Immunomodulatory effects and clinical application of exosomes derived from mesenchymal stem cells

Abstract

Exosomes (Exos) are extracellular vesicles secreted by cells and serve as crucial mediators of intercellular communication. They play a pivotal role in the pathogenesis and progression of various diseases and offer promising avenues for therapeutic interventions. Exos derived from mesenchymal stem cells (MSCs) have significant immunomodulatory properties. They effectively regulate immune responses by modulating both innate and adaptive immunity. These Exos can inhibit excessive inflammatory responses and promote tissue repair. Moreover, they participate in antigen presentation, which is essential for activating immune responses. The cargo of these Exos, including ligands, proteins, and microRNAs, can suppress T cell activity or enhance the population of immunosuppressive cells to dampen the immune response. By inhibiting lymphocyte proliferation, acting on macrophages, and increasing the population of regulatory T cells, these Exos contribute to maintaining immune and metabolic homeostasis. Furthermore, they can activate immune-related signaling pathways or serve as vehicles to deliver microRNAs and other bioactive substances to target tumor cells, which holds potential for immunotherapy applications. Given the immense therapeutic potential of MSC-derived Exos, this review comprehensively explores their mechanisms of immune regulation and therapeutic applications in areas such as infection control, tumor suppression, and autoimmune disease management. This article aims to provide valuable insights into the mechanisms behind the actions of MSC-derived Exos, offering theoretical references for their future clinical utilization as cell-free drug preparations.

Key Words: Mesenchymal stem cells; Exosomes; Immunomodulatory effects; Clinical application; Therapeutic potential

Core Tip: Exosomes (Exos) are extracellular vesicles secreted by cells, and they serve as crucial mediators of intercellular communication, playing a pivotal role in the pathogenesis, progression, and therapeutic interventions for various diseases. Given the immense therapeutic potential of Exos derived from mesenchymal stem cells (MSC-Exos), this review article comprehensively explores mechanisms underlying their immune regulation as well as their therapeutic applications in infection control measures and tackling tumors or autoimmune diseases among others. This article aims to provide valuable insights into further investigations regarding the mechanism behind MSC-Exo actions while offering theoretical references for future clinical utilization of MSC-Exos as cell-free drug preparations.

INTRODUCTION

Exosomes (Exos) were first discovered in 1983 as 50-nm vesicles released by reticulocytes carrying transferrin receptors extracellularly[1]. Since then, the understanding of the mechanisms and functions of Exos has exponentially expanded[2]. Exos are extracellular vesicles (EVs) with a diameter ranging from 40 to 160 nm (mean, ~100 nm) that originate from endosomes[3]. Depending on the specific cell type that they derive from, Exos contain various components such as DNA, RNA, lipids, metabolites, cytoplasmic contents, and cell-surface proteins[4]. The exact physiological role of Exo production by cells remains elusive and requires further investigation[5]. Due to their functional and targeted nature as cellular constituents, Exos play a crucial role in intercellular communication[6]. Mesenchymal stem cells (MSCs) are a class of adult stem cells first identified by Friedenstein in mouse bone marrow and characterized by their multilineage differentiation potential. Caplan subsequently coined the term “mesenchymal stem cells”[7], although their definitive stem cell properties have yet to be rigorously demonstrated in vivo[8]. MSCs have been shown to originate from perivascular and pericytic progenitors in almost all tissues[9]. These cells possess trilineage differentiation potential, enabling them to differentiate into osteoblasts, adipocytes, and chondrocytes. They express positive cell surface markers CD90, CD105, and CD73, while lacking CD45, CD34, CD14, CD79a, and HLA-DR[10]. Under specific conditions, MSCs can also differentiate into other cell types, such as neurons and cardiomyocytes[11].

MSCs possess a certain self-renewal capacity and can be propagated through multiple generations in vitro while maintaining their phenotype and differentiation potential[12]. They secrete a variety of cytokines and growth factors, which inhibit the proliferation of B cells and T cells, suppress monocyte maturation, and promote the generation of regulatory T cells (Tregs) and M2 macrophages[13]. These characteristics endow MSCs with immunomodulatory functions, forming the foundation for their application in treating various immune-related diseases. The primary clinical value of MSCs appears to stem from secreted EVs (including Exos) and a range of cytokines and growth factors[14]. The immunoregulatory effects of these secreted Exos have demonstrated therapeutic potential in numerous clinical studies. These studies encompass conditions such as myocardial infarction, stroke, graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn’s disease, acute lung injury, chronic obstructive pulmonary disease, liver cirrhosis, multiple sclerosis (MS), amyotrophic lateral sclerosis, and diabetes[15].

