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Mesenchymal stromal cell-derived exosomes in cardiac regeneration and repair

  • Si-Jia Sun
    Affiliations
    Cardiology Division, Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong SAR, China
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  • Rui Wei
    Affiliations
    Cardiology Division, Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong SAR, China
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  • Fei Li
    Affiliations
    Cardiology Division, Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong SAR, China
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  • Song-Yan Liao
    Correspondence
    Corresponding author
    Affiliations
    Cardiology Division, Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong SAR, China

    Shenzhen Institutes of Research and Innovation, the University of Hong Kong, Hong Kong SAR, China
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  • Hung-Fat Tse
    Correspondence
    Corresponding author
    Affiliations
    Cardiology Division, Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong SAR, China

    Shenzhen Institutes of Research and Innovation, the University of Hong Kong, Hong Kong SAR, China

    Research Center of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong SAR, China

    Hong Kong-Guangdong Joint Laboratory on Stem Cell and Regenerative Medicine, the University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong SAR, China
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Open AccessPublished:June 10, 2021DOI:https://doi.org/10.1016/j.stemcr.2021.05.003
      Mesenchymal stromal cell (MSC)-derived exosomes play a promising role in regenerative medicine. Their trophic and immunomodulatory potential has made them a promising candidate for cardiac regeneration and repair. Numerous studies have demonstrated that MSC-derived exosomes can replicate the anti-inflammatory, anti-apoptotic, and pro-angiogenic and anti-fibrotic effects of their parent cells and are considered a substitute for cell-based therapies. In addition, their lower tumorigenic risk, superior immune tolerance, and superior stability compared with their parent stem cells make them an attractive option in regenerative medicine. The therapeutic effects of MSC-derived exosomes have consequently been evaluated for application in cardiac regeneration and repair. In this review, we summarize the potential mechanisms and therapeutic effects of MSC-derived exosomes in cardiac regeneration and repair and provide evidence to support their clinical application.

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      Key words

      Main text

      Background

      Cardiovascular diseases, in particular coronary heart disease (CHD), remain the leading cause of morbidity and mortality worldwide (
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      Heart disease and stroke statistics—2020 update: a report from the American Heart Association.
      ). Among all CHDs, acute myocardial infarction (MI) is a major cause of death. Consequent complications such as heart failure contribute to a significant medical, social, and financial burden. A broad spectrum of therapeutic reperfusion strategies is available, such as thrombolytic therapy or primary percutaneous coronary intervention, as well as anti-remodeling medications such as angiotensin-converting enzyme drugs and β-blockers. Nonetheless there is no effective pharmacological intervention that prevents cardiomyocyte death due to myocardial ischemia/reperfusion (I/R) injury (
      • Heusch G.
      • Gersh B.J.
      The pathophysiology of acute myocardial infarction and strategies of protection beyond reperfusion: a continual challenge.
      ). This I/R injury may also contribute to cardiac fibrosis, myocardial remodeling, cardiac arrhythmia, and, eventually, heart failure (
      • Frank A.
      • Bonney M.
      • Bonney S.
      • Weitzel L.
      • Koeppen M.
      • Eckle T.
      Myocardial ischemia reperfusion injury: from basic science to clinical bedside.
      ). At the end stage, the only available therapy for heart failure is heart transplantation or permanent left ventricular (LV) support. There is therefore tremendous interest in and need for novel therapies for post-MI LV remodeling and dysfunction.
      In the last few years great advances have been made in cell-based therapies. The ultimate aim of such therapies is to generate sufficient functional cardiomyocytes to compensate for those lost following MI, either with exogenous cells or by activating endogenous regeneration and repair mechanisms (
      • Menasche P.
      Cell therapy trials for heart regeneration—lessons learned and future directions.
      ). Mesenchymal stem cells (MSCs) are multipotent stem cells that reside in various organs and can be derived from multiple tissues or cells including bone marrow (BM) stroma, adipose tissue, muscle, skin, fallopian tissue, umbilical cord blood, menstrual blood, and even induced pluripotent stem cells (iPSCs) (
      • Caplan A.I.
      Mesenchymal stem cells.
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      Potential therapeutic applications of muscle-derived mesenchymal stem and progenitor cells.
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      • et al.
      Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.
      ). Their strong paracrine effects make MSCs a promising candidate for endogenous regeneration and repair pathways. There is evidence that intravenous, intracoronary, or intramyocardial administration of MSCs can improve LV function in an MI model (
      • Bagno L.
      • Hatzistergos K.E.
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      Mesenchymal stem cell-based therapy for cardiovascular disease: progress and challenges.
      ;
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      A large-scale investigation of hypoxia-preconditioned allogeneic mesenchymal stem cells for myocardial repair in nonhuman primates: paracrine activity without remuscularization.
      ;
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      • Lian Q.
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      Potent immunomodulation and angiogenic effects of mesenchymal stem cells versus cardiomyocytes derived from pluripotent stem cells for treatment of heart failure.
      ). Moreover, our previous study showed that pre-transplantation systemic intravenous administration of MSCs improved retention and therapeutic efficacy of intramyocardially transplanted exogenous cells (
      • Sun S.-J.
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      • Jiang Y.
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      • Lian Q.
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      • Tse H.-F.
      Immunomodulation by systemic administration of human induced pluripotent stem cell-derived mesenchymal stromal cells to enhance the therapeutic efficacy of cell-based therapy for treatment of myocardial infarction.
      ).
      Accumulating evidence shows that MSC-derived exosomes can ameliorate cardiac function and improve cardiac regeneration and repair (
      • de Abreu R.C.
      • Fernandes H.
      • da Costa Martins P.A.
      • Sahoo S.
      • Emanueli C.
      • Ferreira L.
      Native and bioengineered extracellular vesicles for cardiovascular therapeutics.
      ). In this review, we discuss the therapeutic effects of MSC-derived exosomes in cardiac regeneration and repair following MI and describe the potential mechanisms underlying the benefits of MSC-exosome-based therapies. Additionally, we describe potential approaches to improve the efficacy and production of MSC-derived exosomes.

      The advantages of MSC-derived exosomes over their parent MSCs

      Exosomes are extracellular vesicles with a diameter of 30–150 nm. Previous studies have shown that they are secreted by endosomes, stored in the multivesicular endosomes (MVEs), and released through exocytosis (
      • Doyle L.M.
      • Wang M.Z.
      Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis.
      ). The destiny of exosomes is either release into the extracellular space through fusion of MVEs with the cell plasma membrane or degradation by lysosomes along with MVEs (
      • Doyle L.M.
      • Wang M.Z.
      Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis.
      ). The exosome transfers messages of the target cells through three possible pathways (Figure 1). First, the membrane receptors as well as protein or microRNA (miRNA) composition can be transferred to recipient cells by membrane fusion. Second, there are several protein ligands such as heat-shock proteins in the exosome membrane that can activate recipient cells through receptors in recipient cells. Third, the composition of exosomes such as protein and miRNAs can be transferred to recipient cells by endocytosis (
      • Loyer X.
      • Vion A.-C.
      • Tedgui A.
      • Boulanger C.M.
      Microvesicles as cell-cell messengers in cardiovascular diseases.
      ). Exosomes have been considered one of the main mediators that regulate cell-cell communication.
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      • Gherghiceanu M.
      • Popescu L.M.
      • Moccetti T.
      • Vassalli G.
      Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.
      observed that exosomes secreted by MSCs were absorbed by cardiomyocytes. This ultrastructural evidence indicated that there is indeed an MSC-cardiomyocyte communication modulated by exosomes and that MSCs exerted their cardioprotective effect partly through releasing exosomes. Administration of exosomes derived from MSCs has been shown recently to recapitulate the cardioprotective effect of MSCs (
      • de Abreu R.C.
      • Fernandes H.
      • da Costa Martins P.A.
      • Sahoo S.
      • Emanueli C.
      • Ferreira L.
      Native and bioengineered extracellular vesicles for cardiovascular therapeutics.
      ). There are several advantages of using exosomes rather than cell-based therapies. First, the risk of tumorigenicity in MSC-based therapies has been observed in previous studies (
      • Barkholt L.
      • Flory E.
      • Jekerle V.
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      • Bisset L.
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      • et al.
      Risk of tumorigenicity in mesenchymal stromal cell-based therapies—bridging scientific observations and regulatory viewpoints.
      ), whereas no tumor formation has been observed in exosome-based therapies. Second, inflammatory cytokine interferon-γ (IFN-γ) can increase expression of HLA and immune rejection of transplanted MSCs (
      • Sun Y.Q.
      • Zhang Y.
      • Li X.
      • Deng M.X.
      • Gao W.X.
      • Yao Y.
      • Chiu S.M.
      • Liang X.
      • Gao F.
      • Chan C.W.
      • et al.
      Insensitivity of human iPS cells-derived mesenchymal stem cells to interferon-gamma-induced HLA expression potentiates repair efficiency of hind limb ischemia in immune humanized NOD scid gamma mice.
      ), whereas no such effects have been reported following transplantation of MSC-derived exosomes. Third, MSC-derived exosomes are more stable to store compared with MSCs (
      • Lener T.
      • Gimona M.
      • Aigner L.
      • Borger V.
      • Buzas E.
      • Camussi G.
      • Chaput N.
      • Chatterjee D.
      • Court F.A.
      • Del Portillo H.A.
      • et al.
      Applying extracellular vesicles based therapeutics in clinical trials—an ISEV position paper.
      ). Fourth, the production cost of MSC-derived exosomes is lower than that of their parent cells. Finally, as the particle size of exosomes is much smaller than that of their parent cells, exosomes could travel across capillaries without plugging. Therefore, MSC-derived exosomes are more efficacious than their parent cells after intravenous administration. In summary, mounting evidence demonstrates that exosomes not only recapitulate the beneficial effects of their parent cells but also overcome some of the limitations of parent cells (Table 1).
      Figure thumbnail gr1
      Figure 1Pathways for exosomes to target toward and fuse with the receiving cells
      First, membrane fusion: activated receptors as well as the composition of exosomes can be transferred to the target cells by membrane fusion. Second, binding receptors: exosomes can activate target cells through binding their protein ligands to the receptors in recipient cells. Third, endocytosis: when exosomes are taken up by the target cells, the protein or miRNA composition that they carry can be released into target cells. MSC, mesenchymal stromal cell.
      Table 1The advantages of MSC-derived exosomes over their parent cells
      MSCMSC-derived exosomes
      Risk of tumorigenicitylowno
      Immune rejectionlowno
      Stabilitylowhigh
      Production costhighlow
      Therapeutic efficacy after systemic deliverylowhigh
      MSC, mesenchymal stromal cell.

