Mitochondrial transfer from human embryonic stem cell-derived mature cardiomyocytes to mesenchymal stem cells

Characterization of mitochondria in MSCs, immature, and mature hESC-CMs

To explore the utility of Toma-CMs as donor cells for mitochondrial transplantation, we assessed the structure and function of mitochondria in Toma-CMs, control CMs, and MSCs. H9 hESCs were differentiated into hESC-CM and subsequently matured using tomatidine. Transmission electron microscopy was employed to examine the mitochondrial structure in MSCs, control CMs, and Toma-CMs. Tomatidine treatment not only increased mitochondrial number but also enhanced mitochondrial cristae formation in hESC-CMs (Fig. 1A). In addition, the ratio of mitochondrial DNA to nuclear DNA was significantly elevated in Toma-CMs compared to control CMs (Fig. 1B). Mitochondrial area, perimeter, and circularity were also greater in Toma-CMs than in control CMs or MSCs (Fig. 1C). To evaluate mitochondrial ROS and membrane potential, cells were stained with MitoSOX and TMRM. Toma-CMs demonstrated higher levels of mitochondrial ROS and increased membrane potential than those in MSCs or control CMs (Fig. 1D). Fluorescence-activated cell sorting (FACS) analysis validated the rise in mitochondrial ROS and membrane potential in Toma-CMs (Fig. 1E). Moreover, the mitochondrial oxygen consumption rate was higher in Toma-CMs than in control CMs and MSCs (Fig. 1F). These findings indicate that tomatidine treatment enhances mitochondrial quantity and functionality in hESC-CMs.

Isolation of mitochondria from MSCs and hESC-CMs

To isolate mitochondria from hESC-CMs and MSCs, cells were lysed and mitochondria were purified using differential centrifugation as outlined in Materials and Methods. Western blot analysis revealed that the expression level of cardiac troponin-T (cTnT), a cardiomyocyte specific protein, in the cell lysate of Toma-CMs exceeded that in control CMs and was minimally detectable in the isolated mitochondrial fractions from Toma-CMs and control CMs (Fig. 2A). Additionally, the expression levels of mitochondria-specific markers such as mitofilin (MIC60), cytochrome C oxidase subunit 4 (COX IV), and TOM20 were higher in the mitochondrial fractions compared to the cell lysates of MSCs, control CMs, and Toma-CMs (Fig. 2A). The expression of nuclear marker (lamin B1), endoplasmic marker (calnexin), and cytoplasmic markers (FAK and GAPDH) were minimally present in the mitochondrial fractions, suggesting a high enrichment of mitochondria. Consistently, the protein levels in the mitochondrial fractions derived from Toma-CMs surpassed those from MSCs and control CMs (Fig. 2B). To examine the metabolic activity, ATP levels were measured in the mitochondrial fractions, revealing that Toma- CMs had elevated ATP levels compared to those from control CMs and MSCs (Fig. 2C). These results suggest that Toma-CMs are effective donor cells for mitochondrial transplantation.

Transplantation of isolated mitochondria to recipient MSCs

To track mitochondrial transplantation, mitochondria were pre-labeled with MitoTracker Green FM (MTG) in donor MSCs. An MTG-positive signal was subsequently detected in the mitochondrial fraction (Fig. 3A), indicating that the MTG-positive fluorescence of mitochondria was retained after isolation. To investigate whether mitochondria isolated from donor MSCs could transfer to recipient cells, the endogenous mitochondria of the recipient cells were labeled with TMRM and then incubated with MTG-labeled donor mitochondria for one day. After a 24-hour incubation period, the MTG-positive exogenous mitochondria (green color) co-localized with the TMRM-positive endogenous mitochondria (red color) of the recipient MSCs (Fig. 3B), demonstrating the successful transfer of mitochondria from donor to recipient cells. To further verify this co-localization, z-stack confocal images of recipient MSCs transplanted with MTG-positive donor mitochondria were analyzed (Fig. 3C), revealing a high correlation between MTG-positive and TMRM-positive fluorescence signals in recipient cells, suggestive of co-localization between the transplanted exogenous and endogenous mitochondria.

