AMPK inhibition in HSPC controls mitochondria transfer to BM MSC. (A) Primary BM MSC were irradiated (9.5 Gy or 20 Gy), and 48 hours postirradiation, were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice for 16 h at 37°C, and the transfer of mitochondria in MSC was analyzed. (B) Primary BM MSC or CD45⁺ cells isolated from Dendra2-mito WT mice were pretreated with Rejuvesol for 4 hours and washed with intact media. Rejuvesol-pretreated stromal or CD45⁺ cells were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice or WT primary MSC, respectively, for 16 hours, and the transfer of mitochondria in MSC was analyzed. (C) Primary BM MSC were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice with/without Rejuvesol and P2RX7 receptor inhibitor for 16 hours and the transfer of mitochondria in MSC was analyzed. (D-E) Primary BM MSC were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice with/without AMPK inhibitor (BML-275) or AMPK activator (AICAR) for 16 hours, and the transfer of mitochondria in MSC was analyzed. Representative plots (D) and quantification summary (E) shows the effect of AMPK inhibitor and activator on mitochondria transfer from HSPC to BM MSC. (F) Primary BM MSC or CD45⁺ cells isolated from Dendra2-mito WT mice were pretreated with AMPK inhibitor for 4 hours and washed with intact media. AMPK inhibitor–pretreated stromal and CD45⁺ cells were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice or WT stromal cells, respectively, for 16 hours and the transfer of mitochondria in MSC was analyzed. (G) Primary BM MSC were cocultured with CD45⁺ cells isolated from Dendra2-mito WT mice with/without AMPK inhibitor, P2RX7 receptor inhibitor and Rejuvesol for 16 hours, and the transfer of mitochondria in MSC was analyzed. (H-I) Schematic illustration of lethally irradiated congenic WT CD45.1⁺ mice transplanted with WT or Cx43^Δ/Δ Dendra2-mito HSPC and treated with vehicle control or AMPK inhibitor (BML-275, 10 mg/kg, interperitoneally) on days 5, 6, and 7 posttransplantation, and analyzed 2.5 hours after the last dose (H). Transfer of Dendra2⁺ mitochondria from donor HSPC to BM Lin⁻/CD45⁻/PDGFRα⁺/Sca-1⁻ cells in WT and H-Cx43^Δ/Δ chimeric mice treated with vehicle control or AMPK inhibitor (I). n = 8 mice per group, 2 independent experiments. All in vitro data are the average of 4 to 6 independent experiments. Data represented as mean ± SEM and statistical significance was assessed using 1-way ANOVA. *P < .05, **P < .01 and ***P < .001.