      The potential role of MSC-derived exosomes in cardiac regeneration and repair

      Use of exosomes for cardiac repair is still at an early stage, and clinical studies of MSC-derived exosomes in the treatment of cardiovascular disease are ongoing. Table 2 summarizes the findings of pre-clinical studies (
      • Arslan F.
      • Lai R.C.
      • Smeets M.B.
      • Akeroyd L.
      • Choo A.
      • Aguor E.N.E.
      • Timmers L.
      • van Rijen H.V.
      • Doevendans P.A.
      • Pasterkamp G.
      • et al.
      Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.
      ;
      • Bian S.
      • Zhang L.
      • Duan L.
      • Wang X.
      • Min Y.
      • Yu H.
      Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model.
      ;
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      ;
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ;
      • Ju C.
      • Shen Y.
      • Ma G.
      • Liu Y.
      • Cai J.
      • Kim I.-M.
      • Weintraub N.L.
      • Liu N.
      • Tang Y.
      Transplantation of cardiac mesenchymal stem cell-derived exosomes promotes repair in ischemic myocardium.
      ;
      • Liu X.
      • Li X.
      • Zhu W.
      • Zhang Y.
      • Hong Y.
      • Liang X.
      • Fan B.
      • Zhao H.
      • He H.
      • Zhang F.
      Exosomes from mesenchymal stem cells overexpressing MIF enhance myocardial repair.
      ;
      • Luther K.M.
      • Haar L.
      • McGuinness M.
      • Wang Y.
      • Lynch T.L.
      • Phan A.
      • Song Y.
      • Shen Z.
      • Gardner G.
      • Kuffel G.
      • et al.
      Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells.
      ;
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ,
      • Ma T.
      • Chen Y.
      • Chen Y.
      • Meng Q.
      • Sun J.
      • Shao L.
      • Yu Y.
      • Huang H.
      • Hu Y.
      • Yang Z.
      • et al.
      MicroRNA-132, delivered by mesenchymal stem cell-derived exosomes, promote angiogenesis in myocardial infarction.
      ;
      • Mao Q.
      • Liang X.-L.
      • Zhang C.-L.
      • Pang Y.-H.
      • Lu Y.-X.
      LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis.
      ;
      • Ni J.
      • Liu X.
      • Yin Y.
      • Zhang P.
      • Xu Y.-W.
      • Liu Z.
      Exosomes derived from TIMP2-modified human umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction possibly by the Akt/Sfrp2 pathway.
      ;
      • Shao L.
      • Zhang Y.
      • Lan B.
      • Wang J.
      • Zhang Z.
      • Zhang L.
      • Xiao P.
      • Meng Q.
      • Geng Y.-j.
      • Yu X.-y.
      • et al.
      MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair.
      ;
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      ,
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ;
      • Xiao C.
      • Wang K.
      • Xu Y.
      • Hu H.
      • Zhang N.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Li Q.
      • Zhu D.
      • et al.
      Transplanted mesenchymal stem cells reduce autophagic flux in infarcted hearts via the exosomal transfer of miR-125b.
      ;
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ;
      • Yu B.
      • Gong M.
      • Wang Y.
      • Millard R.
      • Pasha Z.
      • Yang Y.
      • Ashraf M.
      • Xu M.
      Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.
      ;
      • Zhao J.
      • Li X.
      • Hu J.
      • Chen F.
      • Qiao S.
      • Sun X.
      • Gao L.
      • Xie J.
      • Xu B.
      Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization.
      ) on the use of MSC-derived exosomes in cardiac regeneration and repair. Application of exosomes derived from BM-MSCs (
      • Bian S.
      • Zhang L.
      • Duan L.
      • Wang X.
      • Min Y.
      • Yu H.
      Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model.
      ;
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      ;
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ;
      • Liu X.
      • Li X.
      • Zhu W.
      • Zhang Y.
      • Hong Y.
      • Liang X.
      • Fan B.
      • Zhao H.
      • He H.
      • Zhang F.
      Exosomes from mesenchymal stem cells overexpressing MIF enhance myocardial repair.
      ;
      • Luther K.M.
      • Haar L.
      • McGuinness M.
      • Wang Y.
      • Lynch T.L.
      • Phan A.
      • Song Y.
      • Shen Z.
      • Gardner G.
      • Kuffel G.
      • et al.
      Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells.
      ;
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Ma T.
      • Chen Y.
      • Chen Y.
      • Meng Q.
      • Sun J.
      • Shao L.
      • Yu Y.
      • Huang H.
      • Hu Y.
      • Yang Z.
      • et al.
      MicroRNA-132, delivered by mesenchymal stem cell-derived exosomes, promote angiogenesis in myocardial infarction.
      ;
      • Shao L.
      • Zhang Y.
      • Lan B.
      • Wang J.
      • Zhang Z.
      • Zhang L.
      • Xiao P.
      • Meng Q.
      • Geng Y.-j.
      • Yu X.-y.
      • et al.
      MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair.
      ;
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      ,
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ;
      • Xiao C.
      • Wang K.
      • Xu Y.
      • Hu H.
      • Zhang N.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Li Q.
      • Zhu D.
      • et al.
      Transplanted mesenchymal stem cells reduce autophagic flux in infarcted hearts via the exosomal transfer of miR-125b.
      ;
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ;
      • Yu B.
      • Gong M.
      • Wang Y.
      • Millard R.
      • Pasha Z.
      • Yang Y.
      • Ashraf M.
      • Xu M.
      Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.
      ;
      • Zhao J.
      • Li X.
      • Hu J.
      • Chen F.
      • Qiao S.
      • Sun X.
      • Gao L.
      • Xie J.
      • Xu B.
      Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization.
      ), embryonic stem cell (ESC)-MSCs (
      • Arslan F.
      • Lai R.C.
      • Smeets M.B.
      • Akeroyd L.
      • Choo A.
      • Aguor E.N.E.
      • Timmers L.
      • van Rijen H.V.
      • Doevendans P.A.
      • Pasterkamp G.
      • et al.
      Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.
      ), umbilical cord (UC)-MSCs (
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ;
      • Ni J.
      • Liu X.
      • Yin Y.
      • Zhang P.
      • Xu Y.-W.
      • Liu Z.
      Exosomes derived from TIMP2-modified human umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction possibly by the Akt/Sfrp2 pathway.
      ;
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ), adipose (AD)-MSCs (
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ), and cardiac MSCs (
      • Ju C.
      • Shen Y.
      • Ma G.
      • Liu Y.
      • Cai J.
      • Kim I.-M.
      • Weintraub N.L.
      • Liu N.
      • Tang Y.
      Transplantation of cardiac mesenchymal stem cell-derived exosomes promotes repair in ischemic myocardium.
      ) resulted in significantly improved cardiac function in a pre-clinical model of MI. Among all these exosomes, BM-MSC-derived exosomes and extracellular vesicle size from 30 to 180 nm were most widely used for cardiac repair and regeneration. Intramyocardial and intravenous transplantation were the two most commonly used modes of delivery. For studies in mice, 20–50 μg of MSC-derived exosomes is used for intravenous administration while 1–600 μg is used for intramyocardial administration. For studies in rats, 40–400 μg of MSC-derived exosomes is used for intravenous administration while 20–80 μg is used for intramyocardial administration. In most of these studies, exosomes were administered immediately after induction of MI. Other studies demonstrated that when administration occurred 30 min, 48 h, and even 1 week after MI, cardiac function was still improved. Moreover, to define the therapeutic role of MSC-derived exosomes, the following issues needed to be clarified: (1) the source of their parent cells; (2) methods of isolation and particle size distribution; and (3) the components of exosomes. Here, we illustrate the mechanisms that underlie the therapeutic efficacy of MSC-derived exosomes in cardiac regeneration and repair and their potential role in enhancing the therapeutic efficacy of cell-based therapy.
      Table 2Pre-clinical studies of MSC-derived exosomes for cardiac regeneration and repair
      No.AuthorsYearModelCell sourceCell typeSize of EVAdministrationTimeDoseEffect
      1Arslan et al.2013myocardial I/R injury (mouse)humanESC-MSCintracoronary5 min prior to reperfusion to 3 h after0.4 μg/mLreduced MI size, decreased LV dilation, increased cardiac function, decreased ATP loss
      2Yu et al.2013myocardial I/R injury (rat)ratBM-MSCaverage: 100 nmintramyocardialimmediatelyderived from 4 × 106 MSCreduced CM apoptosis, improved cardiac function
      3Bian et al.2014MI (rat)humanBM-MSC47–180 nmintramyocardial30 min after MI80 μgimproved cardiac function, decreased MI size, promoted angiogenesis
      4Feng et al.2014MI (mouse)mouseBM-MSCaverage: 57.4 nmintramyocardialimmediately1 μgdecreased MI size, decreased apoptosis
      5Ma et al.2017MI (rat)humanUC-MSCaverage: 96 nmintravenousimmediately400 μgimproved cardiac function, decreased apoptosis, increased angiogenesis
      6Shao et al.2017MI (rat)ratBM-MSCintramyocardialimmediately20 μgimproved cardiac function, reduced MI size, decreased inflammation
      7Wang et al.2017MI (mouse)mouseBM-MSCintravenousimmediatelyimproved angiogenesis and cardiac function
      8He et al.2018MI (mouse)mouseBM-MSCintravenous48 h after MI20 μgimproved cardiac function, decreased apoptosis, increased angiogenesis
      9Ju et al.2018MI (mouse)mouseC-MSCaverage: 120 nmintramyocardialimmediately50 μgimproved cardiac function, increased angiogenesis
      10Luther et al.2018MI (mouse)mouseBM-MSCpericardial sacimmediately12.5 μgdecreased apoptosis
      11Wang et al.2018MI (mouse)mouseBM-MSC30–150 nmintravenousimmediately50 μgimproved cardiac function, increased angiogenesis, decreased MI size, decreased inflammation
      12Xiao et al.2018MI (mouse)humanBM-MSCintramyocardial30 min after MI5 μgimproved cardiac function, decreased apoptosis
      13Xu et al.2018MI (rat)humanBM-MSC, AD-MSC, UC-MSCBM-MSC: 40–100 nm; AD-MSC: 30–100 nm; UC-MSC: 10–90 nmintramyocardial30 min after MIderived from 1.5 × 106 MSCimproved cardiac function, decreased MI size, decreased apoptosis and inflammation
      14Ma et al.2018MI (mouse)mouseBM-MSC<150 nmintramyocardial1 week after MI600 μgimproved cardiac function, increased angiogenesis
      15Mao et al.2019MI (rat)humanMSC30–150 nmintravenousimmediately40 μgdecreased MI size, decreased apoptosis and inflammation
      16Ni et al.2019MI (rat)humanUC-MSC40–90 nmintramyocardialimmediately50 μgimproved cardiac function, decreased MI size, reduced apoptosis, increased angiogenesis
      17Lv et al.2019MI (rat)ratBM-MSCaverage: 90 nmintramyocardial30 min after MI80 μgimproved cardiac function, reduced MI size, decreased apoptosis and inflammation, increased angiogenesis
      18Zhao et al.2019myocardial I/R injury (mouse)mouseBM-MSC50–150 nmintramyocardialimmediately50 μgdecreased MI size and inflammation
      19Liu et al.2020MI (rat)humanBM-MSCaverage: 50 nmintramyocardialimmediately30 μgimproved cardiac function, decreased apoptosis
      MSC, mesenchymal stromal cell; EV, extracellular vesicle; MI, myocardial infarction; I/R, ischemia/reperfusion; ESC, embryonic stem cell; BM, bone marrow; UC, umbilical cord; AD, adipose; LV, left ventricle; ATP, adenosine triphosphate; CM, cardiomyocyte.

