Stem cell exosomes: new hope and future potential for relieving liver fibrosis
Article information
Abstract
Liver fibrosis is a chronic liver injury resulting from factors like viral hepatitis, autoimmune hepatitis, non-alcoholic steatohepatitis, fatty liver disease, and cholestatic liver disease. Liver transplantation is currently the gold standard for treating severe liver diseases. However, it is limited by a shortage of donor organs and the necessity for lifelong immunosuppressive therapy. Mesenchymal stem cells (MSCs) can differentiate into various liver cells and enhance liver function when transplanted into patients due to their differentiation and proliferation capabilities. Therefore, it can be used as an alternative therapy for treating liver diseases, especially for liver cirrhosis, liver failure, and liver transplant complications. However, due to the potential tumorigenic effects of MSCs, researchers are exploring a new approach to treating liver fibrosis using extracellular vesicles (exosomes) secreted by stem cells. Many studies show that exosomes released by stem cells can promote liver injury repair through various pathways, contributing to the treatment of liver fibrosis. In this review, we focus on the molecular mechanisms by which stem cell exosomes affect liver fibrosis through different pathways and their potential therapeutic targets. Additionally, we discuss the advantages of exosome therapy over stem cell therapy and the possible future directions of exosome research, including the prospects for clinical applications and the challenges to be overcome.
INTRODUCTION
Hepatic fibrosis (HF) is a chronic liver disease attributed to numerous underlying causes, such as viral hepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease, autoimmune hepatitis, and metabolic disorders [1]. In the progression of HF, as it develops from stage F0 to stage F4 (i.e., cirrhosis), the clinical symptoms become increasingly pronounced with the gradual deterioration of the condition [2]. HF development is characterized by pathologic changes, such as hepatocellular necrosis, inflammation, oxidative stress (OS), and extracellular matrix deposition, which may eventually lead to cirrhosis [3]. Chronic liver disease and cirrhosis cause 2 million deaths per year globally [4]. However, HF is reversible, considering it is not at an advanced cirrhotic stage. Liver transplantation is the most effective treatment for severe HF and cirrhosis; however, numerous patients await transplantation, and there are limited donors. An assessment of global liver transplantation statistics suggested a significant increase in transplant numbers since 2015 [5]. In 2021, 9,234 liver transplants were reported in the U.S. Moreover, the post-transplantation mortalities were 13.3%, 18.6%, and 35.9% for 3, 5, and 10 years, respectively [6].
Stem cell therapy has been recognized as a promising therapeutic strategy for HF. This can be attributed to the shortage of liver transplantation donors, complications persisting after transplantation, and relatively high mortality. Stem cells are one of the key hotspots in regenerative medicine because of their self-regenerative and multipotential differentiation abilities. Mesenchymal stem cells (MSCs) are extracted from the adipose tissue, bone marrow, and umbilical cord blood in treatments and studies by comparing the efficiency and immunocompatibility of stem cell extraction and considering the ethical issues [7]. MSCs substantially improve liver function when treating liver diseases [8]. Additionally, they develop into different cell lines exhibiting anti-inflammatory and immunomodulatory capabilities, minimizing the need for immunosuppressive drugs [9]. Stem cell therapy is widely applied to musculoskeletal and neurological disorders, immune disorders, hematologic dysfunctions, and degenerative diseases [7]. However, researchers have not identified effective countermeasures because stem cell transplantation may cause cellular rejection, infusion reactions, and medical tumor implantation [10]. Stem cells act in a paracrine manner through their soluble extracellular vehicles (EVs). Moreover, exosomes are considered the miniature versions of their parent cells [11]. Thus, mesenchymal stem cell-derived exosomes (MSC-Exos) inherit similar therapeutic effects, such as anti-inflammatory, immunomodulatory, and tissue regeneration, from their parental cells [12]. These physiological properties are unique to stem cells and serve as the advantages of exosomes over other cellular exosomes when applied to clinical therapies. Moreover, exosomes exhibit a superior safety profile than MSC-derived cells. This is because they do not replicate and can be easily stored without losing their properties [13]. MSC-Exos are a favorable alternative to stem cell therapy regarding its efficacy and circumvent the drawbacks associated with stem cell therapy.
