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Exosomal miR-202-5p derived from iPSC-MSCs protects against myocardial infarction through inhibition of cardiomyocyte pyroptosis

Stem Cell Research & Therapy volume 16, Article number: 282 (2025) Cite this article

Abstract

Background

NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated pyroptosis of cardiomyocytes is a key contributor to the progression of myocardial infarction (MI). This study aimed to investigate whether exosomes derived from human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSC-EXOs) could protect against MI by inhibiting cardiomyocyte pyroptosis and explore the underlying mechanisms.

Methods

Exosomes from human bone marrow-MSCs (BM-MSC-EXOs) and iPSC-MSCs (iPSC-MSC-EXOs) were collected and intramuscularly injected into the peri-infarct region of a mouse MI model. Cardiac function was assessed four weeks post-injection. Myocardial pyroptosis was evaluated using TUNEL staining and measurement of associated factors. Neonatal mouse cardiomyocytes (NMCMs) exposed to serum deprivation and hypoxia (SD/H) were treated with BM-MSC-EXOs or iPSC-MSC-EXOs. A loss-of-function approach was employed to examine the role of iPSC-MSC-exosomal-miR-202-5p in regulating cardiomyocyte pyroptosis.

Results

Compared to BM-MSC-EXOs, iPSC-MSC-EXOs demonstrated superior improvement in cardiac function in MI mice. Both BM-MSC-EXOs and iPSC-MSC-EXOs reduced cardiomyocyte pyroptosis by downregulating proteins NLRP3, ASC, Caspase-1, and gasdermin D-NT, as well as inflammatory factors in MI mice and SD/H-treated NMCMs. iPSC-MSC-EXOs exhibited greater protective effects. MicroRNA sequencing revealed higher levels of miR-202-5p in iPSC-MSC-EXOs than in BM-MSC-EXOs. The protective effect of iPSC-MSC-EXOs against cardiomyocyte pyroptosis was partially reversed by miR-202-5p knockdown. Mechanistically, miR-202-5p in iPSC-MSC-EXOs inhibited cardiomyocyte pyroptosis by downregulating the TRAF3IP2/JNK pathway.

Conclusions

iPSC-MSC-EXOs protect against MI by inhibiting cardiomyocyte pyroptosis via miR-202-5p-mediated suppression of the TRAF3IP2/JNK axis. These findings suggest a promising therapeutic approach for MI.

Graphical Abstract

Introduction

Myocardial infarction (MI) has become a major cause of morbidity and mortality worldwide [1, 2]. It is caused by the abrupt occlusion of the coronary artery with consequent acute myocardial ischemia and irreversible myocardial cell death. Despite available treatments, including early oxygen supplementation, early thrombus treatment and immediate reperfusion, the mortality of MI remains persistently high [3]. The only cure for this devastating disease is heart transplantation, but this is severely hampered by a shortage of donor organs, high cost and post transplantation immune rejection [4]. It is important to investigate the underlying mechanisms of MI to explore novel strategies that may protect the myocardium against damage.

Pyroptosis, a recently recognized type of pro-inflammatory programmed cell death, is mainly activated in a NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome [5]. The NLRP3 inflammasome, a tightly regulated macromolecular complex composed of NLRP3, apoptosis-associated speck-like protein (ASC), and pro-Caspase-1, detects cellular injury and initiates an inflammatory response by activating Caspase-1. This in turn cleaves gasdermin D (GSDMD) and processes release of pro-inflammatory cytokines such as interleukin (IL)−1β and IL-18, ultimately inducing pyroptosis in cardiomyocytes [6, 7]. There is recent accumulating evidence that pyroptosis plays a pivotal role in the pathogenesis of cardiovascular diseases [8,9,10]. Patients with acute MI exhibit an increased plasma level of NLRP3 and caspase-1 [11]. In vitro oxygen–glucose deprivation/reoxygenation in cardiomyocytes, as well as in vivo ischemia/reperfusion (I/R) models, significantly increased NLRP3 inflammasome formation, thereby exacerbating pyroptosis [12]. In non-ischemic cardiomyopathy, chronic pressure overload activates a pro-inflammatory response in the heart, leading to NLRP3 inflammasome activation in cardiomyocytes that promotes maladaptive left ventricular remodeling and fibrosis in rats [13]. It has been reported that 3,4-benzo[a] pyrene, a common component of air pollution particulate matter, aggravates MI injury by activating NLRP3-related cardiomyocyte pyroptosis via regulation of the PINK1/Parkin-mitophagy-mPTP opening axis [14]. Chlorogenic acid treatment has been shown to ameliorate myocardial ischemia–reperfusion injury in mice via inhibition of Lnc Neat1/NLRP3 inflammasome-mediated cardiomyocyte pyroptosis [15]. Therefore, strategies that target NLRP3-related cardiomyocyte pyroptosis post MI may attenuate heart injury.

Over the past decades, mesenchymal stem cell-derived exosomes (MSC-EXOs) have shown great potential in the treatment of cardiovascular diseases by inhibiting cardiomyocyte death, promoting angiogenesis and ameliorating inflammation via delivery of various critical components including miRNAs, lncRNAs and proteins [16,17,18]. It has been shown that MSC-derived exosomal miR-182-5p improved heart function following ischemia/reperfusion injury in mice by inhibiting GSDMD-dependent cardiomyocyte pyroptosis [19]. Thus, targeting cardiomyocyte pyroptosis is a potential rationale for MSC-EXO-based therapy in MI. Nevertheless, the functions of MSCs isolated from bone marrow (BM) or adipose tissue decline with aging or extensive expansion in vitro, with corresponding reduced function of MSC-EXOs [20]. In our previous study, we reported that MSCs derived from induced pluripotent stem cells (iPSC-MSCs) not only shared phenotypic characteristics with adult MSCs, they exhibited a higher proliferation capacity and stronger immune privilege [21, 22]. Our study also showed that they exerted superior therapeutic efficacy to BM-MSCs for cardiovascular diseases due to better paracrine effects [23]. Nevertheless, whether iPSC-MSC-EXOs protect against MI via targeting of NLRP3-related cardiomyocyte pyroptosis has not been determined. In the current study, transfer of miR-202-5p by iPSC-MSC-EXOs promoted cardiac function recovery in a mouse model of MI by inhibiting NLRP3-related cardiomyocyte pyroptosis through regulation of the TRAF3IP2/JNK pathway.

Methods and materials

Cell culture

All cell cultures in this study were reviewed and approved by the Ethics Committees of Tongji University (Approved project: mesenchymal stem cell-based therapy for myocardial infarction, Approval No. 2016-050, Date of approval: Sep 18, 2016). Written informed consent was obtained from patients for participation in the study and the use of samples. iPSC-MSCs and BM-MSCs were cultured as described previously [24]. Briefly, iPSC-MSCs and BM-MSCs were cultured in DMEM supplemented with 10% FBS (10,270-106, GIBCO), 5 ng/mL bFGF (100-18B, PeProTech) and 10 ng/mL EGF (AF-100–15, PeProTech) in a 5% CO2 incubator at 37 °C. All MSCs used in this study were at passage 3–5. Neonatal mice cardiac myocytes (NMCMs) were isolated from heart tissue of 0–1-day-old neonatal mice and routinely cultured at 37 °C in Claycomb Medium (51,800, Sigma) containing 10% FBS as previously reported[24]. Mouse cardiac fibroblasts were isolated from the heart tissue of 0- to 2-day-old neonatal mice. The ventricles were minced and digested using 0.1% collagenase type II (17,101–015, GIBCO) for 5–7 cycles at 37 °C. The digested cell suspension was filtered through a 70 µm cell strainer and pre-plated for 1 h to allow fibroblasts to adhere. Non-adherent cells were discarded, and adherent fibroblasts cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a 5% CO₂ incubator at 37 °C. Cardiac fibroblasts at passage 2–4 were used for subsequent experiments. Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin in a 5% CO₂ incubator at 37 °C.

Isolation and characterization of MSC-EXOs

Briefly, 1 × 106 iPSC-MSCs or BM-MSCs were cultured in a 10-cm culture dish for 24 h at 37 °C. After washing with PBS three times the culture medium was replaced with DMEM containing 10% exosome-depleted FBS (EXO-FBS-250 A-1, Systems Biosciences) and cells cultured for 48 h. Subsequently, the supernatant was collected and MSC-EXOs isolated using anion exchange chromatography as previously described [25, 26]. Briefly, we packed an Econo-Pac column (Bio-Rad Laboratories, CD63 Hercules, CA, USA) with 4 mL anion-exchange resin (Q Sepharose Fast Flow), equilibrated it with Equilibration Buffer, and loaded it with the supernatant. Following washing with Wash Buffer to remove protein impurities, exosomes were eluted in 1 mL fractions using Elution Buffer. The exosome-containing fractions, identified by Bradford protein assay and CD63 ELISA, were pooled, dialyzed in PBS overnight, and concentrated using a 30 kDa protein concentrator (Thermo Fisher Scientific). Endotoxin-free materials and buffers were used throughout to prevent contamination. Finally, MSC-EXOs were suspended in PBS and stored at − 80 °C for future use. BCA protein assay was used to measure the concentration of MSC-EXOs. The morphology and particle size of MSC-EXOs were evaluated by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. Surface markers including Alix, CD63, TSG101 of MSC-EXOs were determined by Western blotting.

