Experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine, Japan. Animal care was conducted humanely in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Animal Resources and published by the National Institutes of Health (Eighth Edition, revised 2011).
Assessment of in vivo mitochondrial transfer
Transplantation of adipose tissue-derived stem cells (ADSCs) harboring stained mitochondria
A myocardial infarction model in F344/NJcl-rnu/rnu rats (CLEA Japan, Inc., Tokyo, Japan) was generated by permanent ligation of the left anterior descending artery (LAD). Two weeks following LAD ligation, human ADSCs (hADSCs; 1 × 106 cells; Lonza, Tokyo, Japan) were transplanted into the infarct area using fibrinogen and thrombin solution (CSL Behring, Tokyo, Japan). The composition of this graft is shown in Supplementary Table S1 online [17]. The hADSCs were pre-stained with MitoTracker Red (Thermo Fisher Scientific, Waltham, MA, USA) [18]. The rats were euthanized at appropriate time points following transplantation, and heart samples were collected (Fig. 6). Following formalin fixation, paraffin-embedded heart sections were stained with an antibody against phalloidin (Cat. #A12379; Thermo Fisher Scientific) and counterstained with Hoechst 33342 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) (Supplementary material).
Observation of graft-heart boundaries with electron microscopy
Mitochondrial ultrastructure and spatial relationship between hADSCs and rat cardiomyocytes (rCMs) were assessed in tissue samples collected from areas including the boundary between heart and hADSC graft. Contrasted sections were imaged under an H-7500 transmission electron microscope (Hitachi, Tokyo, Japan) (Supplementary material).
Intravital imaging of mitochondrial transfer from hADSC graft to beating myocardium
Myocardial infarction-induced adult male CAG/eGFP transgenic Sprague–Dawley (SD) rats (body weight, 200 – 250 g; Japan SLC, Inc., Shizuoka, Japan) were used as recipients of hADSC transplants. The hADSCs were transfected using CellLightTM Mitochondria-RFP BacMam 2.0 (Thermo Fisher Scientific) (Fig. 2A). Graft composition and transplantation was performed as explained above (Supplementary Table S1 online). The beating heart and the graft were stabilized using a custom-made suction device (Fig. 2B) [19]. Clear in vivo images were obtained by methods reported previously [20]. Observation was initiated immediately following transplantation and continued for maximum possible duration (Supplemental material).
Detection of donor mitochondrial DNA in recipient rCMs
Paraffin-embedded tissues from the heart and hADSC graft were harvested three days post transplantation. Tissue sections were stained with hematoxylin and eosin, and residual rCMs around the fibrotic tissue and just beneath the hADSC graft were collected using a laser-equipped microscope (Leica LMD7000). DNA was isolated, and a human mitochondrial DNA fragment was amplified using a customized primer pair specific for human mitochondria (Eurofins Genomics, Louisville, KY, USA) (Supplementary Table S2 online) [21-23]. PCR conditions and reaction mixtures are listed in Supplementary Tables S3 and S4 online. The PCR products were purified and used as a template for sequencing and further analysis (Supplementary material online).
In vitro observation of mitochondrial transfer and its influence on rCM metabolism
Observation of rCM and hADSC co-culture
Commercially available neonatal rCMs (RCM-561; QBM Cell Science, Ontario, Canada) were stained with MitoTracker Green (Thermo Fisher Scientific), seeded on 4-well glass slide chamber, and cultured under normoxic conditions for 3 – 4 h. The same count of hADSCs, cultured under normoxic condition and stained with MitoTracker Red (Thermo Fisher Scientific), was transferred to the rCM culture, and the two cell populations were co-cultured for 1 h before observation (Fig. 3A)under hypoxic conditions (1 % O2), with or without 10 µM αGA (Sigma-Aldrich, St. Louis, MO, USA), for 24 h. Then, co-cultured cells were fixed and stained with anti-cardiac actinin and anti-human mitochondria antibodies, and three-dimensional images were constructed (Supplementary material).
Measurement of oxygen consumption rate
Oxygen consumption rate (OCR) in living cells was measured using a Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. rCMs (2 × 104 cells/well) were cultured on 96-well Agilent Seahorse XF cell culture microplates under normoxic conditions for 48 h, after which the same number of hADSCs were added to each well and cultured with or without αGA, under normoxic conditions, for 24 h before analysis. The detailed culture conditions for each group are listed in Supplementary Table S5 online [24].