A substantial body of evidence suggests that MSCs exert their immunomodulatory functions primarily through paracrine pathways, particularly via Exos[16]. Exos derived from MSCs (MSC-Exos) exhibit comparable biological activities to MSCs and possess several advantageous characteristics such as rapid passage through capillaries, inherent stability, and robust information transfer capacity compared to MSCs[17]. Furthermore, the utilization of Exos can circumvent issues associated with ectopic osteogenesis, tumor formation, pulmonary capillary blockade, and immune rejection commonly encountered in cell therapy. The low immunogenicity and high stability of Exos make them a promising alternative strategy for cell therapy[18]. Notably, Exos play pivotal roles in inflammation, tumors, and autoimmune diseases, as well as graft rejection[19]. MSC-Exos have achieved several significant breakthroughs in recent years. Research has demonstrated their ability to cross the blood-brain barrier, exert neuroprotective effects, and facilitate nerve regeneration. This approach offers novel insights and potential strategies for the treatment of brain injuries, stroke, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. MSC-Exos play a crucial role in immune regulation by modulating immune responses and mitigating inflammation, which holds potential for treating autoimmune diseases such as RA and SLE[20,21]. Additionally, MSC-Exos are being explored for cancer treatment, including metastatic cancers. Studies have demonstrated their utility as drug delivery vectors to enhance the efficacy of anticancer drugs while minimizing side effects. In cardiovascular disease and skin wound repair, MSC-Exos have exhibited the capacity to promote tissue repair and regeneration, supporting myocardial cell survival and functional recovery, as well as accelerating skin wound healing[22]. Recent technological advancements have improved the production efficiency and functional customization of MSC-Exos. For instance, genetic engineering and optimized culture conditions have enhanced specific therapeutic properties of Exos[23]. These innovations highlight the significant potential of MSC-Exos as a novel biologic therapeutic tool. Several clinical studies on Exos have been approved by the Food and Drug Administration (FDA), including investigations into the molecular mechanisms of Exos in melanoma pathogenesis (FDA lot number: NCT02310451), the clinical correlation of glioma exosomal molecular abnormalities (FDA batch No.: NCT06116903), and the effect of Exo administration in preventing early leakage in patients with low anterior resection rectal cancer (FDA Lot No.: NCT06536712). As research progresses, the application prospects for MSC-Exos will continue to broaden[24]. Therefore, this article comprehensively reviews the mechanisms underlying immune regulation by MSC-Exos along with their therapeutic applications in infection control, tumor management, and autoimmune diseases. This article serves as a valuable reference for further investigations into the mechanisms governing MSC-Exo function while providing theoretical support for future clinical implementation of MSC-Exos as cell-free drug preparations.

BIOLOGICAL CHARACTERISTICS OF EXOS DERIVED FROM MSCS

MSCs can be isolated from various tissues, and Exos derived from different tissues and cell types may carry distinct biomolecules that confer unique immune properties and potential therapeutic applications. MSC-Exos exhibit significant immunomodulatory effects, capable of downregulating inflammatory responses and promoting immune tolerance. They are widely utilized in the treatment of inflammatory diseases and autoimmune conditions such as SLE and RA, as well as in promoting tissue repair and regeneration[25]. Exos derived from tumor cells are frequently utilized as biomarkers and therapeutic targets for developing novel cancer immunotherapies and diagnostic approaches[26]. Exos secreted by cardiomyocytes can modulate cardiac inflammatory responses, enhance cardiomyocyte survival, and facilitate cardiac tissue regeneration. In studies of myocardial infarction and other cardiovascular diseases, these Exos have demonstrated potential therapeutic benefits[22]. Exos from adipose-derived stem cells are employed to treat conditions such as impaired wound healing and skin disorders, promoting the regeneration process. Exos derived from neural stem cells exhibit neuroprotective and anti-inflammatory properties, provide neurotrophic support, and regulate immune responses within the nervous system, showing promise in treating neurodegenerative diseases like Alzheimer’s and Parkinson’s[27,28]. These cells can be derived from diverse tissues depending on their origin. Exos are small vesicles released into the extracellular space following fusion between intracellular multivesicular bodies and the cellular membrane[29]. This secretory process involves recognition and trafficking of specific proteins and lipids[30], such as the ESCRT system (endosomal sorting complex)[31]. Exos typically range in size from 40 to 160 nm[32] with a lipid bilayer membrane structure that provides stability and protection[33], conferring them potential therapeutic utility due to their stable structure and precise targeting ability[34,35]. Exos transport distinct proteins and RNA molecules that mirror the cellular origin and exert an impact on the functionality of recipient cells[36]. They assume pivotal roles in intercellular communication by transferring informational molecules to regulate immune response, inflammation, and tissue repair, among other processes[37]. Consequently, owing to their inherent structural stability and precise targeting capabilities, Exos are regarded as promising therapeutic tools[38] (Figure 1).

Figure 1 Exocytosis of exosomes derived from mesenchymal stem cells. The diagram illustrates the process of exosome biogenesis: From early endosomes to multivesicular bodies, and finally the secretion of exosomes containing various components such as nucleic acids, proteins, lipids, cell adhesion molecules, and transmembrane proteins. MSC: Mesenchymal stem cells.

