Generation of HA-iMSCs and HA-iMSC-EVs
iMSC were prepared as described in our previous study [17]. Briefly, iMSC was differentiated from iPSC that was established from WJ-MSC transduced with Yamanaka factors using CytoTune iPS 2.0 Sendai Reprogramming kit (Thermo Fisher Scientific, Waltham, MA, USA). Established iMSCs were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone, Chicago, IL, USA) supplemented with 15% Fetal Bovine Serum (FBS; HyClone) and 1% antibiotic–antimycotic solution (Thermo Fisher Scientific) at 37 °C in 5% CO2 and 95% humidified air. Upon reaching 90% confluence, the cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded at a density of 10,000 cells/cm2. Next day, the cells were treated with 40 μg/mL Hyaluronic acid (Sigma aldrich, St Louis, MO, USA) for 24 h, after which the media were aspirated, and the cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS; HyClone). The culture media were replaced with phenol red-free DMEM (Gibco, Waltham, MA, USA) supplemented with 15% EV-depleted FBS. EV-depleted FBS was prepared as described previously [33]. After 3 days of incubation, the culture medium was harvested, and HA-iMSC-EVs were isolated by ultracentrifugation as described previously [34].
Flow cytometry
To confirm whether HA preconditioned iMSCs express the typical cell surface markers for MSCs, HA-iMSCs were trypsinized and washed twice prior to resuspension in PBS containing 2% FBS. Cells were adjusted to 1 × 106 in 100 µL of cell suspension. For cell surface labeling, cell suspensions were incubated at 4 °C for 30 min with antibodies (dilution, 1:20). APC conjugated mouse anti-human CD73, PE-conjugated mouse anti-human CD105, FITC-conjugated mouse anti-human CD45, PE-conjugated mouse anti-human CD31, and APC conjugated mouse anti-human CD34 antibodies were supplied by eBioscience (Waltham, MA, USA) and, APC-Cy7-conjugated mouse anti-human CD90 was supplied by BioLegend (San Diego, CA, USA). To characterize surface proteins of EVs, HA-iMSC-EVs analysis was performed using human MACSPlex Exosome Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Flow cytometric analysis was conducted using an Attune NxT flow cytometer (Thermo Fisher Scientific).
Cryo-transmission electron microscopy
To confirm the morphology and size of EVs, Cryo-transmission electron microscopy (TEM) was performed as described in our previous study [34]. Briefly, the HA-iMSC-EVs suspension was placed on a grid and blotted and were visualized at 36,000 × magnification using a Talos L120C FEI transmission electron microscope (Thermo Fisher Scientific) at 120 kV.
Nanoparticle tracking analysis
To measure the particle size distribution and concentration of EVs, Nanoparticle Tracking Analysis (NTA) was performed using Zetaview®BASIC NTA-Nanoparticle Tracking (Particle Metrix, Inning am Ammersee, Germany). For the analysis, EVs were serial diluted in sterile PBS to reach the optimal volume for NTA. The standard control was set as Sensitivity: 80, Frame Rate: 30, Shutter: 100, Temperature: 23 °C.
Labeling of HA-iMSC-EVs with DiR and DiD and fluorescent imaging
HA-iMSC-EVs were incubated with 1 μg/mL DiR or DiD buffer according to the protocol provided by Lipophilic Tracers (Invitrogen, Waltham, MA, USA) as described previously [34]. 500 μg of DiR-labeled HA-iMSC-EVs were resuspended in 0.05 mL of phosphate buffered saline (PBS) and intravenously or intramyocardially injected into rats. DiR-labeled HA-iMSC-EVs were detected at 6 and 24 h using an In Vivo Imaging System (IVIS, Perkin Elmer, Waltham, MA, USA). Human umbilical venous endothelial cells (HUVECs) or neonatal rat cardiac fibroblasts (NRCFs) were treated with DiD-labeled HA-iMSC-EVs. After 24 h, DiD-labeled HA-iMSC-EVs were observed under a Nikon Eclipse Ti2-U fluorescence microscope (Nikon, Tokyo, Japan).
