Direct administration of mesenchymal stem cell‐derived mitochondria improves cardiac function after infarction via ameliorating endothelial senescence

FIGURE 1. Characterization of human bone marrow‐derived mesenchymal stem cells (MSCs) and MSC‐mt. (ai) Representative fibroblast‐like morphology and (aii) assessment of the trilineage differentiation capacity of MSC. (b) Flow cytometric analysis of surface markers in MSCs. Morphological characteristics of the isolated MSC mitochondria (MSC‐mt). (ci) Representative ZetaView NTA analysis of MSC‐mt. (cii) Representative transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of isolated MSC‐mt. (d) JC‐1 staining showed that isolated MSC‐mt maintained mitochondrial membrane potential. (e) Bioenergetic activity of MSC‐mt determined by seahorse assay.

FIGURE 2. Transplantation of mesenchymal stem cells mitochondria (MSC‐mt) improved heart function following infarction. (ai) Representative images of echocardiography taken at 28 days after myocardial infarction (MI) and (aii) measurements of ejection fraction (EF) and fraction shortening (FS). n = 8. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (bi) Representative images of Masson's trichrome staining and (bii) results from the quantitative analysis of infarction size and infarct wall thickness. n = 8. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (ci) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and (cii) results from the quantitative analysis of apoptosis at 28 days after MI. n = 4. *p < 0.05, ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (di) Representative images and (dii) results from the quantitative analysis of CD31 and α‐SMA density in the border zone of ischemic hearts. n = 4. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by…

FIGURE 3. In vivo and in vitro tracking of mesenchymal stem cells mitochondria (MSC‐mt) in endothelial cells. (a) Representative immunostaining of endothelial marker CD31 and human mitochondria in the ischemic heart at 7 days post‐MI. n = 4. (b) Representative images of isolated MSC‐mt transferred into human umbilical vein endothelial cells (HUVECs) after 6 and 24 h of coculture. n = 4.

FIGURE 4. Mesenchymal stem cells mitochondria (MSC‐mt) protected human umbilical vein endothelial cells (HUVECs) from oxidative stress‐induced damage. (a) Representative flow cytometry results obtained for FB‐mt or MSC‐mt‐treated HUVECs with H2O2 or rotenone treatments. n = 3. **p < 0.01, ***p < 0.001 by a one‐way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (b) Apoptosis was measured in FB‐mt or MSC‐mt‐treated HUVECs by Annexin V‐PI staining. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (c) Mitochondrial reactive oxygen species (ROS) levels in FB‐mt or MSC‐mt‐treated HUVECs were measured by MitoSOX staining. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (d) Mitochondrial membrane potential was assessed in FB‐mt or MSC‐mt‐treated HUVECs by JC‐1 staining. n = 3. MFI, mean fluorescence intensity.

FIGURE 5. Mesenchymal stem cells mitochondria (MSC‐mt) enhanced angiogenesis in human umbilical vein endothelial cells (HUVECs). (a) Tube formation assay and quantification of FB‐mt or MSC‐mt‐treated HUVECs. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (bi) Representative images of the Matrigel plug (top right corner), CD31 staining and quantitative analysis of CD31 density. (bii) Measurement of hemoglobin content of the plugs. n = 3. *p < 0.05, ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (c) SCF concentration in the culture supernatant as determined by ELISA. n = 3. ns, not significant; *p < 0.05, **p < 0.01 by a one‐way ANOVA followed by Bonferroni post hoc test. (d) Measurement of ATP content in FB‐mt or MSC‐mt‐treated HUVECs. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (e) Cell proliferation was determined by CCK‐8 assay in FB‐mt or MSC‐mt‐treated HUVECs. n = 3. *p < 0.05 by a one‐way ANOVA followed by Bonferroni post hoc test. (f) Colony formation assay was performed at 2 weeks after FB‐mt or MSC‐mt treatment. n = 3.…

FIGURE 6. Mesenchymal stem cells mitochondria (MSC‐mt) inhibited endothelial senescence via ERK pathway. (a) Representative co‐staining images of P21 and CD31 in infarcted hearts at 7 days post‐MI. n = 4. ***p < 0.001 by an unpaired t‐test. (b) Senescent β‐Gal activity determined by flow cytometry. n = 3. *p < 0.05, **p < 0.01 by a one‐way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (c) Representative images and quantification of SA‐β‐Gal staining. n = 3. ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test. (d) Inflammatory cytokine expression determined by protein array. (e) Representative immunoblot images and quantitative analysis in MSC‐mt‐treated human umbilical vein endothelial cells (HUVECs) w/o pathway inhibitors. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 by a one‐way ANOVA followed by Bonferroni post hoc test.

FIGURE 7. Scheme summarizing the proposed mechanism of action behind the mesenchymal stem cells mitochondria (MSC‐mt)‐mediated protective effects following myocardial infarction (MI).