Mitochondria transfer from healthy hematopoietic progenitors boosts stromal cell proliferation and hematopoietic recovery following irradiation and transplantation. (A) Proliferation rates in WT and H-Cx43^Δ/Δ chimeric mice that were injected with BrdU for 1 hour before analysis. Proliferation of BM Lin⁻/CD45⁻/PDGFRα⁺/Sca-1⁻ MSC receiving (positive) or not (negative) Dendra2⁺ mitochondria from donor hematopoiesis was measured at 10, 17, and 28 days posttransplantation. Data are the average of 4 mice per group. (B) Dendra2⁺ mitochondria isolated from WT and Cx43^Δ/Δ Dendra2-mito Lin-negative cells were cocultured over irradiated (7.5 Gy) WT stroma for 24 and 48 hours. Brdu uptake by MSC containing or not containing Dendra2⁺ mitochondria were analyzed at indicated times of coculture. Data are the average of 3 to 6 independent experiments. Dendra2⁺ mitochondria isolated from WT HSPC (WT mito), Dendra2⁺ mitochondria isolated from Cx43^Δ/Δ HSPC (Cx43^Δ/Δ mito). (C-D) Dendra2⁺ mitochondria isolated from WT and Cx43^Δ/Δ Lin-negative cells were cocultured over irradiated (7.5 Gy) WT stroma. After 24 hours, MSC containing (Dendra2-mito⁺) or not (Dendra2-mito⁻) extracellular Dendra2 mitochondria were FACS sorted and grown for 3 days. CFU-F (C), and apoptosis as measured by Annexin V staining (D) in Dendra2-mito⁺ and Dendra2-mito⁻ MSC are shown. Data are the average ± SEM of 3 independent experiments. (E-I) Schematic illustration of lethally irradiated congenic WT CD45.1⁺ mice that were transplanted with WT or Cx43^Δ/Δ Dendra2-mito Lin⁻/CD51⁻ cells and analyzed at days 10, 17, and 28 posttransplantation (E). Representative example (F) and the frequency of BM CFU-F and CFU-Ob in donor, WT, and H-Cx43^Δ/Δ chimeric mice at indicated time posttransplantation (G). BM ST-HSC (Lin⁻/c-Kit⁺/Sca-1⁺/CD48⁻/CD150⁻) content in WT and H-Cx43^Δ/Δ chimeric mice at days 10, 17, and 28 posttransplantation (H). Peripheral blood counts for leukocytes, neutrophils, and platelets in WT and H-Cx43^Δ/Δ chimeric mice at indicated times posttransplantation (I). Data are presented as average ± standard deviation from 2 independent experiments using 4 to 8 mice per group. (J-L) Lethally irradiated WT mice transplanted with WT or Cx43^Δ/Δ Dendra2-mito HSPC were treated with vehicle control or AMPK inhibitor (BML-275, 10 mg/kg, intraperitoneally) on days 5, 6, and 7 posttransplantation and analyzed. CFU-F (J) and CFU-Ob (K) in vehicle or AMPK inhibitor–pretreated WT and H-Cx43^Δ/Δ chimeric mice on day 7 posttransplantation. Peripheral blood platelet and neutrophil counts in WT and H-Cx43^Δ/Δ chimeric mice treated with vehicle control (DMSO) or AMPK inhibitor (BML-275) at indicated time points posttransplantation (L). Data are the average of 4 to 6 mice per group, 2 independent experiment. All data represented as mean ± SEM. Statistical significance was assessed using 2-tailed Student t test except in panels A, C-D where 1-way ANOVA was used. *P < .05, **P < .01, ***P < .001. Scale bar, 10 µm.