Exosomes in Myocardial Repair: Advances and Challenges in the Development of Next-Generation Therapeutics

Abstract

Myocardial disease is a leading cause of morbidity and mortality worldwide. Given the limited regenerative capacity of the human heart following myocardial injury, stem cell-based therapies have emerged as a promising approach for improving cardiac repair and function. The discovery of extracellular vesicles including exosomes as a key component of the beneficial function of stem cells has generated hope for their use to advance cell-based regenerative therapies for cardiac repair. Exosomes secreted from stem cells are membranous bionanovesicles, naturally loaded with various proteins, lipids, and nucleic acids. They have been found to have anti-apoptotic, anti-fibrotic, as well as pro-angiogenic effects, all of which are crucial to restore function of the damaged myocardium. In this brief review, we will focus on the latest research and debates on cardiac repair and regenerative potential of exosomes from a variety of sources such as cardiac and non-cardiac stem and progenitor cells, somatic cells, and body fluids. We will also highlight important barriers involved in translating these findings into developing clinically suitable exosome-based therapies.

In this issue of Molecular Therapy, Adamiak and Sahoo summarize recent findings on the properties and role of exosomes as therapeutic tools in the context of cardiovascular disease. The authors also discuss current challenges and opportunities for translation of exosomes research into clinical practice.

Main Text

According to the World Health Organization (WHO), cardiovascular diseases (CVDs) represent a group of disorders associated with the loss of cardiac function and remain a major cause of morbidity and mortality worldwide. In spite of therapeutic intervention advances, 17.7 million people die each year of CVDs, an estimated 31% of all deaths worldwide.¹ Most strikingly, CVDs claim more lives each year than all forms of cancer and chronic lower respiratory disease combined.² Because of unhealthy ways of life (tobacco use, unhealthy diet, physical inactivity, and the harmful use of alcohol) and the phenomenon of population aging, which contribute to increased risk factors, the number of deaths from CVDs is expected to rise in the near future.

Although the events leading to CVDs are miscellaneous, a fundamental problem is the presence of scar tissue and the irreversible loss of cardiac muscle cells, which dynamically contribute to alterations in ventricular contractility and relaxation. Since the first study reported in 1998 indicating the ability of skeletal muscle to repair the heart,³ a spectrum of stem cells has been investigated for treatment of CVDs. The most studied cell types are skeletal myoblasts (SKMs), bone marrow-derived cells (BMCs), induced pluripotent stem cells (iPSCs), endothelial progenitor cells (EPCs), and cardiac progenitor cells (CPCs). Cardiac stem cell (CSC) therapy possesses several major challenges for researchers and clinicians, which include immune rejection after transplantation, poor engraftment and survival of the transplanted cells, occurrence of ventricular arrhythmias, and risk for tumor formation.⁴ Nevertheless, alternative options, such as a cell-free approach, have emerged as potential new therapeutic strategies that mimic the benefit of cell therapy without the need for cell transplantation. In particular, exosome-based therapy has become a new focal point for the treatment of CVDs.

Here, we discuss the utility of exosomes as cell-free therapeutic candidates that can mediate cardiac repair. To provide the reader with the recent progress in the field, we present the most relevant results, as well as current limitations and challenges associated with exosome-based therapy.

Exosomes in the Context of Myocardial Repair

Exosomes: Nanocarriers of Biological Messages

In multicellular organisms, cells can exchange information with neighboring or distant cells by sending out signals composed of single molecules or via membrane vesicles called extracellular vesicles (EVs). EVs are composed of a lipid bilayer containing transmembrane proteins and enclosing cytosolic proteins and various nucleic acids (including mRNAs, microRNAs [miRNAs], and other non-coding RNAs [ncRNAs]).5, 6, 7 Cells can secrete different types of EVs that have been classified according to their subcellular origin. Current research interest in the field of EVs focuses primarily on vesicles of endosomal and plasma membrane origin called exosomes and microvesicles (or ectosomes), respectively.⁸