MSC-Exos not only express protein markers commonly found in all Exos[39], but also exhibit specific membrane surface molecules on MSCs[40]. These Exos contain bioactive molecules such as microRNAs (miRNAs), mRNA, and proteins that play a crucial role in regulating gene expression and function in target cells. Lai et al[41] first investigated the role of MSC-Exos in a mouse model of myocardial ischemia-reperfusion injury in 2010, followed by studies conducted across various disease models[42]. MSC-Exos have been shown to facilitate tissue regeneration through intercellular communication, particularly following kidney, liver, cardiovascular, and nervous system injuries[43]. By secreting Exos, MSCs modulate immune responses by reducing inflammatory factors while increasing anti-inflammatory factor levels[44]. These Exos carry miRNAs and proteins that regulate apoptotic pathways to enhance the survival of target cells and mitigate oxidative stress damage[45]. In heart disease models specifically, MSC-Exos improve cardiac function and morphology by augmenting survival signaling pathways while suppressing inflammatory responses[46]. Therefore, the clinical potential for utilizing MSC-Exos as a cell replacement therapy is extensive. However, MSC-Exos encounter several practical challenges during the isolation process. For instance, Exo production can be influenced by multiple factors such as the diversity of cell sources, variations in culture conditions, and differences in cell states. This variability introduces uncertainty in Exo yield for each isolation procedure, thereby impacting its consistent supply[47]. Additionally, efficiently extracting high-purity Exos from other cellular components remains a significant challenge. Existing separation techniques may introduce impurities, compromising the efficacy and safety of research outcomes and clinical applications. Scaling up from laboratory to clinical use presents numerous difficulties. To address these issues, it is essential not only to optimize the production process to enhance yield and purity but also to ensure stringent quality control and standardization to meet rigorous clinical application requirements. Overcoming these challenges may involve optimizing culture conditions, refining separation techniques, and developing innovative processes to facilitate the widespread and effective utilization of MSC-Exos in practical applications.

ROLE OF MSC-EXOS IN IMMUNE REGULATION

MSC-Exos play a pivotal role in immunomodulation, which has been associated with their capacity to influence various immune cell functions[48]. MSC-Exos possess the ability to regulate both innate and adaptive immune responses. In terms of innate immunity, they modulate the polarization and cytokine secretion of macrophages and dendritic cells (DCs) through interactions with these cells, thereby suppressing inflammatory responses[49]. Specifically, MSC-Exos can attenuate inflammatory damage by polarizing macrophages towards an anti-inflammatory M2 phenotype rather than a pro-inflammatory M1 phenotype[50]. Concerning adaptive immunity, MSC-Exos achieve immunosuppression by regulating the activity of T cells and B cells. They inhibit the proliferation and cytotoxic functions of T cells while promoting the generation of Tregs, which are crucial for maintaining immune tolerance and preventing autoimmune diseases[51]. Through these mechanisms, MSC-Exos are capable of maintaining immune tolerance and preventing or treating autoimmune diseases. This capability is especially valuable in scenarios where excessive immune responses must be modulated in disease states such as RA and other autoimmune conditions[52]. MSC-Exos also have the ability to modulate humoral immune responses by influencing the antibody-producing function of B cells[53]. The miRNAs, proteins, and other bioactive molecules encapsulated within MSC-Exos can transmit signals between cells via various pathways including direct targeting of specific mRNAs to regulate gene expression or activation/inhibition of immune signaling pathways[54]. MiRNAs are a class of short non-coding RNA molecules that regulate gene expression. MiRNAs encapsulated in Exos can modulate protein expression levels by targeting specific mRNAs, leading to their inhibition or degradation[55]. Specific miRNAs can influence both pro-inflammatory and anti-inflammatory pathways. For instance, certain miRNAs can downregulate the expression of pro-inflammatory factors in M1-type macrophages, thereby promoting their transition to the M2-type (anti-inflammatory) phenotype[56]. Notably, miRNAs such as miR-223 and miR-146a alter the functional state of macrophages by affecting key signaling nodes like nuclear factor-kappaB (NF-κB), signal transducer and activator of transcription (STAT), and phosphatidylinositol 3-kinase/protein kinase B. NF-κB plays a crucial role in the development, differentiation, and responsiveness of immune cells. Exos can modulate the function of immune cells, including T cells, B cells, and macrophages, by regulating the NF-κB pathway. Exos secreted by tumor cells can promote tumor growth and immune escape through the activation of the NF-κB pathway, whereas Exos derived from stem cells may inhibit this pathway and enhance anti-tumor immunity[57]. By modulating the NF-κB signaling pathway, Exos regulate inflammatory responses. For instance, MSC-Exos can exert anti-inflammatory effects by inhibiting NF-κB activity and reducing the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β[58]. Cytokines within Exos directly influence immune cell behavior; for example, anti-inflammatory cytokines like IL-10 or transforming growth factor-beta (TGF-β) delivered via Exos can modulate immune responses and promote immune tolerance. Additionally, membrane proteins and signaling molecules in Exos can bind to receptors on target cells, initiate intracellular signaling cascades, and regulate cellular behavior. Specific proteins within Exos can directly modulate the immune response, such as by inhibiting immune cell activation or inducing immune cell apoptosis to achieve immune regulatory effects[59]. As natural vectors for signal transduction, MSC-Exos possess significant regulatory capabilities in controlling immune system homeostasis by inhibiting excessive inflammatory responses while promoting tissue repair.