Cell viability and tube formation
HUVECs (3 × 103 in 96 well plates) were treated with 500 μM of H2O2 for 2 h. Culture medium were then replaced with serum-free EGM2 Endothelial Growth Medium (LONZA, Basel, Switzerland) containing 100 μg/mL of iMSC-EVs or HA-iMSC-EVs, and cultured for 48 h at 37 °C and 5% CO2 incubator. Cell Counting Kit-8 (CCK-8; Enzo Life Sciences, Farmingdale, NY, USA) analysis was performed according to the manufacturer’s instructions. For tube-forming assay, the HUVECs were resuspended in a serum-free EGM2 medium and seed in 96 well plates (1 × 104 cells/well) pre-coated with Matrigel (Corning, NY, USA). Tube formation was confirmed after 16 h.
Cytotoxicity and reactive oxygen species assay
Cardiomyocytes derived from iPSCs (iPSC-CMs) were treated with 500 μM H2O2 for 2 h, and subsequently incubated with iMSC-EVs or HA-iMSC-EVs in serum-free DMEM for 24 h. Next, the CellROX® Reagent (Thermo Fisher Scientific) was mixed with serum-free DMEM to a final concentration of 5 μM and added to the culture for 30 min. Following staining, the cells were fixed in 4% paraformaldehyde (Fujifilm Wako Chemicals, Richmond, VA, USA) for 10 min and washed three times with DPBS. Nuclei and cell bodies were counterstained with NucBlue™ Fixed Cell stain or CellTracker™ (Invitrogen), respectively. After this process, all samples were observed using a Nikon Eclipse Ti2-U (Nikon, Tokyo, Japan), and the percentage of ROS-positive cells was analyzed based on nuclear intensity.
Real time PCR
Total RNA was isolated from the M1 or M2 polarized THP-1 macrophages treated with HA-iMSC-EVs 100 µg/mL for 24 h using TRIzol® (Ambion, Waltham, MA, USA). M1 macrophages were induced using 100 ng/mL of lipopolysaccharides (LPS) and 20 ng/mL of interferon-gamma (IFN-γ) after 200 ng/mL of phorbol 12-myristate 13-acete (PMA) treatment of THP-1 cells, and M2 macrophages were induced using 20 ng/mL of interleukin 4 (IL-4) and 20 ng/mL of interleukin 13 (IL-13) after 200 ng/mL of PMA treatment. GAPDH was used as a reference to normalize the differences in mRNA quantity in each sample. The relative gene expression levels were analyzed using the comparative 2−ΔΔCt method. hGAPDH: F; 5′-GTCGGAGTCAACGGATTTGG-3, R; 5′-AGTTGAGGTCAATGAAGGGGTC-3, hTNF-α: F; 5′-GAGCTGAACAATAGGCTGTTCCCA-3, R; 5′-AGAGGCTCAGCAATGAGTGACAGT-3, hCXCL10: F; 5’-TGGCATTCAAGGAGTACCTCTC-3, R; 5′-TGATGGCCTTCGATTCTGGA-3, hTGF-β: F; 5′-CCCAGCATCTGCAAAGCTC-3, R; 5′-GTCAATGTACAGCTGCCGCA-3, IL-10: F; 5′-TGAAAACAAGAGCAAGGCCG-3, R; 5′-GCCACCCTGATGTCTCAGTT-3, hCCL22: F; 5′-GCGTGGTGTTGCTAACCTTCA-3, R; 5′-GGGGAGCAGCTATAATGGCA-3.