Exosomes, typically ranging between 30 and 100 nm in size, originate from the inward budding of the membrane of endosomal vesicles called multivesicular bodies (MVBs) and are exported out of the cell after fusion of MVBs with the plasma membrane.8, 9 Owing to their highly regulated biogenesis, exosomes have been proposed to differ from other EVs by their specific membranous and cytosolic composition. Exosomes bear surface molecules that allow them to be specifically targeted to other cells. Once attached to a recipient cell, vesicles can induce signaling via receptor-ligand interaction or can be internalized by endocytosis and/or phagocytosis, or even fuse with the target cell membrane to deliver their content into cytosol, thereby altering the physiological state of the recipient cell.7, 10, 11

EVs are secreted by almost every cell type, including endothelial cells, neuronal cells, smooth muscle cells, tumor cells, and stem cells, and are detected in most body fluids, such as urine, blood, saliva, cerebrospinal fluid, as well as ascites. Recently, cell- and body fluid-derived EVs have gained attention as highly bioactive, acellular carriers in a wide variety of biomedical applications, including heart tissue repair.12, 13 Table 1 summarizes the utility of exosomes in therapy applications for cardiovascular repair, proven in various animal in vivo models.

Table 1.

Exosome Therapy for Cardiovascular Repair in Animal In Vivo Models

Vesicle Source/Donor Cell Phenotype Modification	Species/Type of Injury or In Vivo Assay	Exosomal miRNA/Proteins Involved	Function of Exosomes/Study Highlights	References
Stem Cell-Derived Exosomes
Murine iPSCs	mouse, I/R injury	global miRNA and proteomic profiling performed	improved LV function, reduced CM apoptosis, and enhanced angiogenesis	14
		miR-21 and miR-210	protection of ischemic CMs from apoptosis; no significant change in cardiac function	65
Murine ESCs	mouse, acute MI	miR-294-3p	improved post-MI cardiac function and neovascularization; augmented CM proliferation and survival; promoted CPC survival, proliferation, persistence, and contribution toward repair processes in the heart	15
Human EnMSCs	rat, MI	miR-21, with downstream effects on the target PTEN-Akt survival pathway	enhanced myocardial salvage and microvessel regeneration, improved cardiac function; note: EnMSCs were injected in this study, inhibitor studies indicated that the actions of EnMSCs were mediated by secreted Exo	16
Human UC-MSCs; Akt gene-modified	rat, acute MI	PDGFD	improved cardiac function and promoted blood vessel formation	17
Murine BM-MSCs; cultured under hypoxic or normoxic conditions	mouse, MI	miRNA-210 (in an nSMase2-dependent way)	improved cardiac function, reduced scar size and fibrosis, decreased CM apoptosis, increased vascular density and recruitment of CPCs in the infarcted heart	18
Rat CDCs	rat, chronic MI	no data available	increased global pump function and vessel density, reduced scar mass; note: Exo-primed or -unprimed dermal fibroblasts were injected in this study	22
Human CDCs	mouse, acute and chronic MI	miR-146a	improved cardiac function, imparted structural benefits, and increased viable mass	23
	pig, I/R injury (IC or IM delivery) and CMI (IM delivery)	no data available	decreased infarct size and preserved LV function (I/R injury, only IM delivery); preserved LV volumes and function, decreased scar size, decreased LV collagen content and CM hypertrophy, increased vessel density (CMI, IM delivery)	24
Human (neonate, infant, child) c-kit+ CPCs; cultured under hypoxic or normoxic conditions	rat, I/R injury	computational modeling of miRNAs performed	improved cardiac function (hypoxic and neonate CPC-derived Exo); improved fibrosis and angiogenesis (hypoxic CPC-derived Exo); improved hypertrophy (CPC-derived Exo)	30
Human CD34+ PBMCs	mouse, LI	miR-126-3p	enhanced therapeutic recovery and vascular angiogenesis	32
	mouse, in vivo Matrigel plug assay	miR-126	stimulated angiogenesis	66
Human CD34+ PBMCs; SHH gene-modified	mouse, acute MI	SHH	reduced infarct size and increased border zone capillary density; note: CD34+ cells were injected in this study, in vitro studies indicated that the actions of CD34+ cells were mediated by secreted Exo	67
Body Fluid- or Somatic Cell-Derived Exosomes
Rat CMs; ischemia-mimetic conditions	mouse, acute MI	miR-222 and miR-143	improved neovascularization	36
Rat plasma, RIC-treated rat plasma	Rat, I/R injury	HSP70 (stimulating TLR4 signaling and leading to the activation of ERK1/2, p38MAPK, and subsequent HSP27 phosphorylation in CMs)	decreased infarct size	33
Human pericardial fluid	mouse, LI	let-7b-5p (inhibiting TGFBR1 gene expression in ECs)	improved post-ischemic blood flow recovery and angiogenesis	34
Human pericardial fluid; patients with acute MI	mouse, acute MI	clusterin	stimulated survival and neoangiogenesis, improved cardiac function, promoted the formation of CPCs; note: a shotgun proteomics analysis identified clusterin in Exo isolated from PFMI, clusterin was administered in the pericardial sac of infarcted murine hearts	68