ORIGIN AND BIOLOGICAL PROPERTIES OF MSC-EXOS
EVs are categorized into exosomes, microvesicles, and apoptotic vesicles based on their size and origin [14]. Exosomes are small vesicles ranging between 40 nm and 150 nm; they are secreted by the cells [15]. Additionally, exosomes are the most well-defined EVs abundant in body fluids, such as blood, saliva, amniotic fluid, urine, and breast milk [16]. They act as a cellular messenger, participating in cellular communication and metabolism [17]. The mode of action of exosomes includes content delivery to the cell membrane through receptor-ligand interactions, endocytosis, or direct fusion, facilitating the mediation and regulation of cellular functions in the target cells [18]. Exosomes encompass several biologically active molecules, including proteins, mRNAs, and non-coding RNAs, such as microRNAs, which can transfer functions across different cell types and even species (Fig. 1) [19]. In addition to directly promoting tissue and organ regeneration, EVs regulate the microenvironment, which is deleterious to tissue injury repair [20]. Exosomes perform important biological functions in an organism.
The nucleic acids and proteins produced by the nucleus and organelles, as well as substances taken up through endocytosis, together form early endosomes, which are then expelled from the cell through exocytosis. There are various functional substances present in the exocrine body and it s surface, such as proteins, DNA, miRNA, etc.
MSC-Exos exhibit some similarities and differences in protein expression and function with cell-secreted exosomes. Similar to other exosome formation, MSC-Exos are produced through “endocytosis-fusion-exocytosis” [21]. Similarly, MSC-Exos express proteins associated with exosome formation, such as membrane-associated proteins (CD9, CD63, CD81, major histocompatibility complex type 1 [MHC-I], and MHC-II), heat shock proteins (HSPs) (Hsp8, Hsp70, and Hsp90), and multivesicular bodies (ALG-2-interacting protein X, tumor susceptibility gene 101, and synthetic proteins) [22]. By contrast, they express several stem cell-specific proteins, such as CD29, CD44, and CD73 [23]. In addition to differential protein expression, MSC-Exos encompass numerous substances related to their biological origins, such as lipid rafts, saturated fatty acids, and specific ncRNAs. These substances are closely related to the regulation of target cells [24]. Regarding preservation, isolated MSC-Exos should be processed or frozen more immediately than other exosomes. This is because the storage temperature exerts a strong influence on its structure [25]. Functionally, MSC-Exos are central to promoting hepatocyte proliferation and maintaining hepatocyte function; they possess regulatory properties that allow the transport of functional proteins or RNAs to the target cells through physiological barriers for communication and regulation [26].
The application of MSC-Exos to regenerative medicine appears to be promising for treating tissue wounds by mediating intercellular communication in skin regeneration. Thus, MSC-Exos are central to skin wound healing by reducing inflammatory responses, promoting proliferation, inhibiting apoptosis, and enhancing angiogenesis [27]. The proteins and miRNAs in exosomes regulate cellular metabolism in the liver, thereby altering the microenvironment in target cells. The mediated cell-cell communication alters tumor growth, cell migration, antiviral infections, and hepatocyte regeneration. Thus, the application of MSC-Exos as a diagnostic or therapeutic tool has excessive potential for liver diseases [28]. In summary, MSC-Exos have good prospects for clinical applications and offer an alternative to stem cell-based therapies. This article elucidates investigations on MSC-Exos in regulating HF through multiple pathways and trends for future research on exosome therapeutics.
MECHANISM OF ACTION UNDERLYING EXOSOMES IN HF TREATMENT
MSC-Exos modulate macrophage polarization to alleviate HF
HF development can be attributed to the activation of hepatic stellate cells (HSC). However, immune cells, particularly macrophages, affect HSC activation. Macrophages are highly plastic and can be functionally divided into two major subpopulations, namely, classically activated macrophages (CAM, M1-macrophages) and alternatively activated macrophages (AAM, M2-macrophages) [29]. In vitro, M1-macrophages are induced by lipopolysaccharides (LPS) and interferon-γ (IFN-γ); they exhibit pro-inflammatory activity. By contrast, M2 macrophages are induced by interleukin (IL)-4 and IL-13; they exhibit anti-inflammatory activity [30]. Macrophages and fibroblasts are central to all types of tissue fibrosis, which begins with the recruitment of numerous pro-fibrotic cytokines and chemokines [31]. For example, M1 macrophages secrete several pro-inflammatory cytokines, such as IL-1b, inducible nitric oxide synthase, and tumor necrosis factor-α (TNF-α) that contribute to fibrosis. Contrarily, M2-macrophages are anti-inflammatory macrophages, which primarily produce anti-inflammatory factors, such as IL-10, transforming growth factor β (TGF-β), and arginase 1. Moreover, they serve as fibrinolytic upon a reduction of the influences that cause inflammation [31].