MSC-Exos regulate immunity by delivering immunoactive substances

In recent years, it has been discovered that MSCs play a crucial role in intercellular communication through the secretion of Exos[45]. These Exos possess the ability to modulate immune cell function and response. Through packaging and transportation of signaling molecules, a diverse range of bioactive compounds can be encapsulated within Exos, ensuring protection against degradation while enabling specific delivery to target cells[60]. Furthermore, Exo surfaces are adorned with various membrane proteins involved in target cell recognition and binding[61]. Upon fusion with the target cell membrane, the contents of Exos are released into the cytoplasm, thereby activating relevant signaling pathways[62]. MSC-Exos exhibit an abundance of miRNAs that specifically target and regulate immune-related gene expression[63]. Certain miRNAs have demonstrated their capability to shift macrophage polarization from proinflammatory M1 phenotype towards an anti-inflammatory M2 phenotype by regulating Toll-like receptor and NF-κB signaling pathways, ultimately leading to inflammation reduction[64].

Proteins present in Exos, such as cytokines and signaling proteins, have a direct impact on the functionality of target cells. Exos can deliver anti-inflammatory cytokines to inhibit the proliferation of T cells and B cells or facilitate the generation of Tregs by delivering immunomodulatory proteins. Moreover, they can transport specific signaling molecules that activate or suppress signaling pathways in target cells, thereby influencing the cellular immune response[65]. This mechanism plays a crucial role in regulating immune system homeostasis and preventing potential damage caused by excessive immune activity[66]. The utilization of MSC-Exos offers novel insights and potential applications for treating various immune-related disorders through efficient delivery of diverse immunoactive substances while modulating immune system responses (Figure 1).

MSC-Exos achieve immunosuppression through different mechanisms

Immunosuppression refers to the process of suppressing or reducing immune system activity through various mechanisms. MSC-Exos modulate cytokines, such as IL-10 and prostaglandin E2, thereby regulating macrophage polarization from a pro-inflammatory M1 to an anti-inflammatory M2 type, which reduces inflammation and promotes tissue repair[67]. The role of MSC-Exos in immune regulation is primarily towards immunosuppression rather than activation[68]. These Exos contain a variety of molecules that can regulate T cell function, including specific miRNAs and proteins that downregulate T-cell receptor signaling, and inhibit T-cell proliferation and activation, thus reducing the intensity of the immune response[69].

Exos contain specific components that can impede the maturation and antigen presentation capacity of DCs, thereby diminishing their potential to activate T cells. Consequently, this effect mitigates the hyperactive response of the immune system towards tissues or grafts[70]. Additionally, MSC-Exos exhibit inhibitory effects on B cell proliferation, differentiation, and antibody production, thus contributing to the amelioration of pathological immune responses. Furthermore, MSC-Exos possess the ability to attenuate inflammation and tissue damage by suppressing the cytotoxic activity of natural killer cells and restraining aggressive behavior within their own tissues[71]. Although Exos can effectively suppress unwanted immune responses and aid in the treatment of autoimmune diseases, prolonged immunosuppression may render the body more susceptible to infections and certain diseases[72]. This is because some protective functions of the immune system may also be compromised. Excessive immunosuppression could negatively impact the body’s immune surveillance function, thereby increasing the risk of cancer or other abnormal cell proliferation. In cases of immune suppression, this function may be weakened. Additionally, different tissues may respond variably to Exo therapy. Improper immune regulation may result in incomplete or aberrant tissue repair. Long-term treatment might lead to a decrease in Exo adaptability and efficacy. In addition, there are dosing and delivery challenges: Determining the appropriate dose to avoid excessive suppression of immune system function remains a challenge. Efficiently delivering Exos to specific target cells is also technically challenging. Therefore, when developing Exo-based therapies, thorough research and clinical trials are essential to evaluate these potential risks and ensure their safety and efficacy for long-term applications. Despite these challenges, Exos remain promising candidates for treating tumor-related diseases and immune disorders such as autoimmune diseases, transplant rejection, and inflammatory diseases (Table 1).

Table 1 Applications of exosomes from different sources in various diseases.