Cellular uptake of MSC-EXOs

To examine the uptake of MSC-EXOs by NMCMs or cardiac fibroblasts, iPSC-MSC-EXOs or BM-MSC-EXOs were labeled with PKH67 (MINI67-1 KT, Sigma) and co-cultured with NMCMs or cardiac fibroblasts for 24 h. After washing with PBS three times, labeled NMCMs or cardiac fibroblasts were fixed with 4% paraformaldehyde at room temperature for half an hour. Finally, the cells were mounted with DAPI and captured using a confocal microscope.

HUVEC tube formation assay

HUVECs were seeded onto Matrigel-coated 96-well plates at a density of 1 × 104 cells per well. The cells were treated with BM-MSC-EXOs (10 µg/mL) or iPSC-MSC-EXOs (10 µg/mL), with each treatment group set up in triplicate wells. After 6 h of incubation, tube formation was imaged, and the total tube length and number of branching points quantified using ImageJ software. All experiments were performed in triplicate.

PI staining

The pyroptosis of cardiomyocytes was evaluated by propidium iodide (PI) staining. Briefly, NMCMs were cultured in 24-well plates with cover slides and treated with PBS, BM-MSC-EXOs (10 μg/mL) or iPSC-MSC-EXOs (10 μg/mL) under a serum deprivation/hypoxia challenge (SD/H) (94% N2, 5% CO2, and 1% O2) for 48 h. Next, the cells were incubated with PI (10 μM) at room temperature for 30 min and then mounted with DAPI. Finally, PI positive cells, stained red, were randomly captured from five different fields of view. The percentage cardiomyocyte pyroptosis was calculated as the ratio of PI-positive cells to total number of NMCMs.

Transfection of miR-202-5p inhibitor and mimic

miR-202-5p mimic, inhibitor and miR-control were purchased from GenePharma (Shanghai, China). Briefly, 1 × 106 NMCMs were seeded on a 10-cm culture dish and transfected with 50 nM miR-control, miR-202-5p mimic or inhibitor by Lipofectamine 2000 transfection reagent according to the protocol (11,668,027, Invitrogen). Subsequently, NMCMs were cultured for 48 h and then harvested for further experiments. To isolate miR-202-5pKD-iPSC-MSC-EXOs, iPSC-MSCs were transfected with 50 nM miR-control or miR-202-5p inhibitor and the EXOs isolated.

Luciferase reporter assay

The 3′-UTR of TRAF3IP2 containing the miR-202-5p target site or the mutation in the seed region of the miR-202-5p binding site was inserted into the pGL3 luciferase reporter vector (Promega, USA). 293 T cells were co-transfected with the reporter plasmid (pGL3-TRAF3IP2-3′-UTR or mutant pGL3-TRAF3IP2-3′-UTR vector) and miR-control or miR-202-5p mimics using Lipofectamine 2000 (11,668,027, Invitrogen). Finally, luciferase activity was determined using a Dual-Luciferase Reporter Assay System Kit (E1910, Promega) at 48 h after transfection.

Quantitative real-time PCR

Total RNA from iPSC-MSC-EXOs, BM-MSC-EXOs and NMCMs with different treatments was extracted by TRIzol reagent (2270 A, Takara). Reverse transcription was performed using a PrimeScript RT Reagent Kit (RR037 A, Takara). RT-PCR for TRAF3IP2 or miR-202-5p was carried out using a One-Step TB Green® PrimeScript™ RT-PCR Kit following the manufacturer’s instructions (RR820 A, Takara). GAPDH and U6 served as the internal reference. Relative expression of TRAF3IP2 or miR-202-5p was analyzed by the 2-ΔΔCt method.

MiRNA sequencing

Total RNA from iPSC-MSC-EXOs and BM-MSC-EXOs was extracted using a miRNeasy® Mini kit (217,004, Qiagen) following the manufacturer’s instructions. The miRNA sequencing was performed by Illumina HiSeqTM 2500 (Genedenovo Co. Ltd, Guangzhou, China). Raw reads were filtered and the expression of miRNAs analyzed to evaluate significant differences between BM-MSC-EXO and iPSC-MSC-EXO data sets. Differentially expressed miRNAs (DE miRNAs) were evaluated by a fold change > 1.5 and Q value < 0.001 with the threshold set for down- and up-regulated genes. Heat maps of DE miRNAs were generated by the RStudio.

Western blotting

Total protein of mouse heart tissue from different groups and NMCMs with different treatments was extracted using a total protein extraction kit (Bestbio, BB-3101). Protein concentrations were determined by a BCA protein assay kit (Thermo, 231,227). A total 30 μg protein from different treatments was separated on SDS-PAGE gel by electrophoresis and transferred to PVDF membranes. Next, the PVDF membranes were blocked in TBST with 5% fat-free milk and incubated at 4 °C overnight with the following primary antibodies: anti-Alix (Abcam, ab186429), anti-TSG101 (Abcam, ab125011), anti-NLRP3 (Abcam, ab263899), anti-ASC (Abclonal, A1170), anti-Pro-Caspase1 (Abcam, ab179515), anti-Caspase1 (Abclonal, A0964,), anti-GSDMD (Abcam, ab209845), anti-GSDMD-NT (Affinity, AF4012), anti-TRAF3IP2 (Proteintech, 26,692-1-AP), anti-p-JNK (Proteintech, 80,024-1-RR), anti-JNK (Proteintech, 66,210-1-Ig), anti-IL-18 (ABclonal, A1115), anti-IL-1β (Proteintech, 26,048-1-AP), anti-ERK (Cell Signaling, 9101), anti-p-ERK (Cell Signaling, 4695), anti-AMPK (Proteintech, 10,929-2-AP), anti-p-AMPK (Affinity, AF3423) and anti-GAPDH (Abcam, ab8245). After washing with TBST three times, the membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, the membranes were developed by ECL system (Beyotime, China) and the density of protein bands analyzed by Image J software.

Animal study

All animal procedures were approved by the Animal Research Committee of Guangdong Provincial People’s Hospital (Approved project: Study on the effect and mechanism of iPSC-MSCs exosomal miR-202-5p on the improvement of myocardial infarction by inhibiting the activation of NLRP3 inflammasome. Approval No.KY-Z-2022-348-01, Date of approval: Nov 28, 2022). This study has been reported in accordance with the ARRIVE guidelines [27]. The mice were anesthetized with 2% isoflurane inhalation and then connected to a small rodent ventilator. A model of MI was induced in male C57BL/6 J mice (20–25 g), 6–8 weeks old by ligation of the left anterior descending coronary artery (LAD) using an 8–0 suture as previously reported [28]. After LAD ligation, mice were randomly divided into four groups: 1) phosphate-buffered saline (PBS) (MI group, n = 12); 2) 20 μg BM-MSC-EXOs (BM-MSC-EXOs, n = 12); 3) 20 μg iPSC-MSC-EXOs (iPSC-MSC-EXOs group, n = 12); 4) 20 μg miR-202-5pKD-iPSC-MSC-EXOs (miR-202-5pKD- iPSC-MSC-EXOs, n = 12). All EXOs were dissolved in 40 μL PBS and then intramuscularly injected into four sites at the border zone of the infarcted mouse heart 30 min following surgery. Mice that underwent thoracotomy without LAD ligation served as a sham group (n = 6). All animal procedures were carried out by a skilled technician who was blinded to group allocation and unable to influence whether an animal would receive PBS, BM-MSC-EXOs, iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs treatment. The mice temperature during surgery was controlled by a heat pad. Carprofen was administered immediately after surgery and daily for three days to alleviate pain and inflammation. Transthoracic echocardiography (Ultramark 9, Soma Technology) was carried out to evaluate cardiac function at baseline (before MI) and on day 28 after MI. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were recorded and calculated.

Masson trichrome staining

After cardiac function measurement at 28 days post-MI, all mice were euthanized by intraperitoneal injection of 2 mL pentobarbital (200 mg/mL, Vetoquinol). Next, heart tissue from different groups was quickly harvested, fixed, embedded in paraffin and sectioned into 5 µm slices. Subsequently, Masson’s Trichrome staining was carried out according to the protocol to detect cardiac fibrosis (Sigma, HT15). Images from six mice for each group were captured by a microscope and analyzed. The percentage infarct size was calculated as the sum of infarcted area/the sum of LV area × 100%.

Immunostaining

Following euthanasia, mouse hearts were collected, fixed in 4% paraformaldehyde, and embedded in paraffin. Heart tissue was sectioned into 5 µm-thick slices for subsequent immunostaining. Briefly, sections were deparaffinized, rehydrated, and subjected to antigen retrieval. TUNEL staining was performed using a TUNEL assay kit (RIBOBIO, C11026-1) to detect apoptotic cells. For cardiomyocyte and immune cell identification, sections were incubated with primary antibodies against Troponin (Abcam, ab209813) and CD45 (Servicebio, GB113886), respectively. After incubation with the appropriate secondary antibodies, the sections were counterstained with DAPI to visualize cell nuclei. For each group (n = 6), three random non-overlapping fields of view were selected per heart section. Images were captured using a fluorescence microscope (Leica) and staining quantified using ImageJ software.

Statistical analysis

All experiments were repeated at least three times and data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9.3.0 Software (GraphPad Software, USA). Comparison between two groups was analyzed by unpaired Student’s t-test and between multiple groups by one-way ANOVA followed by the Bonferroni test. A p value < 0.05 was considered statistically significant.