MSC-derived extracellular vesicles (MSC-EVs) alleviate HF through multiple pathways, including the modulation of M1-macrophage polarization to M2-macrophages. Exosomes have been identified as effective paracrine mediators of the antifibrotic effects of MSCs. Researchers have proposed miR-148a as a potential therapeutic target for HF. Mechanistically, in exosomes, miR-148a targets KLF6 directly to regulate macrophage polarization via the Janus kinase/signal transducers and activators of the transcription pathway [32]. Moreover, MSC-EVs effectively switch the polarization state of macrophages from the M1 to the M2 phenotype to alleviate HF [32]. Additionally, MSC-Exos consist of IL-10, which acts as an anti-inflammatory agent in hepatic macrophages and reduces liver injury [33]. The intravenous transplantation of human umbilical cord MSCs (HucMSCs) into methionine- and choline-deficient (MCD) NASH mice improves the appearance of MCD-induced body weight loss and liver injury. This is because exosome-induced macrophages produce anti-inflammatory factors and reduce inflammatory cytokines in the liver tissues, thus alleviating HF and improving the body state in mice [34].
Apart from the direct application of MSC-Exos to the liver, stem cells can be pretreated using cytokines. Stem cells act as a “drug reservoir” for exosomes, yielding more therapeutic exosomes upon cytokine action. For example, HucMSC pretreatment with interleukin 6 significantly upregulates anti-inflammatory miR-455-3p levels in the secreted exosomes. Moreover, the exosomes are translocated to macrophages, inhibiting macrophage overactivation and exerting anti-inflammatory activity by targeting phosphoinositide-3-kinase regulatory subunit 1 [35]. Upon activation by inflammatory vesicle agonists, the trans-Golgi network (TGN) disassembles to form the dispersed trans-Golgi network (dTGN), and dTGN-enriched cells are recruited for a nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) [36]. After TNF-α pretreatment, HucMSCs release numerous exosomes enriched in miR-299-3p. Additionally, exosome translocation to macrophages prevents the disassembly of TGNs to form dTGNs, thereby inhibiting the recruitment and activation of NLRP3 inflammatory vesicles to exert anti-inflammatory effects [37]. In summary, regulating the polarization state and anti-inflammatory activity of macrophages using exosomes is central to preventing HF progression (Fig. 2).
LPS and IFN-γ induce M1 macrophages, whereas IL-4 and IL-13 induce M2 macrophages. Exosomes containing specific miRNAs derived from stem cells can modulate the polarization of macrophages. This modulation enables pro-inflammatory M1 macrophages to transform into anti-inflammatory M2 macrophages.
MSC-Exos modulate autophagy to alleviate HF
Autophagy is the process of self-digestion and metabolism that maintains homeostasis in the internal environment. Autophagy involves the breakdown and reuse of organelles, proteins, and macromolecules in the cytoplasm. Moreover, cellular autophagy plays an important physiological role in recycling substances to adapt to metabolic stress [38]. Cellular autophagy plays a dual role in the liver. In the liver, HSC activation promotes HF and plays an anti-fibrotic role by reducing hepatocyte death or regulating macrophages [39]. Autophagy is critical for maintaining intrahepatic homeostasis in both parenchymal (hepatocytes) and nonparenchymal (HSCs), sinusoidal endothelial cells, and Kupffer cells (KCs) cells inside the liver [40]. Collectively, autophagy has been recognized as an anti-HF pathway in the liver because it contributes to liver homeostasis by removing misfolded proteins, damaged organelles, and lipid droplets, which are central to energy homeostasis and cytoplasmic content control [41]. Exosome formation and cellular autophagy share similar signaling pathways and molecular mechanisms; moreover, they demonstrate a substantial interaction [42]. In the liver, exosome formation and cellular autophagy tend to be inversely related. Cellular autophagy inhibits the sustained and enhanced release of EVs, for example, in alcoholic liver disease. Therefore, EV release from the hepatocytes increases, whereas hepatocyte autophagy is inhibited [43]. HSC stimulation using platelet-derived growth factors inhibits HSC autophagy and enhances EV release [44].
