Mesenchymal stem/stromal cell-derived exosomes in regenerative medicine and cancer; overview of development, challenges, and opportunities

Cardiovascular disease (CVD) has become a public health concern because it is the main cause of mortality in the world [110, 136]. The ability of endogenous cardiac injury to repair and regenerate is significantly poor due to the insufficient proliferation of existing myocytes concomitant with the weak recruitment and division of resident cardiac progenitor cells [137, 138]. Emerging evidence has shown the cardio-protective role of MSC exosomes in myocardial damage. Ju et al. reported that the delivery of cardiac MSC exosomes after myocardial infarction (MI) enhances cardiac activity by promoting cardiac angiogenesis and activating cardiomyocyte (CMC) proliferation with an unclear mechanism. Cardiac MSC exosomes may enable ischemic myocardium to release chemokines, such as stromal cell-derived factor (SDF) or vascular endothelial growth factor (VEGF), supporting angiogenesis. These exosomes may also upsurge the proliferation of CMC by transferring miRNAs to the ischemic myocardium and by stimulating specific phases of the cell cycle in target cells [88]. Yu et al. have described that exosomes derived from MSC transduced by the growing family of related transcription factor (GATA)-4 genes have enriched various miRNAs, such as miR-221 and miR-19a, which are capable of generating higher protective effects on CMC and also the regeneration of ischemic injury. The miR-221 could improve the survival of myocytes in ischemic injury by reducing the expression of the P53-upregulated apoptosis modulator (PUMA), a member of the B cell lymphoma 2 (Bcl-2) proapoptotic protein families. Also, miR-19a could attenuate the expression of phosphatase and tensin homolog (PTEN), a mediator of Akt and extracellular signal-regulated kinase (ERK) survival signaling pathway, thereby leading to the growth of CMC and preventing cell apoptosis in ischemic myocardium by activating these signaling pathways [89, 90]. Additionally, MSC exosomes contain a high amount of miR-210 that could diminish the expression of ephrin-A3, an angiogenesis suppressor molecule, in endothelial cells to promote angiogenesis and cardiac function in mouse MI models [91]. Another study suggested that MSC exosomes had a direct effect on cardiac cells and enhanced their bioenergetics and survival by reducing oxidative stress, enhancing the production of ATP and NADH, and stimulating anti-apoptotic pathways through Akt and glycogen synthase kinase-3 (GSK3) phosphorylation, and also mitigation of the activation of the proapoptotic pathways mediated by downregulation of c-Jun NH2-terminal kinase (c-JNK). MSC exosomes also could restore cardiac activity and reduce infarct size in the ischemic/reperfuse mouse model of the myocardium [92]. Furthermore, Teng et al. demonstrated that MSC exosomes could recover heart function and repair post-myocardial infarction via inducing neovascularization and suppressing inflammatory response after ischemic injury [93]. In another study, Zhu et al. reported that exposure of BM-MSC to the hypoxia culture medium altered their exosomal content compared to the normoxia culture medium. The miRNA content was shown to be significantly different between normoxia-exposed BM-MSC exosomes (nor-exosomes) and hypoxia-exposed BM-MSC exosomes (hypo-exosomes). Analysis implied that anti-apoptotic miR-125b-5p was one of the most enriched miRNAs in hypo-exosomes with high therapeutic effectiveness in the treatment of MI. Correspondingly, systemic administration of hypo-exosomes inhibited apoptosis of CMC through inhibition of proapoptotic genes p53 and bcl2-antagonist/killer 1 (BAK1) and consequently improved ischemic heart repair in the rodent MI model [94]. Besides, reports have indicated that cardiac MSC-EVs facilitate strong proangiogenic behaviors as well as enhanced cell proliferation, tube forming, and survival of human umbilical vein endothelial cells (HUVECs). These proangiogenic effects of human cardiac MSC-EVs were mainly mediated by positive regulation of the angiopoietin-1 (Ang1) / tyrosine kinase Tie-2 signaling pathway [139]. Other studies have shown that exposure to PDGF enhanced the proangiogenic activity of EVs secreted by AT-MSCs. In EVs derived from PDGF-treated MSCs, the levels of proangiogenic c-kit and stem cell factor (SCF) improved and consequently resulted in induced angiogenesis in vitro and in vivo [140].