      Anti-inflammatory effects

      Previous studies showed that MSCs have multiple anti-inflammatory effects that include regulating the polarization of macrophages, inhibiting the activation of effector T cells, and suppressing the secretion of B cells (
      • Corcione A.
      • Benvenuto F.
      • Ferretti E.
      • Giunti D.
      • Cappiello V.
      • Cazzanti F.
      • Risso M.
      • Gualandi F.
      • Mancardi G.L.
      • Pistoia V.
      • et al.
      Human mesenchymal stem cells modulate B-cell functions.
      ;
      • Di Nicola M.
      • Carlo-Stella C.
      • Magni M.
      • Milanesi M.
      • Longoni P.D.
      • Matteucci P.
      • Grisanti S.
      • Gianni A.M.
      Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.
      ;
      • Le Blanc K.
      • Mougiakakos D.
      Multipotent mesenchymal stromal cells and the innate immune system.
      ). Recent studies show that MSC-derived exosomes replicate these anti-inflammatory effects of their parent cells. Both intramyocardial (
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Shao L.
      • Zhang Y.
      • Lan B.
      • Wang J.
      • Zhang Z.
      • Zhang L.
      • Xiao P.
      • Meng Q.
      • Geng Y.-j.
      • Yu X.-y.
      • et al.
      MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair.
      ;
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ;
      • Zhao J.
      • Li X.
      • Hu J.
      • Chen F.
      • Qiao S.
      • Sun X.
      • Gao L.
      • Xie J.
      • Xu B.
      Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization.
      ) and intravenous (
      • Mao Q.
      • Liang X.-L.
      • Zhang C.-L.
      • Pang Y.-H.
      • Lu Y.-X.
      LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis.
      ;
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ) transplantation of MSC-derived exosomes can decrease pro-inflammatory immune cell infiltration in the infarcted heart.
      • Sun X.
      • Shan A.
      • Wei Z.
      • Xu B.
      Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy.
      demonstrated that MSC-derived exosomes switched pro-inflammatory macrophages to anti-inflammatory macrophages.
      • Collino F.
      • Bruno S.
      • Incarnato D.
      • Dettori D.
      • Neri F.
      • Provero P.
      • Pomatto M.
      • Oliviero S.
      • Tetta C.
      • Quesenberry P.J.
      • et al.
      AKI recovery induced by mesenchymal stromal cell-derived extracellular vesicles carrying microRNAs.
      showed that miRNAs play a key role in exosome-based therapies. This anti-inflammatory effect may also be mediated by exosomal miRNAs, evidenced by the promotion of switching of M1 macrophages to M2 macrophages in the peri-infarcted area by miR-182-enriched MSC-derived exosomes (
      • Zhao J.
      • Li X.
      • Hu J.
      • Chen F.
      • Qiao S.
      • Sun X.
      • Gao L.
      • Xie J.
      • Xu B.
      Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization.
      ). Moreover, miR-233-enriched MSC-derived exosomes, which were taken up by macrophages, downregulated SEMA3A and STAT3 and reduced the inflammatory response in macrophages (
      • Wang X.
      • Gu H.
      • Qin D.
      • Yang L.
      • Huang W.
      • Essandoh K.
      • Wang Y.
      • Caldwell C.C.
      • Peng T.
      • Zingarelli B.
      • et al.
      Exosomal miR-223 contributes to mesenchymal stem cell-elicited cardioprotection in polymicrobial sepsis.
      ). In addition, MSC-derived exosomes regulated effector T cells and B cells, inhibited T cell proliferation, and induced activated effective T cell apoptosis (
      • Mokarizadeh A.
      • Delirezh N.
      • Morshedi A.
      • Mosayebi G.
      • Farshid A.-A.
      • Mardani K.
      Microvesicles derived from mesenchymal stem cells: potent organelles for induction of tolerogenic signaling.
      ). miR-181c has been regarded as a key miRNA in this exosome-mediated T cell regulation; miR-181c-enriched MSC-derived exosomes were shown to blunt the Toll-like receptor 4 (TLR4) signaling pathway-mediated release of pro-inflammatory factors (
      • Li X.
      • Liu L.
      • Yang J.
      • Yu Y.
      • Chai J.
      • Wang L.
      • Ma L.
      • Yin H.
      Exosome derived from human umbilical cord mesenchymal stem cell mediates MiR-181c attenuating burn-induced excessive inflammation.
      ). A recent study also showed that MSC-derived exosomes fully reproduced the immunosuppressive effects of MSCs on B cell proliferation and immunoglobulin production by mediating the secretion of immunoglobulins (
      • Budoni M.
      • Fierabracci A.
      • Luciano R.
      • Petrini S.
      • Di Ciommo V.
      • Muraca M.
      The immunosuppressive effect of mesenchymal stromal cells on B lymphocytes is mediated by membrane vesicles.
      ). Although the precise mechanism of these anti-inflammatory effects remains unclear, current clinical trials show that intravenous administration of MSC-derived exosomes is a safe and effective treatment for immune disease (
      • Kordelas L.
      • Rebmann V.
      • Ludwig A.K.
      • Radtke S.
      • Ruesing J.
      • Doeppner T.R.
      • Epple M.
      • Horn P.A.
      • Beelen D.W.
      • Giebel B.
      MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease.
      ).