Results

Characterization of EXOs derived from iPSC-MSCs

BM-MSC-EXOs and iPSC-MSC-EXOs were isolated and identified. TEM revealed that both BM-MSC-EXOs and iPSC-MSC-EXOs exhibited a typical cup-shaped morphology (Fig. 1A). The NTA results demonstrated that the size of BM-MSC-EXOs and iPSC-MSC-EXOs was comparable at 30–120 nm (Fig. 1B). Nonetheless the concentration of iPSC-MSC-EXOs was much higher than that of BM-MSC-EXOs (Fig. 1B). Western blotting revealed that both BM-MSC-EXOs and iPSC-MSC-EXOs were positive for EXO-specific markers Alix, TSG101 and CD63 but negative for Calnexin (Fig. 1C). Next, to determine whether MSC-EXOs could be absorbed by NMCMs, PKH67-labeled BM-MSC-EXOs and iPSC-MSC-EXOs were co-cultured with NMCMs for 24 h under SD/H conditions. Confocal images confirmed the presence of PKH67-labeled MSC-EXOs in the cytoplasm of NMCMs (Fig. 1D). Collectively, these data demonstrated that BM-MSC-EXOs and iPSC-MSC-EXOs had been successfully isolated and identified.

Fig. 1
figure 1

Characterization of BM-MSC-EXOs and iPSC-MSC-EXOs. A BM-MSC-EXOs and iPSC-MSC-EXOs showed a cup-shaped morphology determined by TEM. Scale bar = 100 nm, B NTA assay showed the size and concentration of BM-MSC-EXOs and iPSC-MSC-EXOs, C representative images of Western blotting showing surface protein markers of BM-MSC-EXOs and iPSC-MSC-EXOs, D representative images revealed the uptake of PKH67-labelled MSC-EXOs by NMCMs after 24 h of incubation under SD/H condition. Scale bar = 20 μm. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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Injection of iPSC-MSC-EXOs improved cardiac function and reduced infarct area in mice

The cardioprotective effects of iPSC-MSC-EXOs were explored in a mouse model of MI. Representative echocardiographic images at 28 days post MI in mice from different groups are shown in Fig. 2A. The echocardiography results revealed no significant difference in LVEF or LVFS among the different groups prior to MI (Fig. 2B). On the contrary, at 28 days post MI, compared with the sham group, LVEF and LVFS were significantly decreased in the MI group (Fig. 2B). Administration of BM-MSC-EXOs and iPSC-MSC-EXOs improved LVEF and LVFS, with a greater improvement seen with iPSC-MSC-EXOs (Fig. 2B). Similarly, Masson’s trichrome staining results demonstrated that compared with the MI group, the infarct size (calculated as the sum of infarcted area/the sum of LV area × 100%) was notably decreased in the BM-MSC-EXOs group and iPSC-MSC-EXOs group, again to a greater extent in the latter (Fig. 2C, D). In addition, CD45 immunostaining revealed a significant increase in CD45-positive inflammatory cells following MI (Supplementary Fig. 1). Treatment with both BM-MSC-EXOs and iPSC-MSC-EXOs led to a marked reduction in the number of CD45-positive cells, with a greater reduction observed in the iPSC-MSC-EXO group (Supplementary Fig. 1). Taken together, these results showed that injection of iPSC-MSC-EXOs improved cardiac function and reduced infarct area in a mouse model of MI.

Fig. 2
figure 2

Injection of iPSC-MSC-EXOs improved cardiac function and reduced infarct area in mice. A Representative echocardiography images taken at 28 days in sham or mice with MI that received PBS, BM-MSC-EXOs or iPSC-MSC-EXOs, B quantitative analysis of LVEF and LVFS before MI (baseline) and at 28 days post MI in sham or mice with MI treated with PBS, BM-MSC-EXOs or iPSC-MSC-EXOs, C representative images of Masson’s trichrome staining of heart tissues at 28 days post MI in sham or mice with MI treated with PBS, BM-MSC-EXOs or iPSC-MSC-EXOs. Scale bar = 1 mm, D quantitative analysis of infarct size in heart of sham mice or mice with MI that received PBS, BM-MSC-EXOs or iPSC-MSC-EXOs. Data are expressed as the mean ± SEM. n = 6 mice for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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Injection of iPSC-MSC-EXOs ameliorated cardiomyocyte pyroptosis in infarcted hearts of mice

Previous studies have documented that NLRP3 inflammasome-mediated cardiomyocyte pyroptosis plays an essential role in MI [29,30,31]. Therefore, we first performed TUNEL staining to examine cell death and revealed increased TUNEL positive cardiomyocytes in the infarcted border zone although this effect was greatly inhibited by BM-MSC-EXO and iPSC-MSC-EXO treatment (Fig. 3A, B). Notably, compared with the BM-MSC-EXOs group, the percentage of TUNEL positive cardiomyocytes was significantly reduced in the iPSC-MSC-EXOs group (Fig. 3A, B). To further investigate the impact of iPSC-MSC-EXO transplantation on cardiomyocyte pyroptosis, we examined the expression of pyroptosis-related proteins. The elevated expression of NLRP3, ASC, GSDMD-NT, Caspase-1, IL-18 and IL-1β in the hearts of MI mice was greatly reduced in BM-MSC-EXO-treated and iPSC-MSC-EXO-treated mice and further decreased in iPSC-MSC-EXO-treated mice (Fig. 3C). Collectively, these findings suggested that iPSC-MSC-EXO treatment alleviated cardiac injury following infarction, potentially through the modulation of cardiomyocyte pyroptosis.

Fig. 3
figure 3

Injection of iPSC-MSC-EXOs ameliorated cardiomyocyte pyroptosis in infarcted hearts of mice. A Representative images of TUNEL staining in the infarcted border zone of heart tissue at 28 days post MI in sham mice or mice with MI that received PBS, BM-MSC-EXOs or iPSC-MSC-EXOs. Scale bar = 50 μm, B quantitative analysis of TUNEL and Troponin positive cardiomyocytes of hearts from sham mice or mice with MI that received PBS, BM-MSC-EXOs or iPSC-MSC-EXOs, C western blotting and quantitative analysis of the expression level of NLRP3, ASC, GSDMD-NT, Caspase-1, IL-18 and IL-1β in heart tissue of sham mice or mice with MI that received PBS, BM-MSC-EXOs or iPSC-MSC-EXOs. Data are expressed as the mean ± SEM. n = 6 mice for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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iPSC-MSC-EXOs inhibited SD/H-induced cardiomyocyte pyroptosis in vitro

We examined the protective effects of iPSC-MSC-EXOs on SD/H induced-NMCM death in vitro. As shown in Supplementary Fig. 2, PI staining results showed that SD/H induced NMCM death in a time dependent-manner, an effect that plateaued at 48 h (Supplementary Fig. 2A). Therefore, we chose NMCMs subjected to 48 h SD/H in future studies. To further verify whether SD/H induced NMCMs pyroptosis, we treated NMCMs with NLRP3 inhibitor MCC950 and then exposed cells to SD/H challenge for 48 h. Administration of MCC950 significantly inhibited NMCM death, suggesting that SD/H could induce NMCM pyroptosis (Supplementary Fig. 2B). Next, NMCMs were treated with BM-MSC-EXOs or iPSC-MSC-EXOs for 48 h under SD/H challenge. SD/H exposure led to increased PI positive cells (Fig. 4A, B) and enhanced protein level of NLRP3, ASC, GSDMD-NT and Caspase-1 in NMCMs (Fig. 4C). Administration of BM-MSC-EXOs and iPSC-MSC-EXOs greatly inhibited the increased cell death (Fig. 4A, B) and NLRP3, ASC, GSDMD-NT and Caspase-1 level in SD/H-treated-NMCMs (Fig. 4C). More importantly, the number of PI positive NMCMs and the protein level of NLRP3, ASC, GSDMD-NT and Caspase-1 were decreased more in the iPSC-MSC-EXO group compared with the BM-MSC-EXO group (Fig. 4A–C). Nonetheless these protective effects of BM-MSC-EXOs and iPSC-MSC-EXOs on SD/H-treated NMCMs were largely reversed by nigericin (Nig), an NLRP3 inflammasome inducer (Fig. 4A–C). These results indicated that iPSC-MSC-EXOs protected against NLRP3 inflammasome-mediated cardiomyocyte pyroptosis under SD/H challenge.