BM-MSC, bone marrow mesenchymal stem cell; CDC, cardiosphere-derived cell; CM, cardiomyocyte; CMI, convalescent myocardial infarction; CPC, cardiac progenitor cell; EC, endothelial cell; EnMSC, endometrium mesenchymal stem cell; ESC, embryonic stem cell; Exo, exosomes; IC, intracoronary; IM, intramyocardial; iPSC, induced pluripotent stem cell; I/R, ischemia/reperfusion; LI, limb ischemia; LV, left ventricle; MI, myocardial infarction; PBMC, peripheral blood mononuclear cell; PFMI, pericardial fluid from patients with acute MI; RIC, remote ischemic pre-conditioning; Shh, sonic hedgehog; UC-MSC, umbilical cord mesenchymal stem cell.

Myocardial Reparative Potential of Stem Cell-Derived Exosomes

Cell types extensively studied for cardiac repair in experimental models and in clinical trials include embryonic stem cells (ESCs), iPSCs, and multipotent/unipotent adult stem cell lineages such as mesenchymal stem cells (MSCs), CSCs, including cardiosphere-derived cells (CDCs), and EPCs (Figure 1).

Figure 1.

A Scheme Depicting the Discussed Biological Sources of Exosomes for Cardiac Repair, along with Different Strategies for Donor Cell Phenotype Modification

AAV, adeno-associated virus; CDC, cardiosphere-derived cell; CSC, cardiac stem cell; EPC, endothelial progenitor cell; ESC, embryonic stem cell; Exo, exosome; iPSC, induced pluripotent stem cell; MSC, mesenchymal stem cell.

Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have the potential for directed differentiation into all types of body cells and subsequent repair of multiple tissues, and therefore have been intensively investigated as a potential cardiac regenerative therapy. In this regard, Adamiak et al.¹⁴ recently demonstrated that EVs derived from murine iPSCs (miPSC-EVs) confer cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. These beneficial effects of miPSC-EVs may be related to their molecular cargo, which includes numerous miRNAs (e.g., miRNAs from the miR-17-92 cluster) and proteins (e.g., bone morphogenetic protein 4 [BMP4], platelet-derived growth factor alpha [PDGFA], teratocarcinoma-derived growth factor 1 [TDGF1], thrombospondin 1 [THBS1], and vascular endothelial growth factor C [VEGFC]) that have been implicated in cytoprotection and angiogenesis.¹⁴ Previously, Khan et al.¹⁵ presented results showing that mouse ESC-derived exosomes are able to promote endogenous repair and preserve cardiac function when injected intramyocardially immediately after left anterior descending ligation in a murine model of infarction. The authors concluded that the beneficial effects observed with ESC-derived exosomes are mediated, at least in part, by the transfer of exosomal miR-294.

Multipotent MSCs

Accumulating clinical and experimental evidence indicates that multipotent MSCs, which are capable of giving rise to cells of multiple cell lineages, are a promising cell type in the treatment of cardiac dysfunction. More recently, MSC-derived exosomes are being extensively examined for their role in MSC-based cellular therapy aimed at rebuilding damaged heart. The results of Wang and colleagues¹⁶ confirmed the superior cardioprotection, including cell survival and angiogenesis, by human endometrium-derived MSCs (EnMSC). This study identified miRNAs contained in EnMSC-derived exosomes, in particular miR-21, as potential mediators of EnMSC therapy. Ma et al.¹⁷ isolated exosomes from Akt-overexpressing human umbilical cord MSCs and showed that they improve cardiac regeneration and promote angiogenesis through PDGFD activation. Furthermore, it was also demonstrated that the ability of exosomes obtained from mouse bone marrow-derived MSCs to repair infarcted myocardium is enhanced when cells are exposed to hypoxia.¹⁸ Cardioprotective actions of exosomes in these experiments were attributed to miR-210 and neutral sphingomyelinase 2 (nSMase2) activities in hypoxia-treated MSCs and their secreted exosomes.¹⁸