      Anti-apoptotic effects

      Previous studies showed that intramyocardial (
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      ;
      • Liu X.
      • Li X.
      • Zhu W.
      • Zhang Y.
      • Hong Y.
      • Liang X.
      • Fan B.
      • Zhao H.
      • He H.
      • Zhang F.
      Exosomes from mesenchymal stem cells overexpressing MIF enhance myocardial repair.
      ;
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Ni J.
      • Liu X.
      • Yin Y.
      • Zhang P.
      • Xu Y.-W.
      • Liu Z.
      Exosomes derived from TIMP2-modified human umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction possibly by the Akt/Sfrp2 pathway.
      ;
      • Shao L.
      • Zhang Y.
      • Lan B.
      • Wang J.
      • Zhang Z.
      • Zhang L.
      • Xiao P.
      • Meng Q.
      • Geng Y.-j.
      • Yu X.-y.
      • et al.
      MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair.
      ;
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      ;
      • Xiao C.
      • Wang K.
      • Xu Y.
      • Hu H.
      • Zhang N.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Li Q.
      • Zhu D.
      • et al.
      Transplanted mesenchymal stem cells reduce autophagic flux in infarcted hearts via the exosomal transfer of miR-125b.
      ;
      • Xu H.
      • Wang Z.
      • Liu L.
      • Zhang B.
      • Li B.
      Exosomes derived from adipose tissue, bone marrow, and umbilical cord blood for cardioprotection after myocardial infarction.
      ;
      • Yu B.
      • Gong M.
      • Wang Y.
      • Millard R.
      • Pasha Z.
      • Yang Y.
      • Ashraf M.
      • Xu M.
      Cardiomyocyte protection by GATA-4 gene engineered mesenchymal stem cells is partially mediated by translocation of miR-221 in microvesicles.
      ), intravenous (
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ;
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ;
      • Mao Q.
      • Liang X.-L.
      • Zhang C.-L.
      • Pang Y.-H.
      • Lu Y.-X.
      LncRNA KLF3-AS1 in human mesenchymal stem cell-derived exosomes ameliorates pyroptosis of cardiomyocytes and myocardial infarction through miR-138-5p/Sirt1 axis.
      ;
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ), intracoronary (
      • Arslan F.
      • Lai R.C.
      • Smeets M.B.
      • Akeroyd L.
      • Choo A.
      • Aguor E.N.E.
      • Timmers L.
      • van Rijen H.V.
      • Doevendans P.A.
      • Pasterkamp G.
      • et al.
      Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.
      ) or intrapericardial sac (
      • Luther K.M.
      • Haar L.
      • McGuinness M.
      • Wang Y.
      • Lynch T.L.
      • Phan A.
      • Song Y.
      • Shen Z.
      • Gardner G.
      • Kuffel G.
      • et al.
      Exosomal miR-21a-5p mediates cardioprotection by mesenchymal stem cells.
      ) transplantation of MSC-derived exosomes could ameliorate apoptosis of endogenous cardiomyocytes. The infiltrated inflammatory cells released numerous reactive oxygen species that led to programmed cell death in the peri-infarcted area. MSC-derived exosomes may reduce oxidative stress and improve the anti-apoptotic ability of cardiomyocytes in the peri-infarcted area. A recent study showed that administration of MSC-derived exosomes in an I/R mouse model increased the ATP level, reduced oxidative stress, and improved cardiomyocyte survival (
      • Arslan F.
      • Lai R.C.
      • Smeets M.B.
      • Akeroyd L.
      • Choo A.
      • Aguor E.N.E.
      • Timmers L.
      • van Rijen H.V.
      • Doevendans P.A.
      • Pasterkamp G.
      • et al.
      Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury.
      ). Previous studies showed that miRNAs including miR-19a (
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ), miR-22 (
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      ), miR-199a (
      • Zhu G.
      • Pei L.
      • Lin F.
      • Yin H.
      • Li X.
      • He W.
      • Liu N.
      • Gou X.
      Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR-199a-3p.
      ), and miR-214 (
      • Wang Y.
      • Zhao R.
      • Liu D.
      • Deng W.
      • Xu G.
      • Liu W.
      • Rong J.
      • Long X.
      • Ge J.
      • Shi B.
      Exosomes derived from miR-214-enriched bone marrow-derived mesenchymal stem cells regulate oxidative damage in cardiac stem cells by targeting CaMKII.
      ) play key roles in these exosome-mediated anti-apoptotic effects. Exosomes derived from GATA4-overexpressed MSCs exerted anti-apoptotic and cardioprotective effects by delivering anti-apoptotic miRNAs, including miR-19a (
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ). These anti-apoptotic effects were abolished by inhibition of miR-19a (
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ). Researchers attributed the anti-apoptotic effects to downregulation of phosphatase and tensin homolog (PTEN) (
      • Chen Y.J.
      • Huang T.Z.-P.
      • Seok T.H.
      • Ding T.J.
      • Kataoka T.M.
      • Zhang T.Z.
      • Hu T.X.
      • Wang T.G.
      • Lin T.Z.
      • Wang T.S.
      • et al.
      mir-17-92 cluster is required for and sufficient to induce cardiomyocyte proliferation in postnatal and adult hearts.
      ). Nonetheless, the clinical safety of miR-19a-enriched exosomes is still under debate. Although there are limited data on the arrhythmogenic effect of miR-19a-enriched exosomes, overexpression of miR-19a may induce arrhythmias, since miR-19a aberrantly inhibits connexin-43 expression (
      • Danielson L.
      • Park D.
      • Rotllan N.
      • Chamorro-Jorganes A.
      • Guijarro M.
      • Fernandez-Hernando C.
      • Fishman G.I.
      • Phoon C.
      • Hernando E.
      Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis.
      ).
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      showed that after co-culture with cardiomyocytes, MSCs transferred miR-22 into cardiomyocytes through exosome release. Intramyocardial administration of miR-22-enriched MSC-derived exosomes was shown to decrease cardiomyocyte apoptosis in the ischemic area and reduce infarct size (
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      ). In a kidney ischemia/perfusion model, miR-199a-enriched MSC-derived exosomes decreased apoptosis in renal cells by modulating Sema3A and activating Akt and ERK pathways (
      • Zhu G.
      • Pei L.
      • Lin F.
      • Yin H.
      • Li X.
      • He W.
      • Liu N.
      • Gou X.
      Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR-199a-3p.
      ). miR-199a is one of the key miRNAs that blocked negative regulators of cardiomyocyte proliferation in a neonatal rat model (
      • Eulalio A.
      • Mano M.
      • Dal Ferro M.
      • Zentilin L.
      • Sinagra G.
      • Zacchigna S.
      • Giacca M.
      Functional screening identifies miRNAs inducing cardiac regeneration.
      ). Indeed, overexpression of miR-199a exerted cardioprotective effects and promoted cardiac regeneration and repair after MI (
      • Eulalio A.
      • Mano M.
      • Dal Ferro M.
      • Zentilin L.
      • Sinagra G.
      • Zacchigna S.
      • Giacca M.
      Functional screening identifies miRNAs inducing cardiac regeneration.
      ). Exosomes derived from miR-214-enriched MSCs facilitated cardiac stem cell survival via reduction of reactive oxygen species production. On the contrary, depletion of miR-214 by miR-214 inhibitor or miR-214 mimics impaired the anti-apoptotic effects of these miR-214-enriched exosomes (
      • Wang Y.
      • Zhao R.
      • Liu D.
      • Deng W.
      • Xu G.
      • Liu W.
      • Rong J.
      • Long X.
      • Ge J.
      • Shi B.
      Exosomes derived from miR-214-enriched bone marrow-derived mesenchymal stem cells regulate oxidative damage in cardiac stem cells by targeting CaMKII.
      ). miR-214 exerted its anti-apoptotic effects mainly through regulation of sodium/calcium exchanger 1, cyclophilin D, Bcl-2 like protein 11, and PTEN (
      • Aurora A.B.
      • Mahmoud A.I.
      • Luo X.
      • Johnson B.A.
      • van Rooij E.
      • Matsuzaki S.
      • Humphries K.M.
      • Hill J.A.
      • Bassel-Duby R.
      • Sadek H.A.
      • et al.
      MicroRNA-214 protects the mouse heart from ischemic injury by controlling Ca2+ overload and cell death.
      ;
      • Lv G.
      • Shao S.
      • Dong H.
      • Bian X.
      • Yang X.
      • Dong S.
      MicroRNA-214 protects cardiac myocytes against H2O2-induced injury.
      ;
      • Wang Y.
      • Zhao R.
      • Liu D.
      • Deng W.
      • Xu G.
      • Liu W.
      • Rong J.
      • Long X.
      • Ge J.
      • Shi B.
      Exosomes derived from miR-214-enriched bone marrow-derived mesenchymal stem cells regulate oxidative damage in cardiac stem cells by targeting CaMKII.
      ). Nonetheless, miR-214 may induce dilated cardiomyocytes in the long term (
      • Lv G.
      • Shao S.
      • Dong H.
      • Bian X.
      • Yang X.
      • Dong S.
      MicroRNA-214 protects cardiac myocytes against H2O2-induced injury.
      ). This side effect may limit the clinical application of miR-214-enriched exosomes. Although no report is available on miR-210-enriched exosomes, they may be also involved in the exosome-mediated anti-apoptosis effect. miR-210 promoted endogenous cardiac regeneration through upregulation of β-catenin and Bcl-2 and downregulation of adenomatous polyposis coli, p16, and caspase-3 (
      • Arif M.
      • Pandey R.
      • Alam P.
      • Jiang S.
      • Arghya P.
      • Ahmed R.P.
      MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents.
      ).