Interaction between the cellular production of exosomes and autophagy affects HF. thioacetamide stimulation elevates the stress protein Tribbles homolog 3 (TRIB3) and interacts with the selective autophagy receptor Sequestosome 1 (SQSTM1), causing SQSTM1 aggregate accumulation and impeding autophagy. TRIB3-mediated autophagy inhibition not only prevents autophagic degradation but also promotes hepatocyte secretion by exosomes enriched with inhibin subunit beta A/activin A. Eventually, it causes HSC migration, proliferation, and activation [45]. However, disrupted interaction between TRIB3-SQSTM1 and specific helical peptides restores autophagy in the hepatocytes and HSC, exerting a protective effect against HF [45]. Researchers have investigated the protective role of MSC-Exos in the liver against HF by modulating autophagy. Bone marrow mesenchymal stem cell-derived exosomes (BMSCExos) in hepatocytes increase the expression of autophagy marker proteins LC3 and Beclin-1 and autophagosome formation. Additionally, BMSC-Exos attenuate hepatocyte apoptosis by promoting autophagy [46]. Exosomes secreted by adipose-derived-MSCs (AD-MSCs) acted on a mouse model of carbon tetrachloride (CCl4)-induced HF. miR-181-5p in the exosomes reduced HSC activation by directly inhibiting the STAT3/B-cell lymphoma 2/Beclin1 pathway and increased autophagy, thereby reducing HF [47]. In summary, autophagy by MSC-Exo-targeted modulation is a feasible strategy for HF treatment (Fig. 3).
The interaction between exosomes and autophagy significantly impacts the progression of liver fibrosis. (I) To summarize, autophagy occurs in both parenchymal and non-parenchymal cells of the liver. This process can help mitigate liver fibrosis. (II) When autophagy in hepatocytes is inhibited, the release of exosomes increases. (III) When platelet-derived growth factor activates hepatic stellate cells, blocking their autophagy leads to an increase in exosome release. (IV) AMSC-Exosomes and BMSC-Exosomes help prevent liver fibrosis by promoting the process of autophagy.
MSC-Exos regulate pro-inflammatory cytokine secretion to alleviate HF
Inflammatory cytokines are central to HF development. Numerous inflammatory cytokines, such as IL-6, TGF-β, TNF-α, and IL-1, promote HF [48]. They promote the abnormal proliferation and deposition of hepatic connective tissue, thereby exacerbating HF [49]. Therefore, clinicians should control the production and activation of inflammatory cytokines to prevent HF development and progression.
MSC-Exos regulate TGF-β secretion
The biological functions of TGF-β involve tissue repair and embryonic development [50]. TGF-β plays a regulatory role in cell proliferation, senescence, apoptosis, inflammatory response, tissue fibrosis, and aging [51]. It modulates senescence-associated fibrosis by regulating inflammation, DNA damage, OS, and cellular senescence [52]. These processes help maintain the dynamic homeostasis of tissues and organs. Researchers have identified >30 isoforms of TGF-β, a major pro-fibrotic cytokine; TGF-β1 is the most relevant isoform to HF [53]. It accelerates HF by promoting HSC activation through the TGF-β1/Smads signaling pathway [54]. Some findings appear promising for inhibiting TGF-β expression through MSC-Exos’ action. For example, Wharton’s jelly, attached to the umbilical cord, contains abundant MSCs. It can differentiate into adipocytes, osteoblasts, chondrocytes, neurons, and hepatocytes [55]. The mRNA expression levels of NOX1, NOX2, and NOX4 genes, which are associated with TGF-β, were significantly reduced in LX2 cells within 24 h of treatment using exosomes (40 µg/mL or 50 µg/mL) derived from Wharton’s jelly MSCs. Furthermore, TGF-β expression was significantly inhibited under similar conditions [56]. Therefore, MSC-Exos in liver cells will supposedly inhibit TGF-β expression, thus alleviating HF and becoming a novel treatment target.
Exosomes of different cellular origins affect TGF-β expression in HF. For example, umbilical cord-derived MSCs (UCMSCs) inhibit signaling pathways by suppressing TGF-β expression, inhibiting HSC activation, attenuating HF, and reducing protein expression related to cellular senescence and cell proliferation [57]. Exosomes secreted by AD-MSCs comprising overexpressed miRNA-181-5p inhibit TGF-β1-induced upregulation of pro-fibrotic genes, thereby attenuating hepatic injury. Additionally, they significantly downregulate the expression of type I collagen, α-smooth muscle actin, and fibronectin in hepatic tissues, thereby inhibiting HF [47]. In vitro, applying human embryonic MSC-Exos enriched with miR-6766-3p to human HSC (LX2) significantly reduces pro-fibrotic marker expression. miR-6766-3p inhibits TGFβRII expression to reduce Smads signaling, eventually reducing HSC activation and preventing HF onset or development [58]. MSC-Exo attenuates HF by inhibiting TGF-β expression. Additionally, exosomes produced by other cells exert similar effects. For example, in vivo and in vitro experiments have suggested that exosomes derived from natural killer (NK) cells inhibit TGF-β1-induced proliferation, CCl4-induced HF in mice, and the activation of human HSC lineage cells (Fig. 4) [59].