To compare the efficiencies of mitochondrial transplantation among mitochondria isolated from MSCs, Toma-CMs, and control CMs, recipient MSCs were exposed to mitochondrial fractions derived from these three cell types. Mitochondria isolated from all three types were pre-stained with MTG (green) and subsequently treated with these MTG-labeled exogenous mitochondria transferred into TMRM-pre-stained recipient MSCs for 24 h. All MTG-labeled mitochondria from the three donor cell lines co-localized with the endogenous TMRM-positive mitochondria in the recipient MSCs (Fig. 3D). FACS analysis illustrated that the proportion of MTG^＋TMRM^＋ mitochondria in recipient MSCs transplanted with mitochondria from Toma-CMs was significantly higher than in those transplanted with mitochondria from MSCs or control CMs (Fig. 3E), suggesting that Toma-CMs serve as optimal donor cells for mitochondrial transplantation.

Effects of mitochondrial transplantation on mitochondrial function in MSCs

Next, we investigated the effects of mitochondrial transplantation on the expression of mitochondrial genes and functions, including ROS production, mitochondrial membrane potential, and metabolic activity, in recipient MSCs. To assess the impact on mitochondrial proteins, we measured the expression levels of MIC60 and COX IV, which are associated with mitochondria, in recipient MSCs at 24 h and 48 h post-transplantation using western blotting. The expression levels of MIC60 and COX IV increased following the transplantation of mitochondria derived from MSCs and hESC-CMs (Fig. 4A). Particularly, mitochondria isolated from Toma-CMs markedly enhanced the expression of MIC60 and COX IV in recipient MSCs, compared with those isolated from MSCs or control CMs.

To evaluate whether mitochondrial transplantation affects mitochondrial function in recipient MSCs, we analyzed the levels of mitochondrial ROS and membrane potential using mitoSOX and TMRM staining at 24 h post-transplantation. Transplantation with MSC-derived mitochondria elevated mitoSOX-positive ROS levels from 0% to 12.2%, whereas mitochondria from control CMs and Toma-CMs increased mitoSOX-positive signals to 47.5% and 90.1%, respectively (Fig. 4B, upper panels). Additionally, TMRM-positive mitochondrial membrane potential was enhanced by transplantation with mitochondrial fractions derived from MSCs, control CMs, and Toma-CMs (Fig. 4B, lower panels). Consistent with the mitoSOX-positive signals, the highest TMRM activity was observed in MSCs treated with mitochondria isolated from Toma-CMs (Fig. 4B). We also assessed the effects of mitochondrial transplantation on the cell number and viability of MSCs using the WST-8 assay. Treating MSCs with mitochondria from all three cell types significantly increased both cell number and viability, with the most pronounced effects observed following the transplantation of mitochondria isolated from Toma-CMs (Fig. 4C and D). These results imply that the transplantation of mitochondria isolated from Toma-CMs enhances mitochondrial function and metabolic activity in recipient cells.