Source cell	Disease	Effect	Year	Ref.
Bone marrow mesenchymal stromal cells	Acute kidney injury	Promote renal tubular epithelial cell regeneration	2017	[42]
Mesenchymal stem cells	Ischemic myocardium	Cardioprotection	2015	[43]
Bone marrow mesenchymal stromal/stem cell	Acute graft-versus-host disease	Prolonged the survival of acute graft-versus-host disease mice and reduced pathological damage in multiple graft-versus-host disease target organs	2018	[52]
Tumor	Colorectal cancer	Promote liver metastasis	2020	[53]
Mesenchymal stromal cells	Myocardial ischaemia-reperfusion	Attenuate myocardial ischaemia-reperfusion injury	2019	[54]
Bone marrow mesenchymal stem cells	Acute lung injury	Ameliorate LPS-induced acute lung injury	2024	[56]
M1 macrophages	Tumor	Enhance antitumor immunity to inhibit tumor growth	2021	[57]
Mesenchymal stem cells	Multiple sclerosis	Reduce demyelination, decrease neuroinflammation, and increase the number of regulatory T cells in the spinal cord of EAE mice	2019	[63]
Mesenchymal stem cells	Acquired aplastic anemia	Play a key role in immune dysregulation	2023	[65]
Hypoxic mesenchymal stem cells	Bone fracture	Promote bone fracture healing	2020	[66]
Mesenchymal stem cells	Osteochondral defects	Mediate cartilage repair	2018	[67]
Mesenchymal stem cells	Temporomandibular joint osteoarthritis	Alleviate temporomandibular joint osteoarthritis	2019	[69]
Bone marrow mesenchymal stem cells	Ulcerative colitis	Alleviate ulcerative colitis	2019	[74]
Mesenchymal stem cells	Ischemia/reperfusion injury	Alleviate ischemia/reperfusion injury	2019	[78]
Mesenchymal stromal cells	Pulmonary fibrosis	Alleviated the core features of pulmonary fibrosis and lung inflammation	2019	[79]
Umbilical cord mesenchymal stem cells	Nerve injury-induced pain	Possess anti-inflammatory and neurotrophic abilities	2019	[80]
Mesenchymal stem cells	Intrauterine adhesion	Modify intrauterine adhesion	2022	[81]
Bone marrow-derived mesenchymal stem cells	Prostate cancer	Restrained prostate cancer	2022	[89]
Wharton jelly-derived mesenchymal stem cells	Cervical cancer	As drug delivery systems for cervical cancer	2022	[90]
Olfactory ecto-mesenchymal stem cells	Murine Sjögren’s syndrome	Ameliorate murine Sjögren’s syndrome	2021	[96]
Mesenchymal stem cells	Autoimmune uveoretinitis	Inhibit activation of antigen-presenting cells and suppress development of T helper 1 and 17 cells	2017	[97]
Mesenchymal stem cells	Islet transplantation	Improve islet transplantation	2016	[103]
Dendritic cells	Liver transplantation	Negatively regulate CD8+ T cells via inhibition of NLRP3	2022	[104]
Endothelial cells	ARDS	Modulate the therapeutic efficacy of mesenchymal stem cells through IDH2/TET pathway in ARDS	2024	[107]

LPS: Lipopolysaccharide; NLRP3: Nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3; ARDS: Acute respiratory distress syndrome; IDH2: Isocitrate dehydrogenase 2; TET: Ten-eleven translocation.

ROLE OF MSC-EXOS IN DISEASES

Inflammatory diseases

MSC-Exos have the ability to mitigate inflammatory responses by modulating immune reactions in inflammatory diseases. They facilitate the transportation of anti-inflammatory molecules and inhibit the release of pro-inflammatory cytokines, thereby attenuating excessive immune responses triggered by bacterial or viral infections. These Exos express multiple adhesion molecules (CD29, CD44, and CD73), enabling them to home in on injured and inflamed tissues[15]. Several studies have indicated that macrophages are the primary cellular targets of MSC-Exos for reducing colonic inflammation. The activation of the NF-κB signaling pathway in colonic macrophages induced by damage-associated molecular patterns released from damaged epithelial cells leads to increased expression of inducible nitric oxide synthase, as well as elevated levels of inflammatory factors such as TNF-α, IL-1β, nitric oxide, and chemokines involved in lymphocyte and monocyte recruitment (CCL-17 and CCL-24)[55]. Macrophages are considered pivotal cells responsible for initiating colonic inflammation[73]. Cao et al’s study demonstrated that MSC-derived EVs significantly ameliorated dextran sulfate sodium-induced colitis in mice by inducing polarization towards immunosuppressive M2 phenotype in colonic macrophages[74]. The therapeutic effect exerted by these EVs on ulcerative colitis repair seems to be associated with JAK1/STAT1/STAT6 signaling[74]. Yang et al’s findings suggested that modulation of antioxidant/oxidative balance within the affected gut is accountable for MSC-EV-induced phenotypic and functional effects on macrophages[75]. Additionally, MSC-EV treatment resulted in a reduction in the cleavage of caspases-3, -8, and -9 as well as attenuated release of damage-associated molecular patterns from damaged intestinal epithelial cells. This led to the attenuation of NF-κB signaling pathway activation in colonic macrophages and subsequently promoted the generation of an immunosuppressive M2 phenotype[75].

MSC-Exos exhibit a protective effect on hepatocytes in cases of acute liver injury and fibrosis. In hepatitis, MSC-Exos inhibit natural killer T cells, CD4⁺ T lymphocytes, and hepatic stellate cells, thereby attenuating acute liver failure and fibrosis[76]. Furthermore, they safeguard stem cells by suppressing pyroptosis, inhibiting hepatocyte death, and reducing IL-1β- and IL-18-mediated inflammation[77]. Accumulating evidence suggests that MSC-Exos can shield lung epithelial cells from reactive oxidants and proteolytic enzymes released by infiltrating neutrophils and monocytes[78]. Mansouri et al[79] demonstrated that a single intravenous injection of Exos derived from human bone marrow-derived MSC (BM-MSC) significantly mitigated bleomycin-induced pulmonary fibrosis in mice through modulation of the phenotype and function of alveolar macrophages. Additionally, MSC-Exos alleviate inflammation at the site of nerve injury by inhibiting microglial production of inflammatory factors (TNF-α and IL-1β) while promoting synthesis of anti-inflammatory factors (IL-10 and TGF-β)[80].