Western blot
HA-iMSCs and HA-iMSC-EVs were lysed in NP40 or RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Thermo Fisher Scientific). Protein concentration was measured using the Bradford Assay Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Samples were diluted using 4 × Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) and heated at 100 °C for 10 min. Proteins were loaded and separated on precast polyacrylamide Mini-PROTEAN TGX gels (Bio-Rad Laboratories) and transferred to PVDF membranes (Bio-Rad Laboratories). The membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad Laboratories) for 5 min and then treated overnight with primary antibodies at 4 °C. All primary antibodies were diluted in the EveryBlot Blocking Buffer. Antibodies against GM130, Histone H3 (Cell Signaling Technology, Leiden, The Netherlands), CD63, CD9, calnexin, Cytochrome C (Abcam, Cambridge, UK), TSG101, and CD81 (Invitrogen, Waltham, MA, USA) were used as the primary antibodies. Western blotting for all target proteins, except CD81 was performed under reducing conditions. The membranes were washed for 10 min for five times and then treated with the secondary antibodies for 1 h. After the membranes were washed for 10 min for five times, the target proteins were detected using the ECL Select Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and analyzed using the ChemiDoc Imaging System (Bio-Rad Laboratories). For detecting inflammasome proteins in THP-1 treated with LPS 1 μg/mL, adenosine triphosphate (ATP) 5 mM and HA-iMSC-EVs 400 μg/mL, primary antibodies against NLRP3 (NACHT, LRR, and PYD domain-containing protein 3, also known as cryopyrin), pro-caspase-1, interleukin-1 beta (IL-1β), p65, GAPDH (Abcam, Cambridge, UK), caspase-1 (Novus biologicals, Centennial, CO, USA), alpha-tubulin and phospho-p65 (Cell signaling, Leiden, The Netherlands) were used. The fibrosis-related proteins were detected in NRCF-treated with transforming growth factor-beta (TGF-beta) 1 μg/mL (for 48 h) (Sigma Aldrich, St Louis, MO, USA) and 400 μg/mL HA-iMSC-EVs (for 24 h). Collagen 1, Fibronectin, TIMP-1 (Invitrogen), MMP-2 (Abcam), phospho-SMAD2, and SMAD2/3 (Cell Signaling) were used as primary antibodies. Western blotting of all target proteins, except collagen 1, was performed under reducing conditions.
Bioinformatics
Metascape pathway and process enrichment analysis (http://metascape.org) were used to investigate the potential biological functions and mechanisms of EV proteins. The Reactome gene set was used as an ontology source, and enriched terms with a p-value < 0.01, minimum count of three, and enrichment factor > 1.5 were collected and grouped into clusters. In addition, a Connectivity Map (https://clue.io) was employed to analyze HA-iMSC-EV-specific proteins and identify approved drugs that were similar to HA-iMSC-EVs. Based on this analysis, the potential indications for EVs were predicted.
Myocardial ischemia–reperfusion injury and HA-iMSC-EVs treatment
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of The Catholic University of Korea (approval number: CUMC-2020–0063-03). To produce an ischemia–reperfusion (I/R) model, male Fischer 344 rats (8-weeks old, 160–180 g, Koatech, South Korea) were anesthetized with 2% inhaled isoflurane and intubated via the trachea using an 18-gauge intravenous catheter. The proximal portion of the left anterior descending artery was surgically occluded for 1 h through ligation with a 7–0 PROLENE suture, followed by coronary reperfusion through the release of the tie. 50 μL of HA-iMSC-EVs were injected into the myocardium at a dose of 10 mg/kg or 20 mg/kg, and 500 μL of HA-iMSC-EVs were intravenously injected at a dose of 20 mg/kg, 5 min before releasing the tie. In the group that received two injections, the material was delivered to the myocardium using the same method 7 days after the first injection.
Measurement of myocardial infarct size
2,3,5-Triphenyltetrazolium chloride (TTC) and Evan’s Blue staining was performed to assess the early cardioprotective effects of HA-iMSC-EVs. Rats were anesthetized and mechanically ventilated according to previously described methods. After 60 min of ischemia and 24 h of reperfusion, the suture thread around the LAD artery, previously used in surgery, was retired, and Evan’s blue dye (9% in PBS) was administered intravenously. After 15 min, the heart was excised and immediately incubated for 10 min at—4 °C. The groups were divided into two according to the timing of the injection: (1) under ischemic conditions, the EVs were injected 5 min before reperfusion, and (2) under reperfusion conditions, the EVs were injected 5 min after reperfusion. And each group was divided into two groups according to the route of the injection: intramyocardial (IM) or intravenously (IV) injection. The heart was sliced into 3 sections, each approximately 2 mm in thickness, and incubated with 2% TTC for 30 min in a dark, humid environment at 37 °C. Following three washes, tissues were fixed in 4% paraformaldehyde. Evan’s blue staining indicated non-infarcted myocardium, which was stained deep blue, whereas the viable myocardium was stained red by TTC. The necrotic myocardium appeared white on TTC staining. The ImageJ software was used to quantify the area at risk (AAR) and necrotic areas.