Materials

RPMI 1640 (#LM-011-01), DPBS without calcium and magnesium (#LM-001-01), and Trypsin-EDTA Solution 10× (#LS-015-02) were acquired from Welgene (Gyeongsanbuk-do, Gyeongsan, Republic of Korea). The mTeSR1 (#85850) was sourced from STEMCELL Technologies (Vancouver, Canada). Matrigel (#354277) was obtained from Corning (New York, USA). CHIR99021 (#CT-99021) was procured from Selleck Chemicals (Houston, TX, USA). IWR1-endo (#72562) was purchased from Tocris Biosciences (Bristol, UK). Hank’s Balanced Salt Solution (HBSS; #4891), Y27632 (#Y0503), gelatin (#G9391), dimethyl sulfoxide (DMSO; #D5879), anti-α-Tubulin antibody (#T9026), BSA (#A7946), Trypan Blue solution (#T8154), and Tris buffer (#T1378) were obtained from Sigma Aldrich (St. Louis, Missouri, USA). α-minimum essential medium (α-MEM; #12000-022), DMEM/F12 (#11330-032), RPMI1640 (no Glucose, #11879-020), StemPro Accutase (#A11105-01), fetal bovine serum (FBS; #16000-044), B27 minus insulin (#A18956-01), B27 supplement (#17504-044), Nuclease free water (#AM9938), 0.05% trypsin-EDTA (#25300-054), penicillin-streptomycin (#15140-122), MitoSOX Red mitochondrial superoxide indicator (#M36008), MitoTracker Green FM (#9074S), tetramethyl rhodamine (TMRM; #T668), and antibodies against Lamin B1 (#A25547), FAK (#3285S), COX IV (#4850S), and calnexin (#2433S) were acquired from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH (#SC-47724) and anti-TOM20 (#SC-177764) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-cardiac Troponin T (cTnT; #ab45932) and anti-mitofilin (MIC60; ab110329) antibodies were purchased from Abcam PLC (Cambridge, UK). 0.5 M EDTA (pH 8.0; C-9007) was sourced from Bioneer (Daejeon, Republic of Korea). Sucrose (#31365-0350) and L-lactic acid (#129-02666) were obtained from JUNSEI Chemical (Kyoto, Japan) and FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), respectively. The CellTiter-Glo Luminescent Cell Viability Assay kit (#G7573) was purchased from Promega (Madison, WI, USA). Vectashield Antifade mounting solution containing 4’,6-diamidino-2-phenylindole (DAPI; #H-1200) was sourced from VECTOR Laboratories Ltd. (Peterborough, UK).

Culture of mesenchymal stem cells

MSCs derived from human tonsil tissue were isolated from a patient with chronic tonsillitis undergoing tonsillectomy, as previously described (31). The ethical approval for studies involving human tonsils was granted by the Institutional Review Board of Pusan National University Hospital (1801-033-062). MSCs were subjected to a five-day culture in α-minimum essential medium (α-MEM) (#12000-022, Thermo Fisher Scientific) enriched with 10% fetal bovine serum (FBS) and 1% Pen-Strep (#15140-122, Thermo Fisher Scientific) at 37°C in an atmosphere of 5% CO₂ and 95% humidity. The culture media were refreshed every two days, and upon attaining 80-90% confluence, the cells were harvested using trypsin-EDTA solution (#LS-015-02, Welgene) for reseeding at a 1:5 split ratio.

Differentiation of human ESCs to CMs

H9 hESCs were purchased from WiCell Research Institute (Madison, WI), and the study was approved by the public Institutional Review Board (P01-201410-ES-02). The hESCs were maintained on Matrigel-coated culture dishes in mTeSR1, with subculturing every five days. For cardiomyocyte induction, hESCs were initially exposed to RPMI 1640 medium supplemented with B27 minus insulin and 12 μM CHIR99021, a specific GSK3 inhibitor and promoter of Wnt-signaling, for 24 hours. This was followed by maintenance in a differentiation medium combining RPMI/B27 minus insulin with 5 μM IWR-1 endo and 50 μg/ml L-ascorbic acid for 48 hours. On day 3, the cells received additional treatment with RPMI containing 5 μM IWR-1 endo and B27 minus insulin. From days 6 to 10, differentiation was continued with RPMI/B27 supplement medium. In the final stage of differentiation, the cells were cultured in glucose-free DMEM (#11879-020, Thermo Fisher Scientific) supplemented with 4 mM L-lactic acid, 500 μg/ml human albumin, and 213 μg/ml L-ascorbic acid for 4 days. To enhance CM maturation, cells were exposed to 1 μM tomatidine (Sigma) from day 6 of the differentiation process, as previously reported (16).

Mitochondrial labeling

To visualize mitochondria, cells were labeled with either MTG FM or tetramethylrhodamine (TMRM) according to the manufacturer’s protocol 24 hours before isolation and delivery of mitochondria. Briefly, cells were incubated in growth medium containing 400 nM MTG FM or 50 nM TMRM for 30 minutes at 37°C in 5% CO₂. After staining, cells were washed three times with HBSS and resuspended in fresh, pre-warmed medium. Cells were either harvested or plated as per the manufacturer’s instructions.