Exosomes of different cellular origins can alleviate liver fibrosis via regulating TGF-β/Smad pathway.
Regulating IL-6 secretion by MSC-Exos
IL-6 is the predominant cytokine associated with inflammatory diseases [60]. As a key pro-inflammatory cytokine, it has numerous physiological functions, such as regulating cell proliferation and differentiation [61]. IL-6 exerts a bidirectional effect on HF, promoting inflammation production that causes hepatocyte necrosis and apoptosis and protecting the hepatocytes against injury at appropriate concentrations [62]. Therefore, IL-6 can be applied to liver lesion treatment by selectively inhibiting the IL-6 pathway and decreasing expression [63]. However, MSC-Exos can regulate various cytokines, such as IL-6, thereby intervening in HF onset and progression [64].
MSC-Exos regulate inflammatory cytokine secretion by modulating the degree of gene expression [65]. Adipose-derived mesenchymal MSC-Exos were administered to an LPS/D-Gal-induced acute liver failure mouse model. miR17 in exosomes reduced inflammatory marker expression, such as TNF-α, IL-6, IL-1β, and IFN-γ, by regulating the expression of genes related to inflammatory cytokines, thereby attenuating liver injury [66]. Exosomes isolated from UCMSCs, either infused alone or combined with poly sterols, inhibited mRNA expression of IL-6 in a mouse model of liver failure. Furthermore, the combination of UCMSC-derived exosomes with poly sterols is preferable to exosomes alone; both attenuate hepatic injury in HF mice by modulating the IL-6/STAT3 signaling pathway [67]. These pathways facilitate decreasing the secretion of inflammatory cytokines and liver injury and attenuating HF. MSC-Exos can prevent liver injury by regulating IL-6 and its signaling pathway. Moreover, upon applying IL-6 to HucMSCs, the resulting exosomes were enriched in miR-455-3p. This in turn effectively reduced serum inflammatory cytokine levels and attenuated liver injury [35].
In addition to TGF-β and IL-6, MSC-Exos regulate several cytokines, such as chemokines, IL-7, IL-1β, TNF-α, IFN-γ, and other inflammatory cytokines [68]. During HF progression, MSC-Exos interact with inflammatory cytokines and co-regulate a series of changes to suppress inflammation, improve hepatic function, promote hepatocyte regeneration, and alleviate HF in the liver.
MSC-Exos regulate OS to alleviate HF
OS refers to the excessive production of highly reactive molecules, such as reactive oxygen radicals (ROS) and reactive nitrogen radicals (RNS), in the body upon subjecting an organism to harmful stimuli [69]. In OS, oxidation exceeds oxidant scavenging, and the oxidative and antioxidant systems are out of balance, leading to tissue damage [70]. The liver is an important site for ROS production and is continuously exposed to different toxicants or reactive metabolites (both ROS and RNS) [71]. ROS accumulation in the liver induces hepatocyte dysfunction or death, which eventually leads to the release of injury-associated molecules. Simultaneously, nonparenchymal cells, such as KCs, HSCs, and newly recruited immune cells, are activated and produce pro-fibrotic and pro-inflammatory mediators [72]. Therefore, OS is one of the chief causative factors of liver diseases and can lead to common liver diseases [73]. MSC-derived exosomes are rich in bioactive molecules with numerous bioregulatory functions that suggestively improve the efficacy of treating OS-related diseases [74].