Several studies have also revealed that MSC exosomes possess the ability to rescue liver injury and fibrosis through the delivery of various bioactive molecules to hepatocyte cells and other cells in the liver tissue, and thereby change their function and/or phenotype. MSC exosomes have substantial hepatoprotective effects related to exosomal miRNAs, especially miR-223. MiR-223 plays a vital role in the modulation of the immune system as well as the protection of the liver by downregulating various inflammatory genes and proteins, such as cytokines and nucleotide-binding domain and leucine-rich repeat pyrin containing 3 (NLRP3) and also caspase-1. Exosomal miR-223 suppresses NLRP3/caspase-1 signaling pathway and pyroptosis resulting from this signaling pathway, which may minimize liver inflammation and hepatocyte death in the autoimmune hepatitis animal model [95]. Similarly, Yan et al. have already found that glutathione peroxidase 1 (GPX1) containing MSC exosomes could suppress oxidative stress and apoptosis in carbon tetrachloride (CCl4)-and H2O2-induced hepatic damage and finally restore liver function in vivo. The GPX1 is a main antioxidant enzyme that reduces the formation of reactive oxygen species (ROS) through different mechanisms in inflamed liver tissue. Too, MSC-derived exosomes could increase the spread of hepatocytes and decrease their apoptosis through various pathways [96]. Tan et al. reported that MSC exosomes improved hepatic regeneration of CCl4-induced liver damage in a mouse model through promoting hepatocyte proliferation and inhibiting their apoptosis. They found that MSC exosomes stimulate the hepatocyte cell cycle by upregulation of cyclin D1 and proliferating cell nuclear antigen (PCNA) concurrently suppression of the apoptosis of liver cells by increasing the expression of anti-apoptotic Bcl-xL [97]. In parallel, Tamura et al. investigated the inhibitory effect of MSC-derived exosomes on immune-induced liver damage via injection of concanavalin A (con A). They found that MSC-derived exosomes had hepatoprotective and anti-inflammatory competencies through reducing the expression level of proinflammatory cytokines along with promoting the expression of anti-inflammatory cytokines and the frequency of regulatory T cells [98]. To prolong the bioavailability of MSC-derived exosomes in chronic liver disease, Mardpour et al. encapsulated exosomes in polyethylene glycol (PEG) hydrogels to sustain their release in the systemic circulation. They found that hydrogel-mediated delivery caused boosted accumulation of exosomes in fibrotic tissue over prolonged periods, enabling superior anti-apoptosis, anti-fibrosis, and regenerative capacity over free-exosome delivery [99]. Besides, evaluation of the potential of MSC exosomes as a therapeutic approach for tissue restoration and functional recovery by modifying inflammatory response and inducing multiple anti-apoptotic genes in the experimental acute hepatic failure model suggested that MSC-derived exosome intravenous injection resulted in upregulated Y-RNA-1 as a non-coding RNA in damaged tissue, which in turn, supported defensive mechanisms in the microenvironment of the injured liver [100].

Concerning the observations, restoring deteriorated neural tissue remains a big problem in regenerative medicine due to a complication of the physiology system and a limited ability to self-regenerate [101, 141]. A wide spectrum of reports has demonstrated that MSC exosomes have promising therapeutic potential in neurological disorders, such as neurodegenerative diseases. Recent researches have shown that MSC exosomes can protect hippocampal neurons by decreasing neuronal oxidative stress and synapse damage in an animal model of Alzheimers disease (AD). Exosomes contain multiple active enzymes, in particular catalases, which can reduce the formation of ROS in hippocampal neurons, and thereby protect them from AD and other neurodegenerative conditions [142]. As well, Xin et al. proposed that MSC exosomes induced the remodeling of neuritis and subsequently facilitated functional regeneration in the rat stroke model via miR-133b transfer to adjacent astrocytes and neuron cells [102]. In another study, intravenous administration of MSC exosomes improved neuro-functional recovery and boosted post-stroke neurogenesis and neurovascular remodeling in the rat model. Liu et al. indicated that BM-MSC exosomes could enhance neurovascular development and attenuate neuronal apoptosis. Moreover, these exosomes inhibited neuroinflammation and also suppressed the neurotoxic and reactive state of A1 astrocytes following traumatic