Mitochondrial transfer

Mitochondria were isolated from MTG-labeled MSCs and CMs using differential centrifugation after cell lysis. Briefly, donor cells (5 × 10⁶ cells) were harvested and homogenized in 1 ml of mitochondrial isolation buffer (0.25 M sucrose, 0.025 M Tris, and 1 mM EDTA, pH 7.4). The homogenate was first centrifuged at 1000 × g for 10 minutes, then the supernatant was further centrifuged at 9000 × g for 10 minutes. The pellet was resuspended in mitochondrial isolation buffer and centrifuged at 4000 × g for 10 minutes. The mitochondrial pellet was resuspended again in mitochondrial isolation buffer, and protein concentration was assessed using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA ). Before mitochondrial transfer, recipient MSCs (1 × 10⁶ cells) were seeded onto cell culture dishes coated with 0.1% gelatin gel and prelabeled with TMRM. After staining, the recipient cells were washed three times with HBSS, incubated in fresh α-MEM, treated with the isolated mitochondrial fractions. After incubation of the recipient cells at 37°C for 24 h, the fluorescent images of the recipient cells were collected using a confocal microscope (ZEISS LSM700, Carl Zeiss, Oberkochen, Germany).

Transmission electron microscopy

Cells were rinsed three times with HBSS, then scraped into 0.15 M cacodylate buffer to collect 5 × 10⁶ cells. Cells were centrifuged, the supernatant discarded, and the pellet was incubated for 4 hours with fixation buffer (2% paraformaldehyde and 2.5% glutaraldehyde in 0.15M sodium cacodylate buffer, pH 7.4) at 4°C. After fixation, samples were dehydrated, immersed in resin to prepare ultrathin sections, followed by image acquisition using the transmission electron microscopy (TECNAI G2) was performed at the Brain Research Core Facilities of KBRI.

Flow cytometry (FACS) analysis

After labeling cells with MTG FM, TMRM, and MitoSOX Red mitochondrial superoxide probes, they were rinsed with HBSS and dissociated using trypsin-EDTA Solution 1× for 3 minutes, then resuspended in 300 μl of HBSS. MTG fluorescence was detected with a 488 nm laser and a 530/30 nm filter. Fluorescence of TMRM and MitoSOX was detected with the same laser and a 574/26 nm filter. Fluorescence intensity was measured using Attune NxT (Thermo Fisher Scientific) and analyzed with FlowJo (ver 10, Tree Star Inc. Ashland, OR, USA).

Western blot analysis

Cells were harvested after washing with HBSS and then lysed in a lysis buffer (0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM NaCl, 20 mM Tris-HCl, 25 mM β-glycerol phosphate, 30 mM Sodium pyrophosphate, and 1% Triton X-100, pH 7.4), followed by centrifugation at 15,000 × g for 15 minutes at 4°C to collect supernatant (cell lysates). Protein concentrations of the supernatant (cell lysates) and mitochondrial fractions were determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts (5 μg) of lysates or mitochondrial fractions were loaded onto 8-12% tris-glycine SDS-PAGE for protein separation, transferring to PVDF membranes, and subsequent staining with 0.1% Ponceau S solution (#P3504, Sigma) to verify sample location. Following blocking with 5% nonfat milk for 1 hour, membranes were incubated with primary antibodies in 5% blocking buffer overnight, and secondary antibodies were applied for 2 hours at room temperature. Antibodies were detected using an enhanced chemiluminescence Western blotting system (#GE-RPN2106; Amersham Biosciences, Piscataway, NJ, USA). Western blot images were captured using the ImageQuant^TM 800 (Cytiva, Marlborough, MA, USA) and the band intensities were quantified using ImageJ software (Image Processing and Analysis in Java, NIH).