MSC-Exos exert stronger anti-OS effects in the liver and, thus, prevent liver injury in HF treatment. To verify the definite effects of MSC-Exos, researchers have compared the antioxidant effects of human umbilical cord blood MSC-Exo (HucMSC-Exo) with that of bifendate (DDB), a commonly used hepatoprotective agent, in a CCL4-induced liver injury mouse model. HucMSC-Exo exerted more significant antioxidant and hepatoprotective effects, compared with DDB [75]. Moreover, HucMSC-Exo exerted antioxidant and anti-apoptotic effects upon injection into a mouse model of hepatic injury by the tail vein or orally. Additionally, it promoted the recovery of hepatic oxidative damage and prevented hepatic failure by delivering glutathione peroxidase 1 [76]. Oxygen transport and iron homeostasis are closely related [77]. Thus, abundant iron in the cells and tissues disrupts redox homeostasis and catalyzes ROS production, leading to OS. Eventually, it interacts with iron death at the cellular level and is considered an iron-dependent form of cell death [78]. Benzyl chloride 1 (BECN1) is a key regulator of ferroptosis [79]. An intravenous injection of MSC-ex significantly reduced HSC activation and collagen deposition in the fibrotic livers of experimental mice. Mechanistically, BECN1 delivered via MSC-Exos promoted iron death and OS in HSCs, thereby preventing HF [80].
Additionally, the exosomes of cells across different species play a biological role. Similar to exosomes secreted by mammalian cells, edible plant-derived exosome-like nanoparticles serve as extracellular messengers to mediate intercellular communication and regulate interspecies communication through their products (e.g., miRNAs, biologically active lipids, and proteins) [81]. Researchers have screened blueberry-derived exosomes-like nanoparticles; they attenuated rotenone-induced OS in human HepG2 cells and high-fat diet-fed C57BL/6 mice to mitigate liver injury [82]. Exosomes obtained from multiple sources may protect from liver injury by regulating OS as well as HF.
COMPARING THE ADVANTAGES OF EXOSOME AND STEM CELL THERAPIES
Clinical trials have elucidated issues with the safety and stability of BMSCs that affect clinical treatment progression, clinical staging, and determining infusion volumes upon using BMSCs for the treatment of chronic liver disease, including HF and cirrhosis [83]. Several researchers have investigated the safety of stem cell therapy, which primarily focuses on topical treatments, such as ocular administration [84]. No substantial abnormality has been identified in its local administration, and the therapeutic effect has been satisfactory. However, the safety of systemic MSC injection in humans remains unclear. In a few experiments on stem cell infusion therapy, substantial MSCs accumulated in the lungs of humans and animals upon infusion into the body; most MSCs were eliminated within the subsequent 3 to 6 days. Moreover, <1% of MSCs were distributed in various organs, including the spleen, liver, and bone marrow, after 1 week [85]. Thus, stem cells do not remain stable in the body for a long duration after infusion because of rapid clearance. Moreover, stem cell therapy has drawbacks and negative effects. For example, UCMSC therapy reduces pro-inflammatory cytokine production, such as TNF-α, IFN-γ, IL-6, IL-1β, TGF-β1, and IL-4. By contrast, it increases anti-inflammatory cytokine production, such as IL-10 [86]. Furthermore, it poses the risk of medically derived tumors, such as post-transplant lymphoproliferative disease. This transplantation complication is the leading cause of cancer-related deaths after solid organ or allogeneic hematopoietic stem cell transplantation [87]. In summary, despite several examples of MSC application in clinical studies, maintaining constant and effective quantity and quality control is challenging.
Using exosomes for disease treatment can effectively avoid some issues associated with stem cell therapies and achieve good therapeutic effects. For example, in a study involving 40 patients with chronic kidney disease, 20 were randomly selected to receive stem cell exosome treatment, while the others received a placebo. After a one-year monitoring period, it was found that members of the treatment group showed significant improvements in kidney function and inflammatory status compared to the control group [88]. For acute or chronic graft-versus-host disease (GvHD) caused by stem cell transplantation, researchers used exosomes derived from the supernatant of BM-MSCs to treat GvHD patients, resulting in significant improvement in symptoms; the amount of diarrhea was significantly reduced, and skin and mucosal damage showed marked improvement [89]. At the same time, to observe whether exosome therapy has adverse reactions, in a clinical trial, 24 healthy volunteers inhaled ASC-Exos via nebulization, followed by multiple biological examinations such as blood parameters, liver and kidney function, lactate dehydrogenase, immunoglobulin concentration, and electrocardiograms. All volunteers showed good tolerance to the infusion, and no adverse reactions occurred [90]. Furthermore, as of now, there are still several clinical trials regarding exosomes that are in the monitoring phase, and their application potential is gradually being confirmed (Table 1) [91].