      Pro-angiogenic effects

      Previous research showed that intravenous (
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      ;
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ;
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      ,
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ) and intramyocardial (
      • Bian S.
      • Zhang L.
      • Duan L.
      • Wang X.
      • Min Y.
      • Yu H.
      Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model.
      ;
      • Ju C.
      • Shen Y.
      • Ma G.
      • Liu Y.
      • Cai J.
      • Kim I.-M.
      • Weintraub N.L.
      • Liu N.
      • Tang Y.
      Transplantation of cardiac mesenchymal stem cell-derived exosomes promotes repair in ischemic myocardium.
      ;
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Ma T.
      • Chen Y.
      • Chen Y.
      • Meng Q.
      • Sun J.
      • Shao L.
      • Yu Y.
      • Huang H.
      • Hu Y.
      • Yang Z.
      • et al.
      MicroRNA-132, delivered by mesenchymal stem cell-derived exosomes, promote angiogenesis in myocardial infarction.
      ;
      • Ni J.
      • Liu X.
      • Yin Y.
      • Zhang P.
      • Xu Y.-W.
      • Liu Z.
      Exosomes derived from TIMP2-modified human umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction possibly by the Akt/Sfrp2 pathway.
      ) administration can improve angiogenesis during cardiac regeneration and repair. Our study showed that iPSC-MSCs ameliorated limb ischemia through both vascular differentiation and paracrine secretion, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (
      • Lian Q.
      • Zhang Y.
      • Zhang J.
      • Zhang H.K.
      • Wu X.
      • Zhang Y.
      • Lam F.F.
      • Kang S.
      • Xia J.C.
      • Lai W.H.
      • et al.
      Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.
      ). Recently,
      • Hu G.-W.
      • Li Q.
      • Niu X.
      • Hu B.
      • Liu J.
      • Zhou S.-M.
      • Guo S.-C.
      • Lang H.-L.
      • Zhang C.-Q.
      • Wang Y.
      • et al.
      Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.
      reported that intramuscular administration of exosomes derived from iPSC-MSCs partly replicated the pro-angiogenic effects of their parent cells. In their in vitro study, iPSC-MSC-derived exosomes promoted human umbilical vein endothelial cell migration, proliferation, tube formation, and angiogenesis-related gene and molecule expression (
      • Hu G.-W.
      • Li Q.
      • Niu X.
      • Hu B.
      • Liu J.
      • Zhou S.-M.
      • Guo S.-C.
      • Lang H.-L.
      • Zhang C.-Q.
      • Wang Y.
      • et al.
      Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.
      ). miRNAs and proteins packaged in the MSC-derived exosomes may mainly account for these angiogenic effects. Endothelial cells treated with MSC-derived exosomes demonstrated an increased ability for proliferation, migration, and tube formation. Intravenous administration of these exosomes significantly increased cardiac function and angiogenesis in a rat model of MI (
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ). Researchers concluded that the pro-angiogenic effects were due to increased expression of platelet-derived growth factor D (PDGF-D). As well as PDGF-D, several miRNAs are involved in MSC-derived exosome-mediated pro-angiogenic effects. Evidence suggests that miR-126- and VEGF-enriched MSC-derived exosomes promote pro-angiogenic mRNA expression in ischemic tissues, while the pro-angiogenic effects were weakened in exosomes derived from miR-126 knockdown MSCs in the treated group (
      • Du W.
      • Zhang K.
      • Zhang S.
      • Wang R.
      • Nie Y.
      • Tao H.
      • Han Z.
      • Liang L.
      • Wang D.
      • Liu J.
      • et al.
      Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.
      ).
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      showed that MSC-derived exosomes promoted angiogenesis in the mouse model of MI through delivery of miR-210. Exosome-derived MSCs with miR-210 silence demonstrated a significantly decreased pro-angiogenic effect both in vitro and in vivo (
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      ). Interestingly, preconditioning MSCs with ischemic stroke serum significantly increased miR-20a expression (
      • Kim E.H.
      • Kim D.H.
      • Kim H.R.
      • Kim S.Y.
      • Kim H.H.
      • Bang O.Y.
      Stroke serum priming modulates characteristics of mesenchymal stromal cells by controlling the expression miRNA-20a.
      ). Nonetheless, the effects of miR-20a on neovascularization are still under debate. Inhibition of miR-20a has been suggested to exert pro-angiogenic or anti-angiogenic effects (
      • Doebele C.
      • Bonauer A.
      • Fischer A.
      • Scholz A.
      • Reiss Y.
      • Urbich C.
      • Hofmann W.-K.
      • Zeiher A.M.
      • Dimmeler S.
      Members of the microRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic function in endothelial cells.
      ;
      • Pin A.-L.
      • Houle F.o.
      • Guillonneau M.v.
      • Paquet E.R.
      • Simard M.J.
      • Huot J.
      miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF.
      ;
      • Suárez Y.
      • Fernández-Hernando C.
      • Yu J.
      • Gerber S.A.
      • Harrison K.D.
      • Pober J.S.
      • Iruela-Arispe M.L.
      • Merkenschlager M.
      • Sessa W.C.
      Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis.
      ).