Tumor immunity and cancer therapy

The role of MSC-Exos in tumor immunity is dual, primarily determined by the bioactive molecular composition of the Exos and their interaction with the tumor microenvironment[81]. They can deliver immunosuppressive molecules that facilitate immune evasion by tumors, while also containing specific miRNAs and proteins that potentially exert immunostimulatory effects[82], enhancing immune recognition and cytotoxicity against tumor cells. MSC-Exos possess the ability to modulate the immune balance within the tumor microenvironment and shape its characteristics[83]. Consequently, their impact on tumor immunity is intricate and multifaceted, potentially providing both anti-tumor support or promoting tumorigenesis[84]. Biswas et al[85] demonstrated in vivo that MSC-Exos upregulate programmed death-ligand 1 expression in bone marrow cells while downregulating programmed cell death-1 expression in T cells, thereby suppressing protective antitumor immunity specifically in breast cancer models. Furthermore, MSC-Exos have been shown to suppress proinflammatory responses and oxidative stress mediated by immune system cells as well as humoral factors under both in vitro and in vivo conditions, creating a conducive environment for tissue regeneration[86].

Exos derived from human adipose-derived MSCs have been shown to exert inhibitory effects on ovarian cancer cells by inducing cell cycle arrest and activating mitochondrial-mediated apoptotic signaling pathways[87]. Exo miR-187 derived from human BM-MSCs was found to suppress malignant characteristics in prostate cancer cells[88] by targeting CD276, thereby inhibiting the JAK3-STAT3-Slug pathway in PCa[89]. Exos derived from human umbilical cord MSCs were utilized for paclitaxel loading and their effect on cervical cancer cell line (HeLa) was evaluated. These Exos accelerated cancer cell death through modulation of Bax, BCL2, cleaved Caspase-3, and cleaved Caspase-9 levels, while reducing chemotherapy resistance via regulation of epithelial-mesenchymal transition-related proteins such as TGF-β and catenin-β[90]. MSC-Exos have emerged as a valuable tool for cancer suppression, exhibiting the potential to inhibit hepatocellular carcinoma progression through blockade of the C5orf66-AS1/miR-127-3p/DUSP1/ERK axis[91].

Autoimmune diseases

It has been observed that MSC-Exos exhibit significant inhibitory effects on various effector cells involved in both innate and adaptive immune responses. Remarkable progress has been achieved in the treatment of autoimmune disorders, such as MS, SLE, type 1 diabetes mellitus, uveitis, and RA[92]. MSC-Exos demonstrate the ability to replicate MSC functionality while surpassing the limitations associated with conventional cell therapies.

MS is the most prevalent inflammatory disease of the central nervous system (CNS). Microglia, as the primary immune cells in the CNS, play a crucial role in maintaining tissue homeostasis under normal physiological conditions[93]. Glial cells contribute to both neurodestructive and neuroprotective functions[94]. An imbalance between M1/M2 phenotypes inhibiting neuroprotective functions can promote MS development. Isik et al[95] demonstrated that treatment with BM-MSC-Exos suppressed microglial polarization towards the M1 phenotype while promoting polarization towards the M2 phenotype, leading to secretion of anti-inflammatory cytokines such as IL-10 and TGF-β. Notably, BM-MSC-Exos treatment significantly ameliorated neurobehavioral symptoms of experimental autoimmune encephalomyelitis and alleviated inflammation and demyelination within the CNS.

Uveitis, a leading cause of global visual impairment, is believed to be primarily driven by autoimmunity. Numerous studies have demonstrated the beneficial effects of MSC-Exos on inflammatory ocular diseases. In autoimmune uveitis mice, retinal photoreceptors exhibited severe damage accompanied by infiltration of inflammatory cells; however, when MSC-Exos were administered via tail vein injection in autoimmune uveitis mice, their retinas resembled those of normal mice with minimal structural damage and inflammatory infiltration[96,97]. Notably, T helper 1 (Th1) and Th17 cells play crucial roles in the pathogenesis of autoimmune uveitis. Treatment with MSC-Exos resulted in significantly reduced numbers of interferon-gamma+CD4⁺ cells (Th1) and IL-17+CD4⁺ cells (Th17) compared to phosphate buffered saline-treated mice[98]. These findings suggest that MSC-Exos possess the ability to suppress the development of autoimmune uveitis through inhibition of Th1 and Th17 cell responses.

RA is a chronic inflammatory disease characterized by synovial hyperplasia and immune-cell infiltration, leading to joint destruction[99]. Studies have found that exosomal miRNAs also play an important role in alleviating the development of RA. MSC-Exos expressing miRNA-150-5p reduced the migration and invasion of RA fibroblast-like synoviocytes and downregulated human umbilical vein endothelial cell tube formation by targeting matrix metalloproteinase 14 and vascular endothelial growth factor[100]. MSC-EV provides new insights into the treatment of RA and may provide new opportunities and strategies for the treatment of this autoimmune disease. SLE is a chronic autoimmune disease characterized by immune inflammation and multiple organ damage. The pathogenesis of SLE is extremely complex. With the help of follicular helper T (Tfh) cells, antinuclear antibodies are produced, leading to the deposition of immune complexes in important organs. It has been found that the infusion of human BM-MSCs into a mouse model of lupus nephritis improved the survival of mice and alleviated the clinical symptoms of glomerulonephritis by inhibiting the development of Tfh cells and reducing the levels of autoantibodies[87].