Fig. 4
figure 4

iPSC-MSC-EXOs inhibited the cardiomyocyte pyroptosis induced by SD/H in vitro. A Representative images of PI staining in control, SD/H, SD/H + BM-MSC-EXOs, SD/H + iPSC-MSC-EXOs, SD/H + BM-MSC-EXOs + Nig, and SD/H + iPSC-MSC-EXOs + Nig-treated NMCMs. Scale bar = 100 μm, B quantitative analysis of PI positive NMCMs in control, SD/H, SD/H + BM-MSC-EXOs, SD/H + iPSC-MSC-EXOs, SD/H + BM-MSC-EXOs + Nig, and SD/H + iPSC-MSC-EXOs + Nig-treated NMCMs, C western blotting and quantitative analysis of the expression level of NLRP3, ASC, GSDMD-NT and Caspase-1 in control, SD/H, SD/H + BM-MSC-EXOs, SD/H + iPSC-MSC-EXOs, SD/H + BM-MSC-EXOs + Nig, and SD/H + iPSC-MSC-EXOs + Nig-treated NMCMs. Data are expressed as the mean ± SEM. n = 3 biological replicates for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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miR-202-5p is a critical effector for iPSC-MSC-EXO protection against SD/H-induced cardiomyocyte pyroptosis

Numerous studies have demonstrated that MSC-EXOs exert their biological activity by delivering specific miRNAs, affecting the function of the recipient cardiomyocytes [32, 33]. To explore the mechanisms underlying iPSC-MSC-EXO protection against NLRP3 inflammasome-mediated cardiomyocyte pyroptosis in MI, we performed miRNA sequencing followed by bioinformatics analysis to compare the distinct miRNA expression profile of BM-MSC-EXOs and iPSC-MSC-EXOs. The miRNA heatmap analysis revealed the differential expression of miRNAs in BM-MSC-EXOs and iPSC-MSC-EXOs (Fig. 5A). Based on the top 10 miRNAs enriched in iPSC-MSC-EXOs, and a search for miRNAs known to be involved in either pyroptosis or cardiac repair, miR-202-5p emerged as a probable candidate effector for cardiac repair and pyroptosis (Fig. 5B). It has been reported that tetrandrine treatment attenuated myocardial injury in a rat model of ischemia reperfusion by elevating the level of miR-202-5p and this effect was partially abrogated by miR-202-5p inhibitor [34], suggesting that miR-202-5p exerts a beneficial effect on heart injury. We concluded that miR-202-5p in iPSC-MSC-EXOs could be an effective factor for MI treatment. The expression of miR-202-5p in iPSC-MSC-EXOs was further verified by RT-PCR (Fig. 5C). To further examine the protective effects of exosomal miR-202-5p in iPSC-MSC-EXOs on SD/H-induced cardiomyocyte pyroptosis, we treated iPSC-MSCs with miR-202-5p inhibitor to knockdown miR-202-5p and then isolated the EXOs. Indeed, miR-202-5p inhibitor treatment significantly downregulated the level of miR-202-5p in iPSC-MSCs (Fig. 5D) and iPSC-MSC-EXOs (miR-202-5pKD-iPSC-MSC-EXOs) (Fig. 5E). Next, we examined the effects of miR-202-5pKD-iPSC-MSC-EXOs on SD/H-induced NMCM pyroptosis. PI staining showed that the increased number of PI positive cells induced by SD/H was reduced by iPSC-MSC-EXO treatment, and the effect was reversed by miR-202-5pKD-iPSC-MSC-EXOs (Fig. 5F). To further validate the role of miR-202-5p, we performed rescue experiments by reintroducing miR-202-5p using a miR-202-5p mimic in the miR-202-5pKD-iPSC-MSC-EXO-treated NMCMs. The results showed that the expression of TRAF3IP2, p-JNK/JNK, NLRP3, ASC, and GSDMD-NT was significantly downregulated in the rescue group compared with the miR-202-5pKD-iPSC-MSC-EXO group (Supplementary Fig. 3A). Additionally, PI staining supported these findings, where the percentage of PI-positive cells was significantly higher in the miR-202-5pKD-iPSC-MSC-EXO group compared with the iPSC-MSC-EXO treatment group. Reintroduction of miR-202-5p with a miR-202-5p mimic partially rescued this effect, with a notable reduction in the percentage of PI-positive cells compared with the miR-202-5pKD-iPSC-MSC-EXO group (Supplementary Fig. 3B). To further verify the functional role of miR-202-5p in recipient cardiomyocytes independently of exosomes, we directly transfected NMCMs with miR-202-5p mimics. This overexpression significantly attenuated SD/H-induced cell death, confirming the intrinsic cardioprotective role of miR-202-5p. However, the protective effect was less pronounced than that observed with iPSC-MSC-EXO treatment (Supplementary Fig. 4), suggesting that additional exosomal cargo may act synergistically with miR-202-5p to achieve full therapeutic benefit. These data supported miR-202-5p as a critical effector for iPSC-MSC-EXO protection against SD/H-induced cardiomyocyte pyroptosis.

Fig. 5
figure 5

miR-202-5p is a critical effector for iPSC-MSC-EXO protection against SD/H-induced cardiomyocyte pyroptosis. A RNA-seq analysis demonstrated the differential expression of miRNAs between BM-MSC-EXOs and iPSC-MSC-EXOs, B venn diagram showing miRNAs upregulated in iPSC-MSC-EXOs that are involved in pyroptosis modulation or cardiac repair. C the expression of miR-202-5p in BM-MSC-EXOs and iPSC-MSC-EXOs was measured by qPCR, D quantification of miR-202-5p expression in iPSC-MSCs and miR-202-5pKD-iPSC-MSCs, E quantification of miR-202-5p expression in iPSC-MSC-EXOs and miR-202-5pKD-iPSC-MSC-EXOs, F representative images of PI staining in NMCMs treated with iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs under SD/H challenge. Scale bar = 100 μm, G quantitative analysis of PI positive cells in NMCMs treated with iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs under SD/H challenge. Data are expressed as the mean ± SEM. n = 3 biological replicates for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Enriched miR-202-5p in iPSC-MSC-EXOs alleviated cardiomyocyte pyroptosis by targeting TRAF3IP2 to inhibit the JNK pathway

We attempted to determine the downstream mechanism by which exosomal miR-202-5p in iPSC-MSC-EXOs might regulate cardiomyocyte pyroptosis in MI. Gene prediction tools showed a binding site between miR-202-5p and TRAF3 Interacting Protein 2 (TRAF3IP2) 3’UTR (Fig. 6A). Luciferase reporter assay demonstrated that miR-202-5p mimic greatly downregulated the luciferase activity of TRAF3IP2-WT without altering that of TRAF3IP2 mutant in HEK-293 T cells (Fig. 6B). We used miR-202-5p inhibitor or mimic to treat NMCMs and found that the level of miR-202-5p was increased in miR-202-5p mimic-treated NMCMs but decreased in miR-202-5p inhibitor-treated NMCMs (Fig. 6C). Furthermore, the mRNA and protein level of TRAF3IP2 was elevated in NMCMs with miR-202-5p inhibitor treatment but downregulated in those with miR-202-5p mimic treatment (Fig. 6D, E). Collectively, these data showed that TRAF3IP2 was a target for miR-202-5p. It has been well documented that TRAF3IP2/JNK signaling contributes to heart injury in various cardiovascular disorders [35, 36]. Therefore, we examined the protein level of TRAF3IP2 and p-JNK in the heart of MI mice exposed to BM-MSC-EXO or iPSC-MSC-EXO treatment. Compared with the sham group, the expression of TRAF3IP2 and p-JNK was greatly increased in the MI group but decreased in the BM-MSC-EXO and iPSC-MSC-EXO group, to a greater degree in the latter (Supplementary Fig. 5). To determine whether exosomal miR-202-5p in iPSC-MSC-EXOs inhibited cardiomyocyte pyroptosis via regulation of the TRAF3IP2/JNK signaling pathway, we treated NMCMs with miR-202-5pKD-iPSC-MSC-EXOs under SD/H challenge. The expression of TRAF3IP2, p-JNK/JNK, NLRP3, ASC, GSDMD-NT and Caspase-1 was greatly downregulated in SD/H-treated NMCMs by iPSC-MSC-EXO treatment (Fig. 6F). Nonetheless compared with the iPSC-MSC-EXO group, the expression of TRAF3IP2, p-JNK/JNK, NLRP3, ASC, GSDMD-NT and Caspase-1 was much higher in the miR-202-5pKD-iPSC-MSC-EXO group and these effects were partially abrogated by JNK inhibitor (Fig. 6F). These data indicated that enriched miR-202-5p in iPSC-MSC-EXOs alleviated cardiomyocyte pyroptosis by regulating the TRAF3IP2/JNK pathway.

Fig. 6
figure 6

Enriched miR-202-5p in iPSC-MSC-EXOs alleviated cardiomyocyte pyroptosis by targeting TRAF3IP2 to inhibit JNK pathway. A Bioinformatics analysis predicted the binding sites between miR-202-5p and TRAF3IP2. B luciferase reporter assay showed TRAF3IP2 is a target of miR-202-5p, C the level of miR-202-5p in control, miR-202-5p inhibitor or miR-202-5p mimic-treated NMCMs was measured by qPCR, D the mRNA level of TRAF3IP2 in control, miR-202-5p inhibitor or miR-202-5p mimic-treated NMCMs was measured by qPCR, E the protein level of TRAF3IP2 in control, miR-202-5p inhibitor or miR-202-5p mimic-treated NMCMs was measured by Western blotting, F western blotting analysis of the level of TRAF3IP2, p-JNK/JNK, NLRP3, ASC, GSDMD-NT and Caspase-1 in control, SD/H, SD/H + iPSC-MSC-EXOs, SD/H + miR-202-5pKD-iPSC-MSC-EXOs and SD/H + miR-202-5pKD-iPSC-MSC-EXOs + JNK inhibitor treated NMCMs. Data are expressed as the mean ± SEM. n = 3 biological replicates for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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Knockdown of miR-202-5p reduced the cardioprotective effects of iPSC-MSC-EXOs on MI in a mouse model

To test whether the protective effects of exosomal miR-202-5p in iPSC-MSC-EXOs demonstrated in in vitro studies applied in vivo, we examined the therapeutic efficacy of miR-202-5pKD-iPSC-MSC-EXOs in a mouse model of MI. Heart function at 28 days post MI in mice that received iPSC-MSC-EXO or miR-202-5pKD-iPSC-MSC-EXO treatment was evaluated (Fig. 7A). Compared with the iPSC-MSC-EXOs group, LVEF and LVFS were notably decreased in the miR-202-5pKD-iPSC-MSC-EXOs group (Fig. 7B). Masson’s trichrome staining demonstrated a significantly enhanced level of myocardial fibrosis in the miR-202-5pKD-iPSC-MSC-EXOs group compared with the iPSC-MSC-EXOs group (Fig. 7C, D). TUNEL staining showed that the percentage of TUNEL-positive cardiomyocytes was also greatly enhanced in the miR-202-5pKD-iPSC-MSC-EXOs group compared (Fig. 7E, F). Furthermore, the protein level of TRAF3IP2, p-JNK, NLRP3, ASC, GSDMD-NT, Caspase-1, IL-18 and IL-1β in the heart tissue of mice in the miR-202-5pKD-iPSC-MSC-EXOs group was much higher (Fig. 7G). Collectively, these results exhibited that knockdown of miR-202-5p reduced the cardioprotective effect of iPSC-MSC-EXOs against MI.