Multipotent Cardiac Stem and Progenitor Cells

Because of the clinical success with MSC therapies for cardiac repair, exosomes derived from other adult stem cells have also become subjects of great attention. Specifically, CPC-derived exosomes are gaining significant attention for the clinical treatment of heart disease due to their remarkable ability to induce ischemic tissue repair and regeneration. Given their cardiac developmental origins, these endogenous cells may represent better candidates for cardiac cell therapy compared with stem cells from other sources such as adipose tissue or bone marrow.

Several CPC populations have been reported in the developing and adult heart including CDCs, which can differentiate into three major cell types present in the heart: cardiomyocytes, endothelial cells, and smooth muscle cells.19, 20 CDCs are intrinsic to the heart, express a distinctive profile of antigens (>98% CD105⁺, <0.5% CD45⁺), and trigger functional recovery and structural improvements in various ischemic and nonischemic models of heart failure.²¹ In the previous work by Tseliou and colleagues,²² CDC-derived exosomes were shown to convert inert dermal fibroblasts into therapeutically active cells capable of reducing scar size and boosting cardiac function in a chronic myocardial infarction model. Exosomes have again been identified as key mediators of regenerative and functional effects caused by CDC transplantation into injured mouse hearts, especially by the direct transfer of exosomal miR-146a into recipient cells.²³ Recently, CDC-derived exosomes isolated by filtration and precipitation were injected into the myocardium of pigs following ischemia and reperfusion, which reduced infarct size and improved cardiac function 4 weeks later.²⁴

As one of the very first CPC populations identified in the adult mammalian heart,²⁵ c-Kit⁺ stem cells have already been employed in a phase I clinical trial for the treatment of heart failure.²⁶ Despite clinical evidence that autologous c-Kit⁺ adult CSCs function to improve cardiac performance, their role in the myocardial repair continues to be questioned.27, 28 Debate over cardiomyogenic capability of c-Kit⁺ cells reminds that stem cell-mediated cardiac repair is still a controversial research topic, and strong disagreement exists among scientists over which cell types are the best candidates for therapeutic transplantation. Given the poor performance of transplanted cells in preclinical and clinical studies, we should not underestimate the hypothesis that cell therapy may function through paracrine mechanisms and should thoroughly investigate cell-free approaches, e.g., those involving administration and/or modulation of EVs.

An important factor affecting the potency of CPCs may be the age of the donor. In a study comparing human adult and neonatal c-Kit⁺ CPCs, those from neonates (nCPCs) proliferated more in vitro and led to greater myocardial recovery in rats when injected into the myocardium after permanent coronary artery ligation.²⁹ Interestingly, nCPC-derived exosomes appeared to be responsible for a large proportion of this benefit, although a non-exosomal fraction also delivered some benefit.²⁹ In a rat model of ischemia reperfusion injury, Agarwal et al.³⁰ demonstrated that donor age and oxygen content in the microenvironment significantly alter the efficacy of human c-Kit⁺ CPC-derived exosomes. They found that c-Kit⁺ CPCs derived from children release exosomes that can repair the infarcted heart, although their efficacy decreases sharply with age. Mechanistically, they showed that subjecting cells to hypoxia restored reparative potential of the exosomes and changed their miRNA profile.³⁰

Other Adult Stem Cell Lineages

Another major type of unipotent adult stem cells being investigated for post-injury treatment of cardiac muscle is EPC. Observations from our laboratory demonstrated that adult human CD34⁺ stem cells purified from mobilized peripheral blood mononuclear cells (PBMCs) secrete exosomes (CD34Exo), and that CD34Exo induce angiogenic activity in isolated endothelial cells and in murine models of vessel growth.³¹ Further, CD34Exo were found to be enriched with pro-angiogenic miRNAs such as miR-126-3p, which mediated the angiogenic and therapeutic benefits associated with CD34⁺ stem cell therapy. Another key discovery was the evidence that CD34Exo are most efficiently internalized by endothelial cells in the ischemic tissue to induce cell-cycle induction, angiogenesis, and proliferation.³²