      Anti-fibrotic effects

      Previous research showed that intravenous (
      • Wang X.
      • Chen Y.
      • Zhao Z.
      • Meng Q.
      • Yu Y.
      • Sun J.
      • Yang Z.
      • Chen Y.
      • Li J.
      • Ma T.
      • et al.
      Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction.
      ) and intramyocardial (
      • Bian S.
      • Zhang L.
      • Duan L.
      • Wang X.
      • Min Y.
      • Yu H.
      Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model.
      ;
      • Lv K.
      • Li Q.
      • Zhang L.
      • Wang Y.
      • Zhong Z.
      • Zhao J.
      • Lin X.
      • Wang J.
      • Zhu K.
      • Xiao C.
      • et al.
      Incorporation of small extracellular vesicles in sodium alginate hydrogel as a novel therapeutic strategy for myocardial infarction.
      ;
      • Ni J.
      • Liu X.
      • Yin Y.
      • Zhang P.
      • Xu Y.-W.
      • Liu Z.
      Exosomes derived from TIMP2-modified human umbilical cord mesenchymal stem cells enhance the repair effect in rat model with myocardial infarction possibly by the Akt/Sfrp2 pathway.
      ;
      • Shao L.
      • Zhang Y.
      • Lan B.
      • Wang J.
      • Zhang Z.
      • Zhang L.
      • Xiao P.
      • Meng Q.
      • Geng Y.-j.
      • Yu X.-y.
      • et al.
      MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair.
      ) transplantation of MSC-derived exosomes could ameliorate cardiac fibrosis during cardiac regeneration and repair. Cardiac regeneration and repair and deposition of extracellular collagen are two parallel processes during the reparative phase. The balance between scar formation and cardiac regeneration is crucial for cardiac repair. Scar formation provides mechanical strength and circumvents ventricular rupture, and is beneficial for cardiac repair in the short term. Nonetheless it is inversely correlated with cardiac regeneration in the long term. Therefore, anti-fibrotic therapies are optimized to target the reactive fibrosis that occurs during ventricular remodeling. These are supported by a previous study wherein systemic inhibition of fibrosis formation signals during the inflammatory phase following MI exacerbated cardiac impairment, while inhibition of systemic fibrosis ameliorated cardiac remodeling and dysfunction in the post-MI reparative phase (
      • Ikeuchi M.
      • Tsutsui H.
      • Shiomi T.
      • Matsusaka H.
      • Matsushima S.
      • Wen J.
      • Kubota T.
      • Takeshita A.
      Inhibition of TGF-beta signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction.
      ). The miRNA in the MSC-derived exosomes ameliorated collagen deposition during cardiac remodeling. Apart from anti-apoptotic effects, miR-19a regulated several extracellular matrix proteins, including thrombospondin-1 and connective tissue growth factor (CTGF) (
      • Wen Z.
      • Zheng S.
      • Zhou C.
      • Yuan W.
      • Wang J.
      • Wang T.
      Bone marrow mesenchymal stem cells for post-myocardial infarction cardiac repair: microRNAs as novel regulators.
      ). Whether miR-19a-enriched exosomes regulated post-MI fibrosis requires further exploration. A previous study showed that miR-29 was expressed in BM-MSCs (
      • Jin M.
      • Wu Y.
      • Wang J.
      • Ye W.
      • Wang L.
      • Yin P.
      • Liu W.
      • Pan C.
      • Hua X.
      MicroRNA-29 facilitates transplantation of bone marrow-derived mesenchymal stem cells to alleviate pelvic floor dysfunction by repressing elastin.
      ). miR-29 is a well-defined miRNA that regulates cardiac fibrosis. Lower miR-29 expression leads to more extracellular matrix deposit, culminating in cardiac fibrosis (
      • van Rooij E.
      • Sutherland L.B.
      • Thatcher J.E.
      • Dimaio J.M.
      • Naseem R.H.
      • Marshall W.S.
      • Hill J.A.
      • Olson E.N.
      Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis.
      ). Although no direct evidence supports the anti-fibrotic effect of miR-29, a pharmacological study showed that MI rats treated with carvedilol had reduced myocardial fibrosis that was miR-29 dependent (
      • Zhu J.-N.
      • Chen R.
      • Fu Y.-H.
      • Lin Q.-X.
      • Huang S.
      • Guo L.-L.
      • Zhang M.-Z.
      • Deng C.-Y.
      • Zou X.
      • Zhong S.-L.
      • et al.
      Smad3 inactivation and MiR-29b upregulation mediate the effect of carvedilol on attenuating the acute myocardium infarction-induced myocardial fibrosis in rat.
      ). Interestingly, swim training ameliorated the downregulated expression of miR-29 and decreased the MI-induced fibrotic gene expression (
      • Melo S.F.S.
      • Fernandes T.
      • Baraúna V.G.
      • Matos K.C.
      • Santos A.A.S.
      • Tucci P.J.F.
      • Oliveira E.M.
      Expression of MicroRNA-29 and collagen in cardiac muscle after swimming training in myocardial-infarcted rats.
      ). These studies highlight the pivotal role of miR-29 in cardiac fibrosis development following MI. A recent study also showed that miR-133-overexpressed MSCs reduced infarct size following MI (
      • Chen Y.
      • Zhao Y.
      • Chen W.
      • Xie L.
      • Yang J.
      • Chen Y.
      • Wei L.
      • Shen Z.
      MicroRNA-133 overexpression promotes the therapeutic efficacy of mesenchymal stem cells on acute myocardial infarction.
      ). The anti-fibrotic effects of miR-133 may be due to regulation of the collagen α1 chain and CTGF (
      • Castoldi G.
      • Di Gioia C.R.T.
      • Bombardi C.
      • Catalucci D.
      • Corradi B.
      • Gualazzi M.G.
      • Leopizzi M.
      • Mancini M.
      • Zerbini G.
      • Condorelli G.
      • et al.
      MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension.
      ;
      • Duisters F.R.
      • Tijsen J.A.
      • Schroen J.B.
      • Leenders E.J.
      • Lentink W.V.
      • Van Der Made G.I.
      • Herias M.V.
      • Van Leeuwen E.R.
      • Schellings E.M.
      • Barenbrug E.P.
      • et al.
      miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling.
      ).
      Taken together, exosomes carrying different contents may elicit different therapeutic responses. Exosomes expressing abundant miR-182, miR-233, and miR-181c elicit beneficial effects mainly through anti-inflammatory responses; exosomes expressing abundant miR-19a, miR-22, miR-199a, miR-214, and miR-210 modulate the survival of cardiomyocytes in the ischemic region; exosomes expressing abundant miR-126, miR-210, and miR-20a improve cardiac regeneration and repair by promoting angiogenesis; and exosomes expressing abundant miR-19a, miR-29, and miR-133 inhibit fibrosis during cardiac regeneration and repair. Post-MI regeneration and repair can be divided into two phases: a pro-inflammatory phase, which features pro-inflammatory cytokine release, inflammatory cell infiltration, and phagocytosis of necrotic cell debris; and a reparative phase, characterized by resolution of inflammation and formation of collagen-based scar (
      • Cahill T.J.
      • Choudhury R.P.
      • Riley P.R.
      Heart regeneration and repair after myocardial infarction: translational opportunities for novel therapeutics.
      ). Therefore, anti-inflammatory and anti-apoptotic exosomes may benefit cardiac regeneration during the pro-inflammatory phase, whereas pro-angiogenetic and anti-fibrotic exosomes promote vascularization and cardiac regeneration during the reparative phase. The miRNAs and downstream pathways relevant to the anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-fibrotic effects are summarized in Table 3.
      Table 3The miRNAs and underlying pathways for MSC-derived exosome-mediated cardiac regeneration
      FunctionRoute of administrationmiRNAPathwayTarget cellReference
      Anti-inflammatoryintramyocardial, intravenousmiR-182TLR4 signalmacrophage
      • Zhao J.
      • Li X.
      • Hu J.
      • Chen F.
      • Qiao S.
      • Sun X.
      • Gao L.
      • Xie J.
      • Xu B.
      Mesenchymal stromal cell-derived exosomes attenuate myocardial ischaemia-reperfusion injury through miR-182-regulated macrophage polarization.
      miR-233SEMA3A; STAT3macrophage
      • Wang X.
      • Gu H.
      • Qin D.
      • Yang L.
      • Huang W.
      • Essandoh K.
      • Wang Y.
      • Caldwell C.C.
      • Peng T.
      • Zingarelli B.
      • et al.
      Exosomal miR-223 contributes to mesenchymal stem cell-elicited cardioprotection in polymicrobial sepsis.
      miR-181cTLR4 signalT cell
      • Mokarizadeh A.
      • Delirezh N.
      • Morshedi A.
      • Mosayebi G.
      • Farshid A.-A.
      • Mardani K.
      Microvesicles derived from mesenchymal stem cells: potent organelles for induction of tolerogenic signaling.
      Anti-apoptoticintramyocardial, intravenous, intracoronary, intrapericardialmiR-19aPTENcardiomyocyte
      • He J.-G.
      • Li H.-R.
      • Han J.-X.
      • Li B.-B.
      • Yan D.
      • Li H.-Y.
      • Wang P.
      • Luo Y.
      GATA-4-expressing mouse bone marrow mesenchymal stem cells improve cardiac function after myocardial infarction via secreted exosomes.
      miR-22MeCP2
      • Feng Y.
      • Huang W.
      • Wani M.
      • Yu X.
      • Ashraf M.
      Ischemic preconditioning potentiates the protective effect of stem cells through secretion of exosomes by targeting Mecp2 via miR-22.
      miR-199aSema3A
      • Zhu G.
      • Pei L.
      • Lin F.
      • Yin H.
      • Li X.
      • He W.
      • Liu N.
      • Gou X.
      Exosomes from human-bone-marrow-derived mesenchymal stem cells protect against renal ischemia/reperfusion injury via transferring miR-199a-3p.
      miR-214sodium/calcium exchanger 1; cyclophilin D; Bcl-2 like protein 11; PTEN
      • Wang Y.
      • Zhao R.
      • Liu D.
      • Deng W.
      • Xu G.
      • Liu W.
      • Rong J.
      • Long X.
      • Ge J.
      • Shi B.
      Exosomes derived from miR-214-enriched bone marrow-derived mesenchymal stem cells regulate oxidative damage in cardiac stem cells by targeting CaMKII.
      miR-210β-catenin; Bcl-2; adenomatous polyposis coli; p16; caspase-3
      • Arif M.
      • Pandey R.
      • Alam P.
      • Jiang S.
      • Arghya P.
      • Ahmed R.P.
      MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents.
      Pro-angiogenicintramyocardial, intravenousmiR-126Spred-1endothelial cell
      • Du W.
      • Zhang K.
      • Zhang S.
      • Wang R.
      • Nie Y.
      • Tao H.
      • Han Z.
      • Liang L.
      • Wang D.
      • Liu J.
      • et al.
      Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.
      miR-210Efna3
      • Wang N.
      • Chen C.
      • Yang D.
      • Liao Q.
      • Luo H.
      • Wang X.
      • Zhou F.
      • Yang X.
      • Yang J.
      • Zeng C.
      • et al.
      Mesenchymal stem cells-derived extracellular vesicles, via miR-210, improve infarcted cardiac function by promotion of angiogenesis.
      miR-20ap38 MAP kinase
      • Pin A.-L.
      • Houle F.o.
      • Guillonneau M.v.
      • Paquet E.R.
      • Simard M.J.
      • Huot J.
      miR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF.
      Anti-fibroticintramyocardial, intravenousmiR-19athrombopondin-1; CTGFfibroblast
      • Wen Z.
      • Zheng S.
      • Zhou C.
      • Yuan W.
      • Wang J.
      • Wang T.
      Bone marrow mesenchymal stem cells for post-myocardial infarction cardiac repair: microRNAs as novel regulators.
      miR-29fibrotic gene
      • Melo S.F.S.
      • Fernandes T.
      • Baraúna V.G.
      • Matos K.C.
      • Santos A.A.S.
      • Tucci P.J.F.
      • Oliveira E.M.
      Expression of MicroRNA-29 and collagen in cardiac muscle after swimming training in myocardial-infarcted rats.
      miR-133collagen α1 chain; CTGF
      • Chen Y.
      • Zhao Y.
      • Chen W.
      • Xie L.
      • Yang J.
      • Chen Y.
      • Wei L.
      • Shen Z.
      MicroRNA-133 overexpression promotes the therapeutic efficacy of mesenchymal stem cells on acute myocardial infarction.
      MSC, mesenchymal stromal cell; TLR4, Toll-like receptor 4; PTEN, phosphatase and tensin homolog; CTGF, connective tissue growth factor.