Fig. 7
figure 7

Knockdown of miR-202-5p reduced the cardioprotective effects of iPSC-MSC-EXOs on MI in mice. A Representative echocardiographic images at 28 days after MI in mice that received iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs, B LVEF and LVFS were analyzed at 28 days in mice with MI and administration of iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs, C representative images of Masson’s trichrome staining of heart tissue from mice with MI that received iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs. Scale bar = 1 mm, D quantitative analysis of infarct size in heart tissue of mice with MI and administration of iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs, E representative images of TUNEL staining and Troponin positive cells in heart tissues from mice with MI and administration of iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs. Scale bar = 50 μm, F quantitative analysis of TUNEL staining and Troponin positive cardiomyocytes in heart tissue of mice with MI and administration of iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs, G the protein level of TRAF3IP2, p-JNK, NLRP3, ASC, GSDMD-NT, Caspase-1, IL-18 and IL-1β in heart tissue from mice with MI that received iPSC-MSC-EXOs or miR-202-5pKD-iPSC-MSC-EXOs. Data are expressed as the mean ± SEM. n = 6 mice for each group. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001

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Discussion

There were several major findings in the current study. First, iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuating cardiac injury in MI-treated mice via inhibition of NLRP3-related cardiomyocyte pyroptosis. Second, compared with BM-MSC-EXOs, a greater potential of iPSC-MSC-EXOs in MI treatment was attributed to a higher level of miR-202-5p. Third, iPSC-MSC-EXOs facilitated cardiac repair in a mouse model of MI via exosomal transfer of miR-202-5p by targeting the TRAF3IP2/JNK pathway. iPSC-MSC-EXOs containing miR-202-5p have promising therapeutic potential in MI treatment.

There is accumulating evidence that MSC-EXOs exert beneficial effects during cardiac repair following infarction as a cell-free therapy due to their higher stability and lower tumorigenic risk and immune rejection compared with MSCs [37, 38]. It has been reported that a microneedle patch loaded with MSC-EXOs functionally improved heart function and prevented cardiac fibrosis following MI [39]. Transplantation of MSC-EXOs attenuated cardiac injury following infarction in mice via promotion of M2 macrophage polarization [40]. These results revealed that MSC-EXOs not only recapitulate the cardioprotective efficacy of their parent cells but also overcome some limitations of using MSCs. Nonetheless adult MSCs, such as BM-MSCs or adipose tissue derived MSC (AD-MSCs), will gradually become senescent after long-term culture in vitro, making large-scale generation of MSC-EXOs for clinical application impossible [26]. Therefore, exploring an alternative cellular source for MSC-EXO generation is urgently needed. We have successfully derived MSCs from iPSCs and shown that iPSC-MSCs exhibit a higher proliferative capacity, immune privilege and lower batch-to-batch variation [21, 22], making them an ideal cell source for EXOs. In the current study, iPSC-MSCs produced a significantly higher yield of exosomes compared with BM-MSCs when cultured under identical conditions. More importantly, compared with BM-MSC-EXOs, transplantation of iPSC-MSC-EXOs greatly improved cardiac function and reduced infarct size in a mouse model of MI, indicating that iPSC-MSC-EXOs exert superior therapeutic efficacy for MI.

The therapeutic efficacy of MSC-EXOs on MI has been predominately ascribed to the transfer of proteins, lipids and/or non-coding RNAs, especially miRNAs [33, 41]. It has been reported that EXOs isolated from hemin-pretreated MSCs attenuated cardiac jury in a mouse model of MI via delivery of miR-183-5p to inhibit cardiomyocyte senescence [25]. EXOs derived from miR-214-overexpressing MSCs exhibited superior therapeutic efficacy for MI treatment via promotion of endothelial cell function and amelioration of cardiomyocyte apoptosis [42]. Since iPSC-MSC-EXOs were superior to BM-MSC-EXOs in attenuating cardiac injury following infarction, we performed RNA-seq of iPSC-MSC-EXOs and BM-MSC-EXOs to identify the potential miRNA effectors. The level of miR-202-5p was much higher in iPSC-MSC-EXOs than BM-MSC-EXOs. Indeed, it has been shown that miR-202-5p protects against heart injury in a rat model of myocardial ischemia reperfusion by downregulating the expression of TRVP2 [43]. These results prompted us to determine whether the therapeutic efficacy of iPSC-MSC-EXOs in MI could be attributed to the high level of miR-202-5p. Thus, loss-of-function study was performed to examine the cardioprotective effects of exosomal miR-202-5p in iPSC-MSC-EXOs on MI. Knockdown of miR-202-5p in iPSC-MSC-EXOs significantly reduced their cardioprotective effects, indicating that these effects were mostly mediated by miR-202-5p.

In addition to inhibition of apoptosis, and upregulation of immunomodulatory effects and angiogenesis, accumulating evidence reveals that MSC-EXOs protect against MI via inhibition of pyroptosis of damaged cardiomyocytes by delivering various non-coding RNAs [44, 45]. Tang et al. reported that transplantation of MSC-EXOs containing a high level of miR-320b could greatly inhibit NLRP3 expression and subsequently inhibit cardiomyocyte pyroptosis, leading to improved heart function in a rat model of ischemia/reperfusion [46]. LncRNA KLF3-AS1 in MSC-EXOs attenuated MI progression via amelioration of pyroptosis of cardiomyocytes by regulating the miR-138-5p/Sirt1 axis [47]. In this study, we primarily focused on the protective effects of iPSC-MSC-EXOs on cardiomyocytes, the central players in cardiac injury and repair following MI. We found that iPSC-MSC-EXOs ameliorated cardiomyocyte pyroptosis as evidenced by downregulation of NLRP3, Caspase-1 and gasdermin D as well as inflammatory factors in MI mice and SD/H-treated NMCMs. These effects were partially abrogated by knockdown of miR-202-5p in iPSC-MSC-EXOs, and were subsequently restored by reintroducing miR-202-5p using a mimic, suggesting that iPSC-MSC-EXOs inhibit cardiomyocyte pyroptosis predominantly through the delivery of miR-202-5p. Although the focus of our study was on cardiomyocytes, we also explored the potential effects of iPSC-MSC-EXOs on other key cell types involved in cardiac repair. The preliminary results revealed that iPSC-MSC-EXOs significantly reduced CD45 + immune cell infiltration following MI (Supplementary Fig. 1), indicating an anti-inflammatory response that could help modulate the inflammatory microenvironment. In addition, iPSC-MSC-EXOs enhanced tube formation in HUVECs (Supplementary Fig. 6A), suggesting a pro-angiogenic effect. We also observed that iPSC-MSC-EXOs could be internalized by cardiac fibroblasts after 24 h of co-culture (Supplementary Fig. 6B), potentially influencing fibroblast activity and contributing to the inhibition of post-MI fibrosis. These findings indicate that iPSC-MSC-EXOs may exert therapeutic effects not only through direct inhibition of cardiomyocyte pyroptosis but also via modulation of inflammation, promotion of angiogenesis, and regulation of extracellular matrix remodeling. Further investigation into these additional mechanisms and their molecular pathways is warranted to fully elucidate the diverse therapeutic potential of iPSC-MSC-EXOs in MI.