Measurement of cellular respiration (Seahorse XFp)

Mitochondrial respiration was measured using an Agilent Seahorse XFp analyzer and Cell Mito Stress Test Kit (#10310-100, Seahorse Bioscience, MI, USA). The assay was conducted following the manufacturer’s instructions. Cardiomyocytes were seeded onto 0.1% gelatin-coated XFp cell culture plates at a density of 30,000 cells/well, with or without tomatidine, for one day in RPMI/B27 medium including 10 μM Y276362. This was followed by 2 additional days of incubation, with or without tomatidine, in RPMI B27 medium without Y27632. An equal number of MSCs were seeded onto different wells coated with 0.1% gelatin in α-MEM medium for 2 days. The day before the assay, the sensor cartridge was hydrated using XF calibrant at 37°C in a non-CO₂ incubator. XF assay medium was prepared by adding 1 mM sodium pyruvate, 4 mM L-glutamine, and 10 mM D-(＋)-galactose; the pH was adjusted to 7.4, and the temperature was set to 37°C. One hour prior to analysis, the cells were washed twice with XF medium, and the medium was replaced with XF assay medium. The cells were then incubated at 37°C in a non-CO₂ incubator. Thirty minutes before the analysis, 1 μM Oligomycin, 0.5 μM FCCP, and 1.5 μM each of a mix of Antimycin A and Rotenone were loaded into A, B, and C injection ports of the hydrated sensor cartridge in volumes of 20 μl, 22 μl, and 25 μl, respectively, using a 10× solution from the XFp Mito Stress Test Kit. After calibration, the cell culture plates were inserted into the instrument.

ATP assay

The ATP assay quantifies living mitochondria based on ATP content in isolated mitochondrial fractions. To measure ATP, either 2 × 10⁴ cells or an equivalent amount of isolated mitochondria in mitochondrial isolation buffer were used. Each assay used 100 μl per well dispensed into a 96-well opaque-multi well plate. To establish the background luminescence, control wells contained only mitochondrial isolation buffer. Equal volumes of CellTiter-Glo (#G7570, Promega) reagent were added to each well. The plates and reaction mixtures were brought to room temperature to stabilize the luminescence signal, which was allowed to react for 10 minutes before luminescence was recorded.

Analysis of mtDNA-to-nDNA ratio

To analyze the mtDNA-to-nDNA ratio, cells were washed with HBSS and 500 μl of lysis buffer (10 mM Tris, 1 mM EDTA, 0.3 M sodium acetate, 1% SDS, pH 8) was added along with 5 μl of proteinase K (#T4850, Sigma). The lysates were then incubated overnight at 37°C. The following day, 0.2 mg/ml RNase A was added to the lysates for 30 minutes at 37°C to degrade all components except DNA. To extract nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), 500 μl of PCI solution (phenol, chloroform, and isoamyl alcohol in a 25:24:1 ratio; #P3803, Sigma) was added to the lysates and then centrifuged at 1500 × g for 5 minutes at room temperature. The upper phase was transferred to a new 1.5 ml tube, to which 500 μl of PCI solution was added, and the process was repeated. DNA was precipitated by adding 2.5 volumes of absolute EtOH containing 0.3 M sodium acetate (pH 5.2) and then washed with 70% ethanol before being dried. In the final step of DNA extraction, the DNA was dissolved in 50 μl of nuclease-free water, and its concentration was determined. To measure the ratio of mtDNA to nDNA, qPCR was performed using an ABI7500 (Applied Biosystems, Foster City, CA) and SYBR Green PCR Master Mix (ABS-4309155, Applied Biosystems) following the manufacturer’s guidelines. Data were normalized to nDNA expression and analyzed using the ∆ (∆ CT) method.

Measurement of viable cell number and cell viability

Viable cell number was determined using the Trypan Blue exclusion assay. To assess cell viability, MSCs were plated in 96-well culture plates at a density of 1 × 10³ cells per well. Mitochondria were then added to each well and incubated for 1 day. Post-mitochondrial transfection, the cells were exposed to 100 μl of growth medium containing diluted WST-8 solution at 37°C and 5% CO₂ for a duration of 0.5-4 hours. Absorbance was recorded at 450 nm with a reference wavelength of 600 nm using a microplate spectrophotometer (TECAN).

Statistical analysis

The data are presented as the mean ± S.D. Statistical significance was evaluated using the Mann-Whitney U or Kruskal-Wallis test (with Dunn’s post-hoc) for n < 30, and one-way ANOVA (with Holm-Sidak’s post-hoc) for n > 30.