Clinical applications of exosomes
The specific advantages of exosome therapy are as follows: Exosomes are physiologically stabilized by a double-layer plasma membrane. Moreover, the component substances are protected from the action of immune cells and digestive enzymes, resulting in low immunogenicity [92]. Exosomes deliver different proteins to the target cells and efficiently deliver numerous growth factors and those that regulate cellular responses to the liver [8]. Exosome targeting can achieve good therapeutic effects at smaller drug concentrations, displaying favorable application value [93]. Additionally, the use of human-derived MSCs encounters ethical issues in some countries, despite no ethical issues with UCMSCs. This is because the umbilical cord is considered biological waste and is discarded after birth [94]. However, these concerns are related to considering MSCs from embryos. Nonetheless, exosomes from stem cells instead of cells pose fewer ethical challenges [95]. Thus, MSC-Exos treatment exerts a more favorable therapeutic effect. Exosome therapy is safer than stem cell therapy because of non-ethical constraints, greater therapeutic stability, and the absence of defects inherent to stem cell therapy. Therefore, it serves as an effective alternative to stem cell therapy.
CHALLENGES AND PROSPECTS FOR EXOSOME THERAPY
Exosomes have been developed as drug delivery vehicles or extensively studied as biomarkers. Particularly, MSC-EVs have been used in several clinical studies. However, researchers should further investigate the long-term effects of MSC-EVs. For example, differences in the therapeutic effects of MSC-secreted exosomes from different sources should be evaluated for immunomodulation, antifibrosis, and liver regeneration potential to determine the optimal therapeutic strategy [98]. Because exosomes are secreted by cells, pretreatment before exosome collection may generate a product with better therapeutic efficacy. In one study, ASCs secreted exosomes with specific functions by overexpressing miRNA-181-5p; the resulting exosomes could be targeted for transport into the fibrotic liver [47]. Thus, exosomes can be used to deliver potent molecules released by MSC, whereas those secreted by genetically altered stem cells can be targeted for delivery [99]. EVs are cellular products involved in almost all physiological activities, including cell proliferation, migration, and apoptosis. They are closely associated with immune surveillance and immune cell activation [100]. Thus, exosomes can regulate disease progression. Researchers are exploring the role of exosomes in diagnosing liver disease. Exosome detection may serve as a novel diagnostic indicator for liver disease by facilitating an understanding of liver pathology [101]. Moreover, MSC-derived exosomes exert anti-inflammatory, immunomodulatory, and therapeutic effects [102]. As a novel antigenic material, MSC displays good immune properties; therefore, MSC-Exos can be applied to vaccine development [103]. MSCs secrete EVs in large quantities, which facilitate exploring MSC-Exos as a novel vaccine vector [104]. Conventional liposome-delivered vaccines can cause adverse immunogenic reactions. By contrast, EV vectors effectively avoid such reactions [105,106].
Research on the clinical potential of exosomes is underway. For example, no unified standard has been proposed for exosome preparation. Moreover, researchers have developed separation methods, including ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, size-exclusion chromatography, and immunoaffinity methods, based on the exosome size, density, and immunophenotype. Nonetheless, the yield and physicochemical properties of separated exosomes depend on the isolation method and cannot be identical [107]. For example, ultracentrifugation is a common method that produces exosomes with high purity but low yield. At the same time, when developing exosome-based therapies, it is crucial to ensure that processes adhering to good manufacturing practices and strict quality control measures are employed to produce exosome preparations that meet clinical standards [108]. Strict quality control is also crucial for reproducible research in academic studies [109]. Recently, the International Society for Extracellular Vesicles (ISEV) has established a set of standards and guidelines for extracellular vesicle research, aimed at providing a standardized framework for the clinical application and scientific research of extracellular vesicles [110]. Thus, MSC-EV separation methods must be standardized in the future [111]. Researchers should improve and standardize established separation protocols to increase the yield and purity of the samples. BMSC-Exos treatment is considered a “double-edged sword” [112]. Despite the therapeutic effects, the safety, adverse effects, and antagonistic outcomes of exosome treatment are unclear [113]. Therefore, MSC-EVs should be applied cautiously in the future because the role of MSC-EVs in disease development has not been elucidated completely. In summary, exosomes are promising for vaccine development, disease diagnosis, and treatment; however, clinicians should consider their immunologic properties. In the future, researchers should explore the application value of exosomes from different cellular sources or those secreted after cellular pretreatment as well as discover more efficient and stable exosome isolation methods to obtain the ideal purity and yield.
