Fig. 4. Demonstration of continuous transfer of exogenous mitochondria in blood of Polg mouse model.

Lineage negative cells ubiquitously expressing red-labeled nucleus (dTomato) and green-labeled mitochondria (Dendra) derived from ROSA^nT-nG/PhAM^excised mice were augmented with Dendra2⁺ mitochondria for 21 h and injected to Polg mice dosed at age 24–27 weeks (males) and 30-34 weeks (females). At 12 h, 1 w, 1 m, and 4.5 m post-injection blood was drawn and BM was extracted and both were analyzed by flow cytometry and confocal microscopy. a Graphical presentation of experimental design and a representative image of confocal microscopy of dTomato⁺Dendra2⁺ (left) and dTomato^-Dendra2⁺ (right) found in bone marrow 1 m post-injection. Scale bar 5 µm. b Representation of flow cytometry analysis, which is expected to detect three types of cells: Polg cells, transplanted/daughter cells (red box, dTomato⁺Dendra2⁺) and Polg cells which received mitochondrial transfer (green box, dTomato^-Dendra2⁺). c Representative figure of flow cytometry analysis of dTomato-Dendra2⁺ cells within CD45⁺ cell population in peripheral blood 12 h, 1 w, 1 m and 4.5 m post-injection (left). Percentages of dTomato⁺Dendra2⁺ cells and dTomato^-Dendra2⁺ cells in peripheral blood (right). d Percentages of CD3⁺ T cells, CD19⁺ B cells and CD11b⁺ myeloid cells within the CD45⁺ and dTomato^-Dendra2⁺ populations in peripheral blood 1 m post-treatment. e Percentages of dTomato⁺Dendra2⁺ cells (left) and dTomato^-Dendra2⁺ cells (right) in the bone marrow. f Representative image of confocal microscopy analysis of BM extracted cells at 4.5 months after treatment, demonstrating Dendra2⁺ mitochondria transfer between adjacent cells. Left, differential interference contrast (DIC); center, merged florescent image from DAPI, Dendra2 and dTomato channels. Scale bar, 5 µm; right, 3D reconstruction of the Z-stack images. BM bone marrow, HSPCs hematopoietic stem and progenitor cells.