Other Biological Sources of Exosomes for Myocardial Repair

Interestingly, exosomes purified from other biological sources, such as body fluids and somatic cells, have also been suggested as novel therapeutic options for various CVDs (Figure 1). It is important to remember that the major populations of exosomes in a body fluid usually originate from the cells that are present in that particular biofluid and that surround the biofluid.

Body Fluids

Vicencio et al.³³ found in vitro and in vivo evidence that exosomes isolated from rat plasma have the ability to protect the heart from ischemia reperfusion injury. The authors proposed that beneficial effect of vesicles is mediated by HSP70 present on exosomes, stimulating TLR4 signaling and leading to the activation of ERK1/2, p38MAPK, and subsequent HSP27 phosphorylation in cardiomyocytes.³³ In another study, exosomes derived from human pericardial fluid (PF) have shown the capacity to promote therapeutic angiogenesis in vivo in a mouse limb ischemia model, suggesting their importance in the context of cardiovascular protection and repair.³⁴ Data obtained by Beltrami et al.³⁴ support the hypothesis that PF exosomes are highly bioactive and enhance formation of new vessels via transfer of a newly defined proangiogenic miRNA, let-7b-5p, to endothelial cells.³⁵

Somatic Cells

Recently, exosomes secreted by rat cardiomyocytes subjected to ischemic conditions (Exo^isch) have been shown to promote angiogenesis, both in vitro and in vivo.³⁶ More precisely, Exo^isch stimulated the formation of new functional vessels in two distinct in vivo angiogenic assays, in ovo and the Matrigel plug assays. Neovascularization was also improved following the delivery of Exo^isch to the myocardium of mice subjected to myocardial ischemia (MI) at the onset of injury. Furthermore, Ribeiro-Rodrigues et al.³⁶ provided strong evidence that miR-222 and miR-143, the relatively most abundant miRNAs in ischemic exosomes, account for the angiogenic process. Among other cell types present in the heart, it is believed based on current knowledge that the exogenous transplantation of telocytes (TCs) or the delivery of TC-derived exosomes might have great potential as future therapeutic strategies to foster cardiac regeneration and repair.³⁷

However, in addition to cardioprotective and regenerative effects, exosomes may also exert deleterious effects on cardiac remodeling. A recent study by Bang et al.³⁸ showed a high amount of miRNA in cardiac fibroblast-derived exosomes, which are shuttled to cardiomyocytes, leading to cellular hypertrophy by affecting target genes. On the basis of miRNA profiling assays, the authors identified fibroblast-derived miR-21-3p as a paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy and demonstrated a potential therapeutic use of miR-21-3p antagonism in this particular disorder of cardiovascular system.³⁸

Novel Approaches

Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be exceptionally efficient biological tools in basic science research. However, potential blocking of AAVs by neutralizing anti-AAV antibodies (Nab) is a major challenge limiting the efficacy of AAV-mediated gene delivery. Recently it was shown that AAVs can associate with exosomes when the vectors are isolated from conditioned media of producer cells, and AAV-containing exosomes (AAVExo) are more resistant to Nab compared with standard AAVs.³⁹ Hudry et al.³⁹ demonstrated that AAVExo are capable of efficient transduction of CNS cells after systemic injection in mice. A further approach is to use AAVExo as superior agents for delivering genes (e.g., SERCA2a) to cardiomyocytes and to the heart. In fact, first attempts to test the therapeutic effects of AAV9Exo in a mouse model of myocardial infarction were made in our laboratory and showed that AAV9Exo outperformed conventional AAV vectors in preserving cardiac function post-MI (Liang et al.⁴⁰ and unpublished data).