SUMMARY
In summary, effective treatments for HF caused by chronic liver injury because of multiple etiologies remain limited. However, HF treatment using MSC-Exos appears as a novel approach with promising clinical applications. EVs secreted by stem cells mediate intercellular communication and contribute to HF onset and progression. HF treatment using MSC-Exo can repair the liver tissue by polarizing M1-type macrophages into M2-type macrophages, which exert their anti-inflammatory effects. Additionally, MSC-Exos reduce the secretion of inflammatory cytokines, such as IL-6, TGF-β, and TNF-α, and inhibit HSC activation by regulating HSC autophagy, thus preventing HF. MSC-Exos can alleviate HF by regulating OS. In summary, MSC-Exos plays a regulatory role in alleviating HF. However, research on the role of MSC-Exos in HF is underway. This warrants additional studies and in-depth investigations to elucidate their therapeutic effects against HF and the complex mechanisms underlying exosome-mediated therapy for HF.
Existing studies have not elucidated the adverse effects of MSC-Exo completely. Thus, we intend to explore their therapeutic effects and adverse effects on HF treatment in the future. Exosome preparation involves high cost and low yield, and the preparation process is not unified. Thus, we intend to study exosome preparations from different species or cell sources. We aim to identify exosome preparations that are abundant, simple, cost-effective, and stable. This study may be important for developing novel therapeutic strategies for HF. Additionally, regulating stem cells to secrete exosomes with targeted therapeutic functions by targeting stem cell genes to produce exosomes with specific functions may offer a novel approach to developing exosome agents. Exosome therapy, an emerging research topic, holds promise for exerting therapeutic effects similar to stem cell therapy while overcoming the associated limitations and adverse effects. In the future, an in-depth exploration of its characteristics can provide a good reference for exosome development.
Exosomes are expected to serve as carriers for delivering drugs. The application of exosomes in vaccine research and development may prevent immunogenic reactions. In the future, we aim to explore the feasibility of their application to vaccine formulations and investigate the immunological properties of exosomes as antigens, which will contribute to developing novel carrier vaccines.
Despite the therapeutic potential of MSC-Exos for HF, further advancements are required for a feasible clinical application. Exploring the specific mechanisms by which exosomes treat HF will provide valuable insights for developing novel therapeutic strategies. Researchers should conduct clinical trials using multiple sources of stem cells and exosomes, providing a theoretical basis for the continued advancement of exosomes in treating liver disease and, eventually, improving patient prognosis. In conclusion, exploring the role of MSC-Exos in HF has substantial clinical value.
Notes
Authors’ contribution
LHL: conceptualization & supervision & writing-review and editing; YJL: conceptualization & writing-original draft; KPW: conceptualization & writing-original draft; JGM: conceptualization & writing-review and editing; ZYW: Draw Figures & writing-review; HJ: conceptualization & writingoriginal draft; CJ: conceptualization & supervision & writing-review and editing. All authors read and approved the final manuscript.
Acknowledgements
This study was supported by Zhejiang Provincial Natural Science Foundation of China [No. LY23H030002], Medical Science and Technology Project of Zhejiang Province [No. 2023KY412] and Liver Cancer Early Warning and Early Intervention Technology Innovation Team of Taizhou Central Hospital (Taizhou University Hospital).
Conflicts of Interest
The authors declare no conflict of interest.
Abbreviations
AD-MSCs
adipose-derived-MSCs
BECN1
Benzyl chloride 1
dTGN
dispersed trans-Golgi network
EVs
extracellular vehicles
GvHD
graft-versus-host disease
HF
hepatic fibrosis
HSC
hepatic stellate cells
HSPs
heat shock proteins
HucMSCs
human umbilical cord MSCs
IFN-γ
interferon-γ
IL
interleukin
KCs
Kupffer cells
LPS
lipopolysaccharides
MHC-I
major histocompatibility complex type 1
MSC-EVs
MSC-derived extracellular vesicles
MSC-Exos
mesenchymal stem cell-derived exosomes
MSCs
Mesenchymal stem cells
NASH
nonalcoholic steatohepatitis
NK
natural killer
NLRP3
nucleotide-binding domain
OS
oxidative stress
RNS
reactive nitrogen radicals
ROS
reactive oxygen radicals
SQSTM1
Sequestosome 1
TGF-β
transforming growth factor β
TGN
trans-Golgi network
TNF-α
tumor necrosis factor-α
TRIB3
Tribbles homolog 3
UCMSCs
umbilical cord-derived MSCs