      Potential role in enhancing therapeutic efficacy of cell-based therapy

      Recently, our group has investigated the immunomodulatory effect of human iPSC (hiPSC)-MSCs on improving the survival and therapeutic efficacy of cells following intramyocardial transplantation into a mouse model of MI (
      • Sun S.-J.
      • Lai W.-H.
      • Jiang Y.
      • Zhen Z.
      • Wei R.
      • Lian Q.
      • Liao S.-Y.
      • Tse H.-F.
      Immunomodulation by systemic administration of human induced pluripotent stem cell-derived mesenchymal stromal cells to enhance the therapeutic efficacy of cell-based therapy for treatment of myocardial infarction.
      ). Our results showed that pre-transplantation systemic administration of hiPSC-MSCs increased systemic regulatory T cell (Treg) activation, decreased the number of splenic natural killer (NK) cells and inflammation, enhanced survival of transplanted hiPSC-MSCs and hiPSC-cardiomyocytes following intramyocardial transplantation, and improved the therapeutic efficacy at 4 weeks post MI. Previous studies showed that MSC-derived exosomes could upregulate CD4+ T cell differentiation into Tregs and downregulate the activation of NK cells (
      • Kordelas L.
      • Rebmann V.
      • Ludwig A.K.
      • Radtke S.
      • Ruesing J.
      • Doeppner T.R.
      • Epple M.
      • Horn P.A.
      • Beelen D.W.
      • Giebel B.
      MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease.
      ;
      • Zhang B.
      • Yin Y.
      • Lai R.C.
      • Tan S.S.
      • Choo A.B.
      • Lim S.K.
      Mesenchymal stem cells secrete immunologically active exosomes.
      ). The immunomodulatory effects of MSCs were orchestrated by immunosuppressive factors including indoleamine 2,3-dioxygenase, transforming growth factor β1, prostaglandin E2, nitric oxide, and interleukin-10 (
      • English K.
      • Ryan J.M.
      • Tobin L.
      • Murphy M.J.
      • Barry F.P.
      • Mahon B.P.
      Cell contact, prostaglandin E2 and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+ CD25 High forkhead box P3+ regulatory T cells.
      ;
      • Maggini J.
      • Mirkin G.
      • Bognanni I.
      • Holmberg J.
      • Piazzón I.M.
      • Nepomnaschy I.
      • Costa H.
      • Cañones C.
      • Raiden S.
      • Vermeulen M.
      • et al.
      Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile (macrophages and MSC).
      ;
      • Nauta A.
      • Kruisselbrink A.
      • Lurvink E.
      • Willemze R.
      • Fibbe W.
      Mesenchymal stem cells inhibit generation and function of both CD34 super(+)-derived and monocyte-derived dendritic cells.
      ;
      • Ren G.
      • Chen X.
      • Dong F.
      • Li W.
      • Ren X.
      • Zhang Y.
      • Shi Y.
      Concise review: mesenchymal stem cells and translational medicine: emerging issues.
      ;
      • van Koppen A.
      • Joles J.A.
      • van Balkom B.W.M.
      • Lim S.K.
      • de Kleijn D.
      • Giles R.H.
      • Verhaar M.C.
      Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease.
      ). Recently, researchers proposed that these immunosuppressive factors were packed in the extracellular vesicles, especially exosomes (
      • Lener T.
      • Gimona M.
      • Aigner L.
      • Borger V.
      • Buzas E.
      • Camussi G.
      • Chaput N.
      • Chatterjee D.
      • Court F.A.
      • Del Portillo H.A.
      • et al.
      Applying extracellular vesicles based therapeutics in clinical trials—an ISEV position paper.
      ). Indeed, subcutaneous administration of MSC-derived exosomes improved the survival of transplanted immortal human keratinocytes in a rat skin wound model (
      • Zhang B.
      • Wang M.
      • Gong A.
      • Zhang X.
      • Wu X.
      • Zhu Y.
      • Shi H.
      • Wu L.
      • Zhu W.
      • Qian H.
      • et al.
      HucMSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing.
      ). As a result, if hiPSC-MSC-derived exosomes can recapitulate the immunomodulatory effect of their parent cells, pre-transplantation systemic administration of hiPSC-MSC-derived exosomes is a promising strategy by which to improve the cell retention of cell-based therapy because of their advantages of low tumorigenicity and superior immune tolerance and stability. Isolation of immunomodulatory exosomes from MSCs may provide a promising means by which to combine cell-based therapies and exosome-based therapies for cardiac regeneration and repair.