Echocardiography
The I/R-induced cardiac injury was functionally evaluated using echocardiography. Rats were anesthetized with 2% isoflurane, and data were recorded using a transthoracic echocardiography system equipped with a 15 MHz L15-7io linear transducer (Philips, Amsterdam, Netherlands, Affiniti 50G). Serial echocardiograms were performed at baseline and 4 h, 1, 2, 3 and 5 weeks after surgery. The experiment was conducted in a blinded manner, with the echocardiography operator unaware of the group allocation. Left ventricular systolic function was assessed by calculating the ejection fraction (EF) and fractional shortening (FS) using the following formulas: EF (%) = [(LVEDV-LVESV)/LVEDV] × 100 and FS (%) = [(LVEDD-LVESD)/LVEDD] × 100.
Hemodynamic measurements
Hemodynamic measurements were conducted at the 5-week endpoint prior to euthanasia. The rats were anesthetized using 2% isoflurane, and thoracotomy was performed without causing bleeding. A 26-gauge needle was used to puncture the left ventricular (LV) apex, and a 2F conductance catheter (Millar, Houston, TX, USA, SPR-838) was inserted into the LV. Continuous recording of the pressure–volume (PV) parameters was accomplished using a PV conductance system (Emka TECHNOLOGIES, Paris, France, MPVS Ultra) connected to a digital converter (ADInstruments, Dunedin, New Zealand, PowerLab 16/35). Load-independent cardiac function measurements were obtained with different preloads induced by inferior vena cava occlusion using a needle holder. To assess the parallel conductance after hemodynamic measurements, hypertonic saline (50 µL of 20% NaCl) was injected into the left jugular vein. Blood was collected from the LV into a heparinized syringe and placed into cuvettes for conversion of the conductance signal to volume using a catheter. Calibration of the parallel and cuvette conductance was used to confirm the absolute volume of the rat.
Immunohistochemical staining
The hearts were fixed with 4% paraformaldehyde overnight, and paraffin blocks were prepared. Cross-sections of the heart, measuring 4 µm, were made using a microtome (Leica, Wetzlar, Germany, LRM2255), starting from the top of the apex. Immunofluorescence was performed to evaluate the capillary density of the injured hearts. After deparaffinization and rehydration, antigen retrieval was performed using a target retrieval solution in a decloaking chamber. The sections were then incubated with a diluted primary antibody at 4 °C overnight. The primary antibodies used were mouse anti-cardiac troponin T (1:200) (Abcam, Cambridge, UK; ab8295) and goat anti-CD31 (1:200) (R&D Systems, Minneapolis, MN, USA; AF3628). After washing the samples four times with phosphate-buffered saline (PBS), the secondary antibody was added and incubated for 90 min at room temperature in the dark. The secondary antibodies used in this study were donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (1:500) (Invitrogen, Waltham, MA, USA, A-21202), and rabbit anti-goat IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 594 (1:500) (Invitrogen, A-11080). Following another wash with PBS, the sections were stained with an anti-fade mounting medium containing 40,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA, H-1200–10) for nuclear staining and then mounted onto slides.
Masson’s trichrome staining
To determine the fibrotic area, wall thickness scar area, and viable myocardium of the injured hearts, Masson’s trichrome staining (Sigma-Aldrich, Saint Louis, MO, USA, HT15) was carried out. Paraffin slides were preincubated overnight at 37 °C before deparaffinization and rehydration. After deparaffinization and rehydration, the paraffin sections were fixed again for 1 h and 30 min in Bouin’s solution at 56 °C, followed by washing with tap water for 20 min. The sections were then stained with Weigert’s iron hematoxylin solution for 15 min at room temperature, followed by staining with Biebrich scarlet acid fuchsin solution for 20 min at room temperature. Finally, sections were counterstained with aniline blue for 15 min and incubated in 1% acetic acid for 2 min at room temperature. The collagen fibers appeared blue, and the viable myocardium appeared red. Heart section imaging was conducted using a slide scanner (3DHISTECH Ltd., Budapest, Hungary, PANNORAMIC MIDI II), and all areas, including the fibrotic area, were quantified using ImageJ software.
Statistical analyses
In vitro statistical analyses were performed by one-way analysis of variance (ANOVA) using GraphPad Prism Software and data were expressed as means ± standard deviation (SD). Alternatively, one-way analysis of variance (ANOVA) performed using SPSS version 18.0 (IBM Corp., Armonk, NY, USA) and the data were expressed as means ± standard error (SE). A value of p < 0.05 were considered statistically significant. In vivo data are presented as the mean ± standard error of the mean (SEM). Statistical significance (p < 0.05) was determined by one-way ANOVA and unpaired t-test using GraphPad Prism Software.
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