Opportunities and Challenges for Developing EV-Based Therapeutics

Advantages of EVs for Therapeutic Applications

Naturally produced membranous vesicles efficiently targeting recipient cells and delivering their bioactive contents may represent potential candidates for therapeutic applications in different areas of regenerative medicine. Indeed, the use of non-living yet bioactive exosomes in mending a broken heart provides several clear advantages over the use of cells. It is worth emphasizing that exosomes can be selectively and quickly taken up by target cells (e.g., cardiac endothelial cells) opening up a plethora of possibilities in cell- and tissue-specific targeting.³² Apart from the above-mentioned benefits, exosomes are smaller, less complex, and less fragile than their parent cells, and thus easier to modulate, manufacture, and store.⁴¹ Furthermore, due to the nature of exosomes, they present no risk for tumor formation, are less immunogenic, and confer protection against enzymatic and non-enzymatic degradation of their molecular cargo.⁴¹

Limitations of EVs for Therapeutic Applications

However, before exosomes can enter the clinic as medicinal products, significant steps need to be taken to address many open questions in the field. A notable challenge lies in developing scalable and reproducible protocols for exosome purification and storage, as well as in improving techniques and criteria for quality analyses. The appropriate isolation method would deliver sterile exosomes with reproducible purity and potency, and would need to attain compliance with good manufacturing practice (GMP) guidelines.42, 43 It is crucial to define and establish appropriate units for determining the dose (protein amount or particle number) of exosomes, dosage regimen (single/multiple applications), and the route of administration.⁴⁴ Strategies must be also designed to successfully identify, quantify, and characterize the main exosomal compound that is causing the biological effect and define the mode of action through qualified potency assays in disease-relevant in vitro and animal models.⁴³

When developing EV-based therapeutics, we need to be aware that exosomes elicit differential effects on recipient cells depending on the physiological state of the parent cell. Cell secretome, including vesicular component, changes significantly in response to cellular stress and microenvironment modifications (e.g., under hypoxic conditions¹⁸). Beneficial effects of exosomes have been also reported to vary according to the donor’s age or the tissue of origin.30, 45 As an example, EVs isolated from bone marrow and umbilical cord MSCs were shown to decrease tumor cell proliferation, while an opposite effect was observed with adipose tissue MSC-derived EVs.⁴⁵ Interestingly, subpopulations of EVs from a single cell type separated based on their density have at least partly different protein, lipid, and RNA content, and might exert different biological functions on target cells.46, 47, 48, 49, 50 The results obtained by Collino et al.⁴⁷ demonstrated heterogeneity in quantity and composition of subpopulations distributed at different densities. The authors concluded that the population of EVs contained in the medium flotation density fraction showed superior activity in promoting renal protection from injury in vitro.⁴⁷ Thus, enhanced therapeutic efficacy may be potentially accomplished not only by carefully selecting the cells with desired properties to produce EVs, but also by working with purer isolates containing only one specific subpopulation of EVs.⁵¹

Current Attempts to Overcome Challenges Associated with Therapeutic Application of EVs

Isolation of Exosomes

Valuable clinical trial with exosome product will only be possible with the improvement of isolation and purification methods, which still remains a significant challenge in the field. The demands of clinical applications involving therapeutics can be potentially met with recently developed strategies for vesicle isolation, including immunoaffinity-based capture, sieving, trapping with porous structures, and acoustic separation.⁵² With the use of such novel techniques, significant reduction in isolation time, sample volume, as well as reagent consumption could be accomplished. Additionally, potential methods for increasing the overall exosome yield, for example, EV production in response to external stress, are being investigated, opening up new perspectives toward the development of innovative protocols for time-saving, cost-effective, and efficient exosome isolation.⁵³

Storage of Exosomes

Another challenge to application of exosome-based therapeutics that continues to be extensively studied involves exosome preservation and storage. Beyond developing standardized techniques and protocols for long-term storage of exosomes, our understanding of storage-induced changes in vesicle biological function has to be diametrically increased. Evidence to date suggests that the most promising mode of storage for exosomes remains −80°C, but alternatives such as lyophilization and the incorporation of additives (e.g., disaccharide stabilizers) might be potentially applied to improve vesicle storage stability.⁵⁴ In the patent owned by Capricor Therapeutics, Kreke et al.⁵⁵ has already demonstrated that lyophilization and rehydration of EVs isolated from conditioned medium did not cause any substantial reduction in bioactivity or miRNA content, and represent a promising approach for producing EV formulations for future therapeutic use. It is beyond doubt that storage conditions alter functional properties of exosomes. However, studies conducted so far provided inconsistent results, and additional analyses are needed given that effects of storage appear to vary with sample source and processing, as well as exosome isolation method.⁵⁴