Organ transplantation

Immune rejection is a crucial factor limiting the prognosis of organ transplantation and represents an urgent problem that needs to be addressed. Previous studies have demonstrated that the inflammatory environment can influence the characteristics and expression of biomolecules, such as proteins and nucleic acids, within Exos. Conversely, stem cells can effectively modulate inflammatory responses by transferring genetic information, including miRNA, via Exos, thereby playing an immunomodulatory role in tissue repair processes. Exosomal miRNAs serve as key regulators of islet cell function encompassing insulin expression, processing, and secretion. These exosomal miRNAs act as valuable biomarkers for assessing islet cell function and survival with significant implications for the outcome of islet transplantation[101]. Furthermore, they may be closely associated with vascular remodeling and immune regulation following islet transplantation. Notably, miR-21-5p derived from BM-MSC-Exos has been found to prevent islet apoptosis by suppressing PDCD4 expression, thus offering potential therapeutic applications as a cell-free agent to minimize beta-cell apoptosis during early stages of islet transplantation[102]. The team led by Mahato successfully delivered Exos into a rat model following islet transplantation, while co-culturing with peripheral blood mononuclear cells. This intervention significantly downregulated the expression levels of Fas and miR-375 in the rat model post-transplantation, thereby enhancing Tregs function through peripheral blood mononuclear cell proliferation. These findings highlight the significant immunomodulatory potential of MSC-Exos in improving immune tolerance after islet transplantation. Consequently, this study suggests that utilizing Exos as a means of immunomodulation holds great promise for advancing research in organ transplantation[103].

Currently, the direct application of MSC-Exos in organ transplantation indicates a promising potential for MSCs in the field of organ transplantation. Graft-infiltrating DCs and CD8⁺ T lymphocytes play crucial roles in immune regulation following liver transplantation (LT). Exos also emerge as a significant factor involved in transplantation immunity. Exos derived from CD80⁺ DCs negatively regulate CD8 T cells by suppressing nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 expression, thereby playing an essential role in attenuating acute LT rejection. These findings unveil a novel function of Exos derived from CD80⁺ DCs associated with the induction of LT tolerance[104].

CLINICAL POTENTIAL AND APPLICATION OF MSC-DERIVED EXOS

MSCs have been extensively utilized in cell therapy due to their potent immunomodulatory and regenerative properties[105]. The paracrine activity of MSCs, particularly the production of various factors through EVs, notably Exos, has been demonstrated as a crucial determinant of their primary efficacy after infusion[106]. MSC-Exos possess significant advantages over MSCs themselves and effectively mitigate adverse side effects such as infusion-related toxicity[107]. Consequently, MSC-Exos are emerging as a promising cell-free therapeutic tool with an increasing number of clinical studies evaluating their therapeutic efficacy in diverse diseases[48]. MSC-Exos exhibit immense potential for clinical application as cell-free agents[108].

MSC-Exos from diverse sources exhibit conserved biological functions; however, they may also display functional disparities[109]. For instance, adipose tissue-derived MSC-Exos demonstrate superior angiogenic capacity compared to those derived from bone marrow[110]. Conversely, BM-MSC-Exos possess immunomodulatory and anti-inflammatory effects by inhibiting interferon-gamma secretion in T cells[111]. The route of administration is another crucial factor influencing the therapeutic efficacy of MSC-Exos, with various routes being evaluated. Although intravenous injection remains the most commonly employed route in preclinical studies[112], alternative approaches such as intraperitoneal and subcutaneous administration result in enhanced accumulation of MSC-Exos within organs like the pancreas[113].

Clinical applications of MSC-Exos in different diseases

MSC-Exos were evaluated as therapeutic agents for various conditions, including acute respiratory distress syndrome, renal disease, GvHD, osteoarthritis (OA), stroke, Alzheimer’s disease, and type 1 diabetes[46].

In neoplastic diseases, MSC-Exos, as natural nanocarriers, can serve as effective delivery vehicles for anticancer drugs and gene therapy tools such as small interfering RNA or miRNA, directly targeting cancer cells. This approach enhances treatment precision and mitigates the side effects associated with conventional chemotherapy. By modulating immune cells within the tumor microenvironment, MSC-Exos can augment the immune system’s ability to recognize and attack tumors. Additionally, MSC-Exos can transport anti-tumor molecules that inhibit tumor cell proliferation or induce apoptosis[114]. Chronic kidney diseases are progressive and irreversible disorders that occur when renal function declines below a certain threshold[115]. Progressive tubulointerstitial fibrosis is a common characteristic of end-stage renal disease caused by chronic kidney disease. Nassar et al[116] utilized umbilical cord blood MSC-Exos to ameliorate the progression of the disease. The umbilical cord blood MSC-Exos group exhibited significant improvements in glomerular filtration rate, serum creatinine level, blood urea nitrogen levels, and urinary albumin-to-creatinine ratio. These improvements may be attributed to an elevation in circulating anti-inflammatory cytokines and a reduction in pro-inflammatory cytokines; specifically, there was a notable increase in plasma TGF-β1 and IL-10 levels in the treatment group. Skin pigmentation is a dermatological disorder that affects skin color through discoloration or darkening of the skin. Studies have investigated the therapeutic effects of Exos derived from adipose tissue-derived MSCs on skin pigmentation by potentially inducing ceramide or sphingosine 1-phosphate synthesis which regulates melanogenesis in melanocytes and reduces melanin content in the treated group[117].

