Mitochondrial transfer from Connexin 43 deficient donor HSPC to BM MSC is decreased. (A) Level of Dendra2-mito transfer from HSPC to BM stromal cells in coculture without contact in trans-wells of 0.3µm. (B-D) WT or H-Cx43^Δ/Δ Dendra2-mito Lin-negative cells were cocultured on WT MSC and the transfer of Dendra2⁺ mitochondria in MSC was analyzed. Mean fluorescence intensity of Dendra2 in PDGFRα⁺ MSC after 2, 4, 8, and 16 hours of coculture (B). Mitochondrial ROS levels (C) and ΔΨm (D) in PDGFRα⁺ MSC containing donor-derived Dendra2⁺ mitochondria at different times of coculture. Data are the average of 3 to 6 independent experiments. (E) Lin-negative cells from WT or Vav1-cre Cx43^fl/fl Dendra2-mito mice were transduced with empty or Cx43-full length (R-Cx43-FL) retrovirus vector, followed by coculture over WT stroma for 16 hours. Overexpression of R-Cx43-FL in Cx43^Δ/Δ Dendra2⁺ HSPC rescue mitochondrial transfer in PDGFRα⁺ MSC. The frequency of mitochondria transfer in PDGFRα⁺ MSC was also increased in R-Cx43-FL transduced WT HSPC. Data are the average of 3 independent experiments. (F-J) Schematic illustration of lethally irradiated congenic WT CD45.1⁺ mice transplanted with WT or Cx43^Δ/Δ Dendra2-mito Lin⁻/CD51⁻ cells and analyzed at days 10, 17, and 28 posttransplantation (F). Representative histograms (G) and bar diagram (H) show the frequency of BM Lin⁻/CD45⁻/PDGFRα⁺/Sca-1⁻ MSC containing Dendra2⁺ mitochondria from donor hematopoiesis at the indicated days posttransplantation. Mitochondrial ROS levels (I), and ΔΨm (J) in WT and H-Cx43^Δ/Δ chimeric mice BM Lin⁻/CD45⁻/PDGFRα⁺/Sca-1⁻ MSC containing donor-derived Dendra2⁺ mitochondria after 28 days posttransplantation are shown. (n = 4-9 mice per group, 2 independent experiments). (K-M) Incorporation of isolated mitochondria from HSPC is independent of the expression of Cx43 in source HSPC. Dendra2⁺ mitochondria were isolated from WT and Cx43^Δ/Δ Dendra2-mito Lin-negative cells and coculture over-irradiated (7.5 Gy) WT primary stroma for 24 and 48 hours (K). Bar graphs show the frequencies of PDGFRα⁺ MSC containing extracellular Dendra2⁺ mitochondria at indicated time points (L), and mitochondrial ROS production in PDGFRα⁺ MSC containing extracellular Dendra2⁺ mitochondria (M). Data are the average of 3 to 5 independent experiments. Dendra2⁺ mitochondria isolated from WT HSPC (WT mito). Dendra2⁺ mitochondria isolated from Cx43^Δ/Δ HSPC (Cx43^Δ/Δ mito). (N-O) BM Lin⁻/CD45⁻ cells containing donor-derived Dendra2⁺ mitochondrial were sorted from WT and H-CX43^Δ/Δ chimeric mice (1 month posttransplantation) and mitochondrial OCR was measured by Seahorse XFe96-Analyzer using sequential injections of oligomycin, FCCP, and Rotenone (N). Quantification summary of mitochondrial OCR in WT and H-CX43^Δ/Δ chimeric mice Lin⁻/CD45⁻ cells containing donor-derived mitochondria (data are the average of 3 independent experiments with 2 to 4 technical replicates) (O). All data represented as mean ± SEM. Statistical significance was assessed using 1-way ANOVA except in panels H, I, J, and O where 2-tailed Student t tests were used. *P < .05, **P < .01, ***P < .001. BHI, bioenergetic health index; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; SRC, spare respiratory capacity.