ mitochondria assessed at 1 month posttransplantation (n = 5 mice per group). All data are presented as mean ± SEM. Statistical significance was assessed using 2-tailed Student t test except in panels C and F, where one-way ANOVA was used. **P < .01, ***P < .001.