TRAF3IP2, a redox-sensitive cytoplasmic adaptor molecule, is a newly identified gene that is closely associated with a variety of diseases including cardiovascular diseases [48, 49]. It has been reported that ischemia/reperfusion significantly enhanced TRAF3IP2 expression in the heart, and conditional gene deletion in cardiomyocytes significantly inhibited ischemia/reperfusion-induced heart injury via suppression of the JNK signaling pathway [35]. Another study demonstrated that angiotensin-II-induced cardiomyocyte hypertrophy and fibrosis was via regulation of TRAF3IP2-dependent NF-κB and JNK/AP-1 activation [50]. More importantly, the JNK signaling pathway plays a critical role in the regulation of cardiomyocyte pyroptosis [51, 52]. Although direct evidence of a causal relationship is lacking, these findings suggest a potential link between the TRAF3IP2/JNK axis and the modulation of cardiomyocyte pyroptosis. In this study, the expression of TRAF3IP2 and p-JNK protein was significantly increased in MI mice and SD/H-treated NMCMs but decreased in iPSC-MSC-EXO-treated MI mice and NMCMs. We hypothesized that TRAF3IP2 acted as the downstream gene of miR-202-5p and mediated cardiomyocyte pyroptosis. Bioinformatic analysis and quantitative assessment confirmed that miR-202-5p directly targets TRAF3IP2 in cardiomyocytes, as validated by luciferase reporter assays. We found that miR-202-5p treatment greatly inhibited TRAF3IP2 expression in NMCMs, further demonstrating that TRAF3IP2 was negatively regulated by miR-202-5p. Furthermore, knockdown of miR-202-5p in iPSC-MSC-EXOs reduced their cardioprotective effects in MI, and increased TRAF3IP2, p-JNK expression and cardiomyocyte pyroptosis, suggesting that miR-202-5p-enriched iPSC-MSC-EXOs may attenuate cardiomyocyte pyroptosis partly through inhibition of the TRAF3IP2/JNK signaling axis. To explore additional pathways that might be involved, we conducted supplementary experiments examining the impact of iPSC-MSC-EXOs on the ERK and AMPK pathways. Under SD/H conditions, downregulation of p-AMPK and upregulation of p-ERK were significantly reversed by iPSC-MSC-EXO treatment (Supplementary Fig. 7). This suggests that beyond the TRAF3IP2/JNK axis, iPSC-MSC-EXOs may exert broader effects on cardiomyocyte pyroptosis through interactions with the AMPK and ERK signaling pathways. Further investigation into these parallel or interacting pathways may provide a deeper understanding of the diverse mechanisms underlying the therapeutic effects of miR-202-5p-enriched iPSC-MSC-EXOs.

There are some limitations in the current study that should be acknowledged. First, although our study focused on miR-202-5p due to its strong association with both pyroptosis and cardiac repair, further in-depth profiling of other miRNAs identified in this study, as well as other noncoding RNAs and proteins, is necessary to better understand their potential contributions to the therapeutic efficacy of iPSC-MSC-EXOs for MI. This notion is further supported by our supplementary findings (Supplementary Fig. 4), where direct overexpression of miR-202-5p in NMCMs partially mimicked the protective effect of iPSC-MSC-EXOs, but with a notably reduced magnitude. These results suggest that, beyond miR-202-5p, other exosomal components likely play synergistic roles in mediating the full cardioprotective effects. Second, in addition to the TRAF3IP2/JNK pathway, whether iPSC-MSC-exosomal-miR-202-5p regulates other downstream signaling cascades to inhibit cardiomyocyte pyroptosis warrants further investigation. Our preliminary data showed that iPSC-MSC-EXOs significantly reversed the downregulation of p-AMPK and the upregulation of p-ERK under SD/H conditions. It remains unclear whether these effects are directly mediated by miR-202-5p or result from other components of the exosomal cargo. Given that miRNAs typically have multiple predicted targets, the possibility of off-target effects or parallel regulatory interactions cannot be excluded. Third, although the broader effects of iPSC-MSC-EXOs on various cell types in the ischemic heart require more exploration, we offer preliminary evidence of their potential multifaceted role. We found that iPSC-MSC-EXOs could be internalized by cardiac fibroblasts, significantly enhance tube formation in HUVECs, and reduce CD45 + immune cell infiltration in the infarcted heart. These results suggest that iPSC-MSC-EXOs may exert therapeutic effects by modulating the activities of fibroblasts, endothelial cells, and immune cells, not just by acting directly on cardiomyocytes. This indicates a potential multi-targeted mechanism involving enhanced angiogenesis, reduced inflammation, and modulation of extracellular matrix remodeling, contributing to an improved cardiac repair process. These preliminary findings warrant further investigation to elucidate the underlying molecular pathways and to better understand how iPSC-MSC-EXOs interact with different cell types in the ischemic microenvironment. Fourth, to exclude the protective effects of endogenous miR-202-5p, a model of MI in cardiomyocyte-deficient miR-202-5p−/− mice should be developed to verify the cardioprotective effects of iPSC-MSC-EXOs.

In addition to the above experimental limitations, several translational challenges should also be considered for future clinical application of iPSC-MSC-EXO–based therapy. First, while our current findings provide clear evidence of the short-term cardioprotective effects of iPSC-MSC-EXOs up to 28 days post-MI, the long-term efficacy remains to be determined. Future studies with extended follow-up will be necessary to determine whether the observed improvements in cardiac function and infarct size translate into sustained myocardial remodeling, preserved contractility, and long-term prevention of adverse outcomes such as heart failure or arrhythmia. These efforts will be crucial for assessing the true translational potential of iPSC-MSC-EXO–based therapy. Second, optimizing both the dosage and delivery route of iPSC-MSC-EXOs is essential. In this study, we used a single dose due to the invasive nature of intramyocardial injection, which limits the feasibility of repeated administration in animal models. However, the optimal therapeutic dose range, minimal effective dose, and potential threshold for saturation or toxicity remain unknown. Future studies should systematically evaluate dose–response relationships to define the most effective and safest dosing strategy. In addition, combining a single local myocardial injection with subsequent non-invasive approaches, such as intravenous or hydrogel-based sustained release, may offer enhanced therapeutic efficacy while improving translational feasibility in clinical settings. Third, assessing the safety profile of iPSC-MSC-EXOs, including their potential immunogenicity and risk of off-target effects, is crucial. Although exosomes are generally considered less immunogenic than cell-based therapies [53], further studies are needed to confirm their safety in humans, potentially using autologous or HLA-matched iPSC sources. Finally, scalability is a key consideration for clinical translation. Our previous research has shown that iPSCs can be expanded indefinitely in vitro and differentiated into MSCs that maintain stable growth, diploid karyotype, consistent gene expression, and unaltered surface marker profiles for over 40 passages [21, 54]. These features support the feasibility of large-scale, standardized production of iPSC-MSC-EXOs. While this study focused on iPSC-MSCs and BM-MSCs, other sources such as adipose- and umbilical cord-derived MSCs have also shown cardioprotective effects in preclinical models. AD-MSCs are particularly accessible and abundant, but issues like donor variability, limited expansion potential, and early senescence may compromise consistency and yield. In contrast, iPSC-MSCs offer a more renewable and uniform platform for exosome production. Future head-to-head comparisons across multiple MSC sources under standardized conditions will be essential to identify the optimal source for clinical application.

Conclusions

In conclusion, our study found that transplantation of iPSC-MSC-EXOs was superior to BM-MSC-EXOs in attenuating cardiac injury in a mouse model of MI due to the higher level of miR-202-5p. Furthermore, iPSC-MSC exosomal miR-202-5p protected the heart against ischemic injury by inhibiting NLRP3-related cardiomyocyte pyroptosis via targeting of the TRAF3IP2/JNK axis. This offers an appealing and promising therapeutic strategy for MI treatment in a clinical setting.

Availability of data and materials

The datasets generated during the current study regarding the miRNA sequencing is available in the figshare repository. miRNA sequencing data: https://figshare.com/articles/dataset/RNA_-seq_of_iPSC-MSC-EXOs_and_MSC-EXOs/28160087?file=51538139. Other datasets used in this study are available from the corresponding author on reasonable request.

Abbreviations

ASC:

Apoptosis-associated speck-like protein

AD-MSCs:

Adipose tissue derived mesenchymal stem cells

EXOs:

Exosomes

GSDMD:

Gasdermin D

HUVECs:

Human umbilical vein endothelial cells

iPSC-MSCs:

Induced pluripotent stem cell-derived mesenchymal stem cells

IL-18:

Interleukin-18

IL-1β:

Interleukin-1β

LVEF:

Left ventricle ejection fraction

LVFS:

Left ventricle fractional shortening

MI:

Myocardial infarction

MSCs:

Mesenchymal stem cells

NLRP3:

NOD-like receptor thermal protein domain associated protein 3

NMCMs:

Neonatal mice cardiac myocytes

TEM:

Transmission electron microscopy

SD/H:

Deprivation and hypoxia

TRAF3IP2:

TRAF3 interacting protein 2

References

  1. Virani SS, Alonso A, Aparicio HJ, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Cheng S, Delling FN, Elkind MSV, Evenson KR, Ferguson JF, Gupta DK, Khan SS, Kissela BM, Knutson KL, Lee CD, Lewis TT, Liu J, Loop MS, Lutsey PL, Ma J, Mackey J, Martin SS, Matchar DB, Mussolino ME, Navaneethan SD, Perak AM, Roth GA, Samad Z, Satou GM, Schroeder EB, Shah SH, Shay CM, Stokes A, VanWagner LB, Wang NY, Tsao CW. Heart disease and stroke statistics-2021 update: a report from the american heart association. Circulation. 2021;143(8):e254–743.

    Article  PubMed  Google Scholar 

  2. Dani SS, Lone AN, Javed Z, Khan MS, Zia Khan M, Kaluski E, Virani SS, Shapiro MD, Cainzos-Achirica M, Nasir K, Khan SU. trends in premature mortality from acute myocardial infarction in the United States, 1999 to 2019. J Am Heart Assoc. 2022;11(1): e021682.