Quantification of cytokines from the hADSC graft
hADSC grafts were cultured under normoxic or hypoxic (1 % O2) conditions, and culture supernatant was collected after 24, 48, and 72 h. The secretion of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), IL-6, and IL-10, which has been reported to be associated with functional improvement, was measured by performing enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA).
Isolation and motion analysis of rCMs with/without hADSC supernatant
The 7–8-week-old male Crl:CD (SD) rats were anesthetized by inhalation of isoflurane, and heart was harvested. The heart was connected to a Langendorff circulation circuit through aortic ligation to the cannula (Fig. 3G). The perfusion buffer (Tyrode’s solution, Supplementary Table S6 online) containing 300 units/mL collagenase type II (Gibco; Thermo Fisher Scientific) was perfused into the coronary arteries. The heart was homogenized on a petri dish, and cardiac cells were pipetted and transferred to a 96-well plate (Fig. 3H-I). The cardiomyocytes were stimulated with either 1 µM isoprenaline, 400 µM propranolol, or hADSC culture supernatant (Fig. 3J). Motion of rCMs was observed and analyzed using the SI8000 Cell Motion Imaging System (Sony, Tokyo, Japan) (Supplementary material online) [25-27].
Evaluation of influence of mitochondrial transfer on cardiac function andmetabolism
Graft survival following transplantation
Luciferase-transduced hADSCs were transplanted into the nude rat myocardial infarction model in the same manner as described above (Fig. 4A). Luminescence intensity of the grafts was measured at the body surface. For histological analysis, hearts with residual graft samples were collected at days 1, 3, 7, and 14, and paraffin-embedded samples were stained using an antibody specific to human mitochondria (Cat. #MAB1273; Merck Millipore, Burlington, MA, USA) (Supplementary material).
Assessment of cardiac function
Following the injections with hADSCs (1 × 105 cells/body (low-dose group), 5 × 105 cells/body (medium-dose group), 1 × 106 cells/body (high-dose group), and 5 × 106 cells/body (max-dose group)), cardiac function was assessed using an echocardiography system equipped with a 12 MHz transducer (SONOS 7500; Philips, Amsterdam, Netherlands). Heart samples were collected on days 3, 7, 14, and 56 following transplantation (Supplementary material).
Two weeks after inducing myocardial infarction, either hADSCs (A group, n = 20), hADSCs combined with αGA (final concentration, 10 μM; A + αGA group, n = 10), or αGA alone (final concentration, 10 μM; αGA group, n = 4) were transplanted. For the control group, sham operation performed (fibrinogen and thrombin solution only; C group, n = 10). Cardiac function was examined using echocardiography. Heart samples were collected on days 3, 7, 14, and 56 following transplantation (Supplementary material).
Histological analysis of the heart
Heart samples were obtained from each group 8 weeks following surgery. To assess rCM diameter and fibrosis, heart sections were stained with periodic acid-Schiff and Picro Sirius red, respectively. The sections were also stained with anti-von Willebrand factor antibody, and capillary density was calculated in the peri-infarct area. For morphological evaluation of mitochondria in the heart, electron microscopy was performed as described above.
Quantification of intramyocardial ATP and mitochondrial DNA
ATP content in the heart samples was assessed using a colorimetric ATP assay kit (ab83355; Abcam, Cambridge, UK). To quantify human and rat mitochondrial DNA in the recipient heart, we performed quantitative PCR. Total DNA was isolated from the infarct region and remote areas, and PCR was performed with SYBR Green (Thermo Fisher Scientific) using specific primer pairs (Supplementary Table S1 online).
Statistical analysis
All values are expressed as means ± SEM. Statistical analyses were performed using JMP Pro 14 software (SAS Institute Inc., Cary, NC, USA). Comparison across multiple groups with normal distribution was performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. Comparison between two groups was performed using an unpaired or paired two-tailed Student’s t-test or Mann–Whitney U test, as appropriate. Comparison across multiple groups with non-normal distribution was performed using Kruskal–Wallis tests followed by Dunn’s multiple comparisons tests. p < 0.05 were considered statistically significant.