spinal cord injury (SCI) in rats, implying their competence to exploit for SCI therapy [103]. Additionally, exosomes derived from MSCs could provide a significant protective role in peripheral nerve damage. In this regard, exosomes derived from UC-MSCs could aggregate in nerve defect, promote the generation of axons and Schwann cells, minimize muscular atrophy, and exert anti-inflammatory reactions in injured nerve tissues by downregulating proinflammatory cytokines (IL-6 and IL-1) concomitant with upregulating anti-inflammatory cytokines (IL-10). Therefore, UC-MSC exosomes could establish a favorable stromal condition for increased functional recovery and amelioration of sciatic nerve defects in rats [104]. Mechanistically, Mao et al. suggested that gingiva MSC exosomes promote regeneration of peripheral nerve via upregulation of genes participated in Schwann cell dedifferentiation or repair, including c-Jun, notch1, glial fibrillary acidic protein (GFAP), and SRY (sex-determining region Y)-box 2 (Sox2) and the stimulation of c-Jun N-terminal kinase (JNK) pathways in Schwann cells. Hence, these findings provide proof of the concept that gingiva MSC exosomes activate repair phenotype in Schwann cells and promote their proliferation and migration [105]. Besides, AT-MSC exosomes demonstrated inhibitory effects on inflammation-mediated demyelinating disease, in particular, multiple sclerosis (MS). Exosomes also greatly modulate microglial activity by altering microglial cell morphology and reducing the number of Iba-1-positive microglial cells in the brains of the Theilers murine encephalomyelitis virus (TMEV)-infected mice. Regarding observations, administration of exosomes intravenously improved immunomodulatory activities by reducing the plasma levels of inflammatory cytokines, substantially the concentration of Th1 cell-generated IFN- and Th17 cell-produced IL-17A, resulted in ameliorated motor deficits of TMEV-infected mice [88]. As well, systemic administration of prostaglandin E2 receptor 4 (EP4) antagonists induced MSC-EV secretion and increased the production of anti-inflammatory cytokines and other proteins such as IL-2, IL-10, RANTES, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) which could alleviate memory and learning deficiencies, improve blood-brain barrier integrity and anti-inflammatory responses, and also elicit functional recovery in a mouse model with hippocampus damage [143].

Preclinical researches have demonstrated that MSC exosomes have a marked capacity to rescue acute and chronic kidney disease via the secretion of various mRNAs, miRNAs, and immunosuppressive factors into the tissue environment [127, 144, 145]. The molecular mechanisms responsible for kidney regeneration rely on the control of immune responses, the prevention of apoptosis and necrosis of epithelial tubular cells, and the reduction of oxidative stress in the kidney tissue. Ju et al. have shown that MSC exosomes improved dedifferentiation and proliferation of damaged tubular cells by transfer of hepatocyte growth factor (HGF) mRNA and enhanced HGF synthesis, thereby accelerating kidney regeneration and delaying fibrosis [89]. Also, another study verified the role of MSC-generated miRNAs in kidney protection by suggesting the anti-inflammatory ability of miR-22 in the relief of I/R-induced acute kidney injury (AKI). In detail, miR-22 suppresses the programmed cell death 4 (PDCD4)/ nuclear factor-B (NF-B) pathways required for dendritic cell (DC) maturation, which consequently attenuates its potential for the secretion of proinflammatory cytokines. MiR-22 also reduces epithelial cell damage via targeting the mammalian target of rapamycin (mTOR)/ hypoxia-inducible factor (HIF) feedback interaction pathway, enabling AKI recovery [106]. Further, Zou et al. found that the exosomes derived from human Whartons Jelly MSC (WJ-MSC) downregulated chemokine C-X3-C motif ligand 1 (CX3CL1) expression and improved kidney function in rats with kidney ischemia-reperfusion injury (IRI). They found that the exosomes could reduce kidney cell apoptosis and raise the proliferation of these cells concomitant with the enhancement of anti-inflammatory responses by decreasing the macrophage frequencies in wounded sites [107]. Moreover, human UC-MSC exosomes could potently improve cisplatin-induced AKI in laboratory models by suppressing oxidative stress upon decreasing oxidant enzymes and increasing antioxidant enzymes cellular levels and also through mitigation of kidney cell apoptosis by inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway [108]. Besides, intra-kidney delivery of MSC