Quality Analyses of Exosomes

The International Society for Extracellular Vesicles (ISEV) and EV-TRACK Consortium have attempted to address another major challenge to the translational applications in exosome research, that is, the lack of published data reliability caused by non-standardized isolation processes and insufficient characterization. Implementation of EV-TRACK⁵⁶ and minimal information for studies of EVs (MISEV) guidelines,57, 58 as well as adoption of the criteria included in so-called ISEV position papers,44, 59, 60 should aid researchers in planning studies, and reporting and comparing their results. In addition, a more rigorous characterization of exosomes, using a combination of methods (e.g., flow cytometry, electron microscopy, atomic force microscopy, as well as RNA, protein, and lipid profiling), is urgently needed to better explore vesicle biology and effectively move the field in a clinically meaningful direction.⁶¹

In Vivo Analyses of Exosomes

Appropriate preclinical in vivo models will help answer questions regarding exosome dosage regimen, route of administration, biodistribution, and half-life times, as well as determine exosome toxicity, immunogenicity, immunotoxicity, or tumorigenicity.⁴⁴ In particular, in vivo imaging of exosomes is crucial for application of vesicle-based therapeutics in various diseases, including myocardial infarction. It is important to realize that visualizing the kinetics of exosomes in the body and quantifying the amount of exosomes delivered to target damaged tissue could potentially reduce unnecessary effort and expenses in clinical trials.⁶² Gangadaran et al.,⁶² in their recent review, summarized strategies of in vivo tracking of EVs (both direct and indirect EV-labeling strategies for bioluminescent, fluorescent, nuclear, and magnetic resonance imaging) and indicated nuclear imaging using direct labeling (radioiodine, technetium-99 m [^99mTc], and other radionuclides) as an exceptionally safe and useful option for translational and clinical applications. Indeed, recently Varga et al.⁶³ demonstrated the capability of the radioisotope labeling of erythrocyte-derived EVs using the ^99mTc-tricarbonyl complex for in vivo biodistribution studies. In this study, the authors found that the intravenously administered ^99mTc-labeled EVs mostly accumulated in the liver and spleen of mice. Smyth et al.⁶⁴ labeled tumor-derived exosomes using ¹¹¹indium-oxine and similarly demonstrated that they are rapidly taken up by the reticuloendothelial system in the liver and spleen when injected intravenously in a mouse model. The authors concluded that the rapid clearance of exosomes inhibits their accumulation in tumor tissue to any significant level, limiting their use as a drug delivery system when injected intravenously.⁶⁴

Academic-Industry Partnership

Furthermore, collaboration between academia and biotech companies is now being considered as an essential part on the road to the exosome-based translational nanomedicine. Through such partnership, industrial partners gain access to ideas and innovation, while academic partners can benefit from know-how required for successful technology transfer for industrial-scale GMP manufacturing.⁴³

Conclusions

Given that exosomes derived from various cellular sources and bodily fluids exert cardioprotective and regenerative effects both in vitro and in vivo, these nanoparticles are definitely worthy of further investigation in our quest for ways to protect the myocardium at risk for severe injury. Although translation to clinical practice would require validation of the safety and efficacy of exosomes, studies conducted so far rationalize a paradigm shift from conventional cell-based therapies to a safer, cheaper, more accessible and potent, cell-free, and off-the-shelf therapy. Such a novel form of a therapeutic intervention, whereby cells could be exclusively used in vitro for the production of EVs, which would then be the only medicinal product given to the patient, would probably aid in overcoming many hurdles associated with conventional cell transplantation (Figure 2).

Figure 2.

Flowchart of GMP-Compliant Production of Exosomes for Clinical Application for Heart Repair

GMP, good manufacturing practice; HPLC, high-performance liquid chromatography; QC, quality control; TFF, tangential flow filtration.

Conflicts of Interest

The authors have no conflict of interest.