Fig. 5

UMSCs transfer mitochondria to PBMCs in AIS patients and restore their mitochondrial function. PBMCs were isolated from AIS patients within 3 d of stroke onset and cocultured with MitoTracker-labeled UMSCs under LPS stimulation ex vivo for 72 h. The effects of UMSCs on MMP and ROS levels in PBMCs were assessed via JC-1 and ROS staining. Experiments were conducted under both direct contact (CC) and indirect contact (TW) conditions (N = 6/group). A, B Representative images of PBMCs following mitochondrial transfer in the coculture system. Violet: MitoTracker; blue: DAPI (cell nuclei); scale bars: 20 and 50 μm. B Merged image of stained exogenous mitochondria overlaid with visible microscopy under the CC coculture system. The black arrow indicates UMSCs, and the white arrow indicates PBMCs. C Representative flow cytometry plots and statistical graphs of mitochondrial fluorescence expression in T cells. D Fluorescence microscopy of PBMCs stained with JC-1 after coculture. The images depict JC-1 aggregates (red fluorescence), JC-1 monomers (green fluorescence), and merged images. The accumulation of JC-1 dye in the mitochondrial matrix results in red fluorescence; as the MMP decreases, JC-1 remains in its monomeric form, producing green fluorescence. E Quantified JC-1 fluorescence intensity data obtained through flow cytometry. F Fluorescence microscopy of PBMCs stained for ROS after coculture. The images show ROS (green fluorescence), DAPI (blue fluorescence), and merged images. G ROS levels measured by flow cytometry, presented as the percentage of ROS-positive cells (left) and the fluorescence intensity of ROS (right). Statistical data are presented as scatter plots with mean values and SD. T-tests (C) and the Welch T-test with Dunnett's post hoc test (E, G) were used for group comparisons, with p < 0.05 considered statistically significant. ns indicates not significant or p ≥ 0.05; *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations: Pos: LPS stimulated; Neg: negative stimulated; Ctrl: control group (without UMSCs); ROS, reactive oxygen species; MFI, mean fluorescence intensity