      Approaches to improve the production and therapeutic effects of MSC-derived exosomes

      Although MSC-derived exosomes represent a promising candidate for cardiac regeneration and repair, their low-level production from routine culture conditions limits their therapeutic efficacy. Different culture conditions may result in altered production of MSC-derived exosomes and even different components in the released exosomes. For example, culture of MSCs with stroke serum, obtained from the blood of mice with middle cerebral artery occlusion, demonstrated remarkably increased miR-20a expression in their released exosomes when compared with MSCs cultured with fetal bovine serum or normal serum (
      • Kim E.H.
      • Kim D.H.
      • Kim H.R.
      • Kim S.Y.
      • Kim H.H.
      • Bang O.Y.
      Stroke serum priming modulates characteristics of mesenchymal stromal cells by controlling the expression miRNA-20a.
      ). It is evident that culture conditions will considerably affect the constituents of its relevant exosomes as well as their production. It is of paramount importance to define the appropriate culture conditions to improve the production of exosomes without compromising their therapeutic effect. Apart from culture medium, cell type also influences the efficacy of exosome-based therapies, since exosomes from different microenvironments may have a different focus on a certain cell type. For example, extracellular vesicles derived from the nervous system are preferentially absorbed by cells in the nervous system (
      • Pegtel D.M.
      • Peferoen L.
      • Amor S.
      Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain.
      ). Whether co-culturing of MSCs with cardiomyocytes can improve the efficacy of MSC-derived exosomes remains to be revealed.
      As mentioned above, the destiny of exosomes is either degradation by lysosomes or release into the extracellular space. Therefore, increased production of MSC-derived exosomes can be realized by decreasing degradation and increasing exosome secretion. For example, inhibition of P2X7 receptors, soluble NSF attachment protein receptors, tumor suppressor-activated pathway 6, rhomboid domain-containing protein 1, or phospholipase D2 compromises exosome release. Overexpressing these mediators may enhance exosome extracellular secretion and prevent exosome degradation (
      • Phan J.
      • Kumar P.
      • Hao D.
      • Gao K.
      • Farmer D.
      • Wang A.
      Engineering mesenchymal stem cells to improve their exosome efficacy and yield for cell-free therapy.
      ). Regulating the key mediators in the exosome degradation or secretion pathways may promote effective MSC-derived exosome secretion, culminating in increased exosome production.
      Genetic modification of their parent cells is another efficient means to improve the therapeutic effect of exosomes. The therapeutic efficacy of MSC-derived exosomes can be improved by overexpression or knockdown of certain RNAs or proteins. Several DNA or RNA sequences have been regarded as cardioprotective or pro-angiogenic. For example, modifying MSCs by transfecting them with a recombinant adenovirus carrying the Akt gene sequence could increase the expression of PDGF-D in its related exosomes (
      • Ma J.
      • Zhao Y.
      • Sun L.
      • Sun X.
      • Zhao X.
      • Sun X.
      • Qian H.
      • Xu W.
      • Zhu W.
      Exosomes derived from Akt-modified human umbilical cord mesenchymal stem cells improve cardiac regeneration and promote angiogenesis via activating platelet-derived growth factor D.
      ). These genetically modified MSCs and their related exosomes have an increased ability to promote neovascularization, cardiac regeneration, and cardiac repair. Both DNA sequences and RNA sequences could be regarded as modified target and non-coding RNAs that were increased in the exosomes derived from genetically modified MSCs, and have been considered to play a pivotal role in cardiac regeneration and repair following MI.
      Although there is no consensus on the optimum delivery route for MSC-derived exosomes, intramyocardial transplantation, especially transendocardial transplantation, may be the most efficacious (
      • Collantes M.
      • Pelacho B.
      • Garcia-Velloso M.
      • Gavira J.
      • Abizanda G.
      • Palacios I.
      • Rodriguez-Borlado L.
      • Alvarez V.
      • Prieto E.
      • Ecay M.
      • et al.
      Non-invasive in vivo imaging of cardiac stem/progenitor cell biodistribution and retention after intracoronary and intramyocardial delivery in a swine model of chronic ischemia reperfusion injury.
      ;
      • Kanelidis J.A.
      • Premer M.C.
      • Lopez M.J.
      • Balkan M.W.
      • Hare M.J.
      Route of delivery modulates the efficacy of mesenchymal stem cell therapy for myocardial infarction: a meta-analysis of preclinical studies and clinical trials.
      ). Nonetheless, previous studies showed that more than 90% of cells were lost within the first few days following transplantation because a large percentage of cells spilled out from the myocardium when they were delivered via direct intramyocardial injection. The same problem exists for administration of exosome-based therapy. An alternative approach is to use cardiac tissue engineering to produce a scaffold and then seed exosomes onto the patch to optimize retention and engraftment.

      Limitations of MSC-derived exosomes

      Although MSC-derived exosomes are a promising substitute for MSCs in regenerative medicine, there are several considerations that should be addressed prior to their clinical application. First, exosomes are currently isolated according to their vesicle size. If different vesicle sizes reflect different components in exosomes, more accurate isolation methods need to be adopted, since different-size-defined exosomes may have variable therapeutic efficacy for cardiac regeneration and repair. Second, exosomes are stored in MVEs before release into intercellular substance. Extracting MSC-derived exosomes from MVEs before they are released may help to isolate purified exosomes, as MSC-derived exosomes isolated from culture media and blood may be contaminated by exosomes from other cell types. Third, a well-defined method for single-exosome analysis is currently unavailable. Investigating cargoes from a single exosome rather than the whole exosome population would provide a better understanding of the exact mechanisms underlying exosome-based therapies. Finally, it is pertinent to define the appropriate microenvironment in which to generate therapeutic exosomes for cardiac regeneration and repair. Recent evidence demonstrates that MSC-derived exosomes may contain different compounds and exert different therapeutic effects in different microenvironments. For example, MSCs may secrete immunomodulatory exosomes in the pro-inflammatory phase after MI and then pro-angiogenic exosomes in the reparative phase of cardiac regeneration and repair. Taken together, difficulty in detecting exosomes in vivo, dynamic secretion and uptake at the tissue level, and lack of an efficient purification route are all challenges for current exosome studies.

      Perspective and conclusion

      Mounting evidence suggests that exosomes derived from MSCs can be a potential therapy to promote cardiac regeneration and repair after MI. The therapeutic role of MSC-derived exosomes is extremely complex. Demonstrating the components in exosomes and their exact interaction with other cells in the infarcted heart may help researchers to understand their therapeutic effects and optimize the effects of exosome-based therapies. Exosome research is still in its infancy although a pre-clinical large animal study is currently ongoing. Studying the therapeutic efficacy of MSC-exosome-based therapy in this porcine model or other pre-clinical large animal model could provide further evidence to support clinical translation of MSC-exosome-based therapy. Moreover, our previous study showed that hiPSC-MSCs demonstrated superior therapeutic efficacy for cardiac repair and regeneration in comparison with BM-MSCs due to their increased survival after transplantation (
      • Lian Q.
      • Zhang Y.
      • Zhang J.
      • Zhang H.K.
      • Wu X.
      • Zhang Y.
      • Lam F.F.
      • Kang S.
      • Xia J.C.
      • Lai W.H.
      • et al.
      Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.
      ). Therefore, it is of great interest to investigate whether hiPSC-MSC-derived exosomes have therapeutic benefits for cardiac repair and regeneration that are superior to those of BM-MSC-derived exosomes. Our previous study also showed that pre-transplantation systemic intravenous administration of hiPSC-MSCs induced immunomodulation and facilitated the survival of intramyocardially transplanted cells to improve cardiac function in MI. Pre-transplantation systemic administration of MSC-derived exosomes may be a promising strategy to improve cell retention of cell-based therapy. In conclusion, studying exosomes provides insight into the exact mechanism of cardiac regeneration and repair, helps optimize cell-based therapies, and promotes the development of precision medicine.

      Author contributions

      S.-J.S.: Literature search, data collection, data analysis, paper writing. R.W.: Data interpretation, figures. F.L.: Literature search. S.-Y.L.: Study design, data analysis, data interpretation, paper revising. H.-F.T.: Study design, paper revising.

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