    Article  PubMed  Google Scholar 

  3. Freedland KE, Rich MW, Carney RM. Improving quality of life in heart failure. Curr Cardiol Rep. 2021;23(11):159.

    Article  PubMed  Google Scholar 

  4. McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, Burri H, Butler J, Čelutkienė J, Chioncel O, Cleland JGF, Coats AJS, Crespo-Leiro MG, Farmakis D, Gilard M, Heymans S, Hoes AW, Jaarsma T, Jankowska EA, Lainscak M, Lam CSP, Lyon AR, McMurray JJV, Mebazaa A, Mindham R, Muneretto C, Francesco Piepoli M, Price S, Rosano GMC, Ruschitzka F, Kathrine SA. 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2021;42(36):3599–726.

    Article  CAS  PubMed  Google Scholar 

  5. Russo HM, Rathkey J, Boyd-Tressler A, Katsnelson MA, Abbott DW, Dubyak GR. Active caspase-1 induces plasma membrane pores that precede pyroptotic lysis and are blocked by lanthanides. J Immunol. 2016;197(4):1353–67.

    Article  CAS  PubMed  Google Scholar 

  6. Zhaolin Z, Guohua L, Shiyuan W, Zuo W. Role of pyroptosis in cardiovascular disease. Cell Prolif. 2019;52(2): e12563.

    Article  PubMed  Google Scholar 

  7. Takahashi M. Role of NLRP3 inflammasome in cardiac inflammation and remodeling after myocardial infarction. Biol Pharm Bull. 2019;42(4):518–23.

    Article  CAS  PubMed  Google Scholar 

  8. Chen X, Tian PC, Wang K, Wang M, Wang K. Pyroptosis: role and mechanisms in cardiovascular disease. Front Cardiovasc Med. 2022;9:897815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Toldo S, Abbate A. The role of the NLRP3 inflammasome and pyroptosis in cardiovascular diseases. Nat Rev Cardiol. 2024;21(4):219–37.

    Article  CAS  PubMed  Google Scholar 

  10. Wang YW, Dong HZ, Tan YX, Bao X, Su YM, Li X, Jiang F, Liang J, Huang ZC, Ren YL, Xu YL, Su Q. HIF-1α-regulated lncRNA-TUG1 promotes mitochondrial dysfunction and pyroptosis by directly binding to FUS in myocardial infarction. Cell Death Discov. 2022;8(1):178.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wang L, Qu P, Zhao J, Chang Y. NLRP3 and downstream cytokine expression elevated in the monocytes of patients with coronary artery disease. Arch Med Sci. 2014;10(4):791–800.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wang K, Zhou Y, Wen C, Du L, Li L, Cui Y, Luo H, Liu Y, Zeng L, Li S, Xiong L, Yue R. Protective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation. Biochem Pharmacol. 2024;229: 116541.

    Article  CAS  PubMed  Google Scholar 

  13. Dang S, Zhang ZY, Li KL, Zheng J, Qian LL, Liu XY, Wu Y, Zhang CY, Zhao XX, Yu ZM, Wang RX, Jiang T. Blockade of β-adrenergic signaling suppresses inflammasome and alleviates cardiac fibrosis. Ann Transl Med. 2020;8(4):127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wu BS, Xiang HQ, Yu YW, Liu S, Song DY, Wu C, Lin ZH, Zhu CX, Xue YJ, Ji KT. 3,4-benzo[a]pyrene aggravates myocardial infarction injury by activating NLRP3-related pyroptosis through PINK1/Parkin-mitophagy-mPTP opening axis. Int Immunopharmacol. 2023;122: 110481.

    Article  CAS  PubMed  Google Scholar 

  15. Chai X, Liang Z, Zhang J, Ding J, Zhang Q, Lv S, Deng Y, Zhang R, Lu D. Chlorogenic acid protects against myocardial ischemia-reperfusion injury in mice by inhibiting Lnc Neat1/NLRP3 inflammasome-mediated pyroptosis. Sci Rep. 2023;13(1):17803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu Y, Wang M, Yu Y, Li C, Zhang C. Advances in the study of exosomes derived from mesenchymal stem cells and cardiac cells for the treatment of myocardial infarction. Cell Commun Signal. 2023;21(1):202.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Li J, Tang Y, Yin L, Lin X, Luo Z, Wang S, Yuan L, Liang P, Jiang B. Mesenchymal stem cell-derived exosomes in myocardial infarction: therapeutic potential and application. J Gene Med. 2023;26:e3596.

    Article  PubMed  Google Scholar 

  18. Ma C, Yang Z, Wang J, She H, Tan L, Ye Q, Wang F, Feng X, Mo X, Liu K, Liu L. Exosomes miRNA-499a-5p targeted CD38 to alleviate anthraquinone induced cardiotoxicity: experimental research. Int J Surg. 2024;110(4):1992–2006.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Yue R, Lu S, Luo Y, Zeng J, Liang H, Qin D, Wang X, Wang T, Pu J, Hu H. Mesenchymal stem cell-derived exosomal microRNA-182-5p alleviates myocardial ischemia/reperfusion injury by targeting GSDMD in mice. Cell Death Discov. 2022;8(1):202.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang Y, Huang X, Sun T, Shi L, Liu B, Hong Y, Fu QL, Zhang Y, Li X. MicroRNA-19b-3p dysfunction of mesenchymal stem cell-derived exosomes from patients with abdominal aortic aneurysm impairs therapeutic efficacy. J Nanobiotechnol. 2023;21(1):135.

    Article  CAS  Google Scholar 

  21. Sun YQ, Zhang Y, Li X, Deng MX, Gao WX, Yao Y, Chiu SM, Liang X, Gao F, Chan CW, Tse HF, Shi J, Fu QL, Lian Q. Insensitivity of human iPS cells-derived mesenchymal stem cells to interferon-γ-induced HLA expression potentiates repair efficiency of hind limb ischemia in immune humanized NOD Scid gamma mice. Stem Cells. 2015;33(12):3452–67.

    Article  CAS  PubMed  Google Scholar 

  22. Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation. 2010;121(9):1113–23.

    Article  PubMed  Google Scholar 

  23. Zhang Y, Liang X, Liao S, Wang W, Wang J, Li X, Ding Y, Liang Y, Gao F, Yang M, Fu Q, Xu A, Chai YH, He J, Tse HF, Lian Q. Potent paracrine effects of human induced pluripotent stem cell-derived mesenchymal stem cells attenuate doxorubicin-induced cardiomyopathy. Sci Rep. 2015;5:11235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang Y, Yu Z, Jiang D, Liang X, Liao S, Zhang Z, Yue W, Li X, Chiu SM, Chai YH, Liang Y, Chow Y, Han S, Xu A, Tse HF, Lian Q. iPSC-MSCs with high intrinsic MIRO1 and sensitivity to TNF-α yield efficacious mitochondrial transfer to rescue anthracycline-induced cardiomyopathy. Stem Cell Rep. 2016;7(4):749–63.

    Article  CAS  Google Scholar 

  25. Zheng H, Liang X, Han Q, Shao Z, Zhang Y, Shi L, Hong Y, Li W, Mai C, Mo Q, Fu Q, Ma X, Lin F, Li M, Hu B, Li X, Zhang Y. Hemin enhances the cardioprotective effects of mesenchymal stem cell-derived exosomes against infarction via amelioration of cardiomyocyte senescence. J Nanobiotechnology. 2021;19(1):332.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fang SB, Zhang HY, Wang C, He BX, Liu XQ, Meng XC, Peng YQ, Xu ZB, Fan XL, Wu ZJ, Chen D, Zheng L, Zheng SG, Fu QL. Small extracellular vesicles derived from human mesenchymal stromal cells prevent group 2 innate lymphoid cell-dominant allergic airway inflammation through delivery of miR-146a-5p. J Extracell Vesicles. 2020;9(1):1723260.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010;8(6): e1000412.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Gao X, Liang X, Liu B, Hong Y, He H, Shen Y, Chen J, Huang X, Hu B, Li W, Li X, Zhang Y. Downregulation of ALKBH5 rejuvenates aged human mesenchymal stem cells and enhances their therapeutic efficacy in myocardial infarction. Faseb j. 2023;37(12): e23294.

    Article  CAS  PubMed  Google Scholar 

  29. Yanpiset P, Maneechote C, Sriwichaiin S, Siri-Angkul N, Chattipakorn SC, Chattipakorn N. Gasdermin D-mediated pyroptosis in myocardial ischemia and reperfusion injury: cumulative evidence for future cardioprotective strategies. Acta Pharm Sin B. 2023;13(1):29–53.

    Article  CAS  PubMed  Google Scholar 

  30. Zhang KZ, Shen XY, Wang M, Wang L, Sun HX, Li XZ, Huang JJ, Li XQ, Wu C, Zhao C, Liu JL, Lu X, Gao W. Retinol-binding protein 4 promotes cardiac injury after myocardial infarction via inducing cardiomyocyte pyroptosis through an interaction with NLRP3. J Am Heart Assoc. 2021;10(22): e022011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ji N, Qi Z, Wang Y, Yang X, Yan Z, Li M, Ge Q, Zhang J. Pyroptosis: a new regulating mechanism in cardiovascular disease. J Inflamm Res. 2021;14:2647–66.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Bhaskara M, Anjorin O, Wang M. Mesenchymal stem cell-derived exosomal microRNAs in cardiac regeneration. Cells. 2023;12(24):2815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nasser MI, Masood M, Adlat S, Gang D, Zhu S, Li G, Li N, Chen J, Zhu P. Mesenchymal stem cell-derived exosome microRNA as therapy for cardiac ischemic injury. Biomed Pharmacother. 2021;143: 112118.