Allogeneic hematopoietic stem cell transplantation is a potentially life-saving treatment for patients with hematological malignancies. One of the most serious complications associated with hematopoietic stem cell transplantation is acute or chronic GvHD. A study utilized Exos derived from bone marrow MSCs in patients with GvHD, resulting in reduced secretion of pro-inflammatory cytokines and significant improvement in symptoms, including a reduction in diarrhea and inhibition of skin and mucosal GvHD within 14 days. However, it should be noted that one patient died of pneumonia seven months after treatment. Nevertheless, these results are promising and demonstrate potential efficacy in the treatment of GvHD[118].

Exos derived from MSCs in infectious diseases mitigate tissue damage caused by excessive immune responses through immune regulation. These Exos can transport antibacterial molecules or regulatory factors that either directly exert antibacterial effects or enhance the body’s innate immunity, thereby enabling a more effective combat against pathogens[119]. Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by the coronavirus severe acute respiratory syndrome coronavirus 2, which was discovered in 2020[120]. Inhalation of Exos is believed to reduce inflammation and lung injury while inducing regenerative processes, suggesting a potential therapeutic role in treating COVID-19. In a clinical study using BM-MSC-Exos, lymphopenia significantly improved and CD3⁺, CD4⁺, and CD8⁺ T cells increased following MSC-Exos injection, indicating the immunomodulatory effects as the therapeutic mechanism of MSC-Exos[121]. Therefore, the authors consider MSC-Exos to hold promise as a treatment for COVID-19.

In autoimmune diseases, MSC-Exos can restore immune homeostasis and mitigate disease symptoms by suppressing hyperactive immune cells, including T cells and B cells, and promoting the generation of Tregs. The bioactive molecules within these Exos can attenuate chronic inflammatory responses in various autoimmune conditions, thereby alleviating symptoms associated with diseases such as arthritis and SLE[118]. OA is an arthritic condition affecting joints that leads to pain and stiffness. BM-MSC-Exos have been investigated as a therapeutic agent for OA across various joints. At six months post-infusion, both Brief Pain Scale scores along with Upper Extremity Function Scale and Lower Extremity Function Scale scores demonstrated improvement following treatment with BM-MSC-Exos. These findings indicate that utilizing BM-MSC-Exos effectively improves OA joint conditions while ensuring safety[122].

CONCLUSION

MSC-Exos have the ability to regulate immune responses through various mechanisms, including modulation of both innate and adaptive immune systems, inhibition of excessive inflammatory responses, and promotion of tissue repair. They exert their effects by delivering immunoactive substances such as miRNAs and proteins directly to target cells, playing a crucial role in maintaining immune system homeostasis. Additionally, Exos play a regulatory role in inflammatory diseases, tumors, autoimmune diseases, and organ transplantation. Due to their outstanding performance in regulating inflammatory responses, inhibiting tumor growth, and promoting immune tolerance, Exos hold great therapeutic potential for various pathological conditions as a cell-free therapy tool that offers higher safety and stability compared to traditional cell therapy (Figure 2).

Figure 2 Mesenchymal stem cell-derived exosomes regulate immune responses in diseases through different cytokines. PGE2: Prostaglandin E2; IL-6: Interleukin 6; TGF-β: Transforming growth factor-beta; TNF-α: Tumour necrosis factor-alpha; IL-10: Interleukin-10; NO: Nitric oxide; DC: Dendritic cell.

However, challenges remain before clinical applications can be fully realized. These challenges include the standardization and characterization of Exos, optimization of preparation processes, and determining the optimal route for administration. Additionally, long-term safety and efficacy need to be thoroughly verified. Future research must focus on bridging these gaps by developing strategies such as engineering Exos to enhance their functionality or to target specific tissues more effectively.

To address these challenges, it will be essential to innovate methods for Exo engineering, enabling precise modulation of their cargo and surface properties for targeted delivery. Techniques such as genetic modification or surface marker manipulation could be employed to direct Exos specifically to disease sites, enhancing therapeutic outcomes. Further, large-scale production techniques need to be optimized to ensure consistency and quality in Exo preparations.

More preclinical and clinical studies are necessary to advance this novel cell-free therapy into clinical use. Future studies should continue exploring the mechanisms of action, optimizing production and preparation processes, and verifying efficacy through more rigorous clinical trials. By addressing these knowledge gaps and developing robust methodologies, the application of Exos as a novel cell-free therapy in clinical treatment can be promoted. Advancing their application in regenerative medicine and other fields will require ongoing research and development to unlock their full therapeutic potential.