    Article  CAS  PubMed  Google Scholar 

  34. Zhao W, Wu Y, Ye F, Huang S, Chen H, Zhou R, Jiang W. Tetrandrine ameliorates myocardial ischemia reperfusion injury through miR-202-5p/TRPV2. Biomed Res Int. 2021;2021:8870674.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Erikson JM, Valente AJ, Mummidi S, Kandikattu HK, DeMarco VG, Bender SB, Fay WP, Siebenlist U, Chandrasekar B. Targeting TRAF3IP2 by genetic and interventional approaches inhibits ischemia/reperfusion-induced myocardial injury and adverse remodeling. J Biol Chem. 2017;292(6):2345–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Valente AJ, Yoshida T, Clark RA, Delafontaine P, Siebenlist U, Chandrasekar B. Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling. Free Radic Biol Med. 2013;60:125–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. de Abreu RC, Fernandes H, da Costa Martins PA, Sahoo S, Emanueli C, Ferreira L. Native and bioengineered extracellular vesicles for cardiovascular therapeutics. Nat Rev Cardiol. 2020;17(11):685–97.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Sun SJ, Wei R, Li F, Liao SY, Tse HF. Mesenchymal stromal cell-derived exosomes in cardiac regeneration and repair. Stem Cell Rep. 2021;16(7):1662–73.

    Article  CAS  Google Scholar 

  39. Yuan J, Yang H, Liu C, Shao L, Zhang H, Lu K, Wang J, Wang Y, Yu Q, Zhang Y, Yu Y, Shen Z. Microneedle patch loaded with exosomes containing MicroRNA-29b prevents cardiac fibrosis after myocardial infarction. Adv Healthc Mater. 2023;12(13): e2202959.

    Article  PubMed  Google Scholar 

  40. Zhu F, Chen Y, Li J, Yang Z, Lin Y, Jiang B, Shao L, Hu S, Shen Z. Human umbilical cord mesenchymal stem cell-derived exosomes attenuate myocardial infarction injury via miR-24-3p-promoted M2 macrophage polarization. Adv Biol (Weinh). 2022;6(11): e2200074.

    Article  PubMed  Google Scholar 

  41. Akbar N, Razzaq SS, Salim A, Haneef K. Mesenchymal stem cell-derived exosomes and their MicroRNAs in heart repair and regeneration. J Cardiovasc Transl Res. 2023;17(3):505–22.

    Article  PubMed  Google Scholar 

  42. Zhu W, Wang Q, Zhang J, Sun L, Hong X, Du W, Duan R, Jiang J, Ji Y, Wang H, Han B. Exosomes derived from mir-214-3p overexpressing mesenchymal stem cells promote myocardial repair. Biomater Res. 2023;27(1):77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li Y, Li Q, Zhang O, Guan X, Xue Y, Li S, Zhuang X, Zhou B, Miao G. miR-202-5p protects rat against myocardial ischemia reperfusion injury by downregulating the expression of Trpv2 to attenuate the Ca (2+) overload in cardiomyocytes. J Cell Biochem. 2019;120(8):13680–93.

    Article  CAS  PubMed  Google Scholar 

  44. Deng J, Zhang T, Li M, Cao G, Wei H, Zhang Z, Hu T. Irisin-pretreated BMMSCs secrete exosomes to alleviate cardiomyocytes pyroptosis and oxidative stress to hypoxia/reoxygenation injury. Curr Stem Cell Res Ther. 2023;18(6):843–52.

    Article  CAS  PubMed  Google Scholar 

  45. Yan B, Zhang Y, Liang C, Liu B, Ding F, Wang Y, Zhu B, Zhao R, Yu XY, Li Y. Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway. Theranostics. 2020;10(15):6728–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tang J, Jin L, Liu Y, Li L, Ma Y, Lu L, Ma J, Ding P, Yang X, Liu J, Yang J. Exosomes derived from mesenchymal stem cells protect the myocardium against ischemia/reperfusion injury through inhibiting pyroptosis. Drug Des Devel Ther. 2020;14:3765–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Mao Q, Liang XL, Zhang CL, Pang YH, Lu YX. LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis. Stem Cell Res Ther. 2019;10(1):393.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wei J, Yan T, Liang Y. Targeting TRAF3IP2 alleviates high glucose-induced cardiomyocyte inflammation and apoptosis. Drug Dev Res. 2022;83(1):167–75.

    Article  CAS  PubMed  Google Scholar 

  49. Sakamuri SS, Valente AJ, Siddesha JM, Delafontaine P, Siebenlist U, Gardner JD, Bysani C. TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis. Mol Cell Endocrinol. 2016;429:84–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Valente AJ, Clark RA, Siddesha JM, Siebenlist U, Chandrasekar B. CIKS (Act1 or TRAF3IP2) mediates Angiotensin-II-induced Interleukin-18 expression, and Nox2-dependent cardiomyocyte hypertrophy. J Mol Cell Cardiol. 2012;53(1):113–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ruan Z, Li Y, Chen Y. HECTD3 promotes NLRP3 inflammasome and pyroptosis to exacerbate diabetes-related cognitive impairment by stabilising MALT1 to regulate JNK pathway. Arch Physiol Biochem. 2022;130:1–12.

    Google Scholar 

  52. You J, Li X, Dai F, Liu J, Zhang Q, Guo W. GSDMD-mediated pyroptosis promotes cardiac remodeling in pressure overload. Clin Exp Hypertens. 2023;45(1):2189138.

    Article  PubMed  Google Scholar 

  53. Clua-Ferré L, Suau R, Vañó-Segarra I, Ginés I, Serena C, Manyé J. Therapeutic potential of mesenchymal stem cell-derived extracellular vesicles: a focus on inflammatory bowel disease. Clin Transl Med. 2024;14(11): e70075.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Lian Q, Zhang Y, Liang X, Gao F, Tse HF. Directed differentiation of human-induced pluripotent stem cells to mesenchymal stem cells. Methods Mol Biol. 2016;1416:289–98.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors declare that they have not used AI-generated work in this manuscript. The authors thank Ms. Sarah Aglionby for editing the manuscript.

Funding

This research was in part supported by National Natural Science Grant of China (No. 82270253 to Y. Zhang, 82072225 to X. Li, 82072139 to B. Hu, 82170073 to Q. Han), the Natural Science Foundation for Distinguished Scholars of Guangdong Province (20022B1515020104 to Y. Zhang), the Foundation for Distinguished Scholars of Guangdong Provincial People’s Hospital (KY0120220132 to Y. Zhang), Natural Science Foundation of Guangdong Province (20022 A1515012501 to Q. Han), Natural Science Foundation of Shanghai (24ZR1459300 to X. Liang) and the National key research and development program intergovernmental key projects (2023YFE0114300).

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Author notes

  1. Jiaqi Chen, Xiaoting Liang and Qian Han have contributed equally to this work.

Authors and Affiliations

  1. Department of Emergency Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China

    Jiaqi Chen, Haiwei He, Xinran Huang, Ying Shen, Jie Qiu, Cong Mai, Ziqi Li, Bei Hu, Xin Li & Yuelin Zhang

  2. Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

    Xiaoting Liang & Kexin Ma

  3. Department of Respiratory Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, Guangzhou, Guangdong, China

    Qian Han

  4. Shanghai Heart Failure Research Center, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China

    Xiaoting Liang, Fang Lin & Kexin Ma

  5. Shanghai East Hospital, Jinzhou Medical University, Jinzhou, China

    Kexin Ma

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  1. Jiaqi Chen
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Contributions

Y. Zhang, X. Li and B. Hu designed the research, analyzed the data and wrote the manuscript. J. Chen, X. Liang and Q. Han performed the research, analyzed the data and wrote the manuscript. H. He, X. Huang, Y. Shen, J. Qiu, F. Lin, C. Mai, Z. Li and K. Ma contributed to the collection of the data and the discussion.

Corresponding authors

Correspondence to Bei Hu, Xin Li or Yuelin Zhang.

Ethics declarations

Ethics approval and consent to participate

All cell cultures in this study were reviewed and approved by the Ethics Committees of Tongji University (Approved project: mesenchymal stem cell-based therapy for myocardial infarction, Approval No. 2016–050, Date of approval: Sep 18, 2016). Written informed consents were obtained from patients and their families for participation in the study and the use of samples. All animal procedures were performed in accordance with the ARRIVE guidelines and approved by the Animal Research Committee of Guangdong Provincial People’s Hospital (Approved project: Study on the effect and mechanism of iPSC-MSCs exosomal miR-202-5p on the improvement of myocardial infarction by inhibiting the activation of NLRP3 inflammasome. Approval No.KY-Z-2022–348-01, Date of approval: Nov 28, 2022).

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The authors declared no competing interest.

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Chen, J., Liang, X., Han, Q. et al. Exosomal miR-202-5p derived from iPSC-MSCs protects against myocardial infarction through inhibition of cardiomyocyte pyroptosis. Stem Cell Res Ther 16, 282 (2025). https://doi.org/10.1186/s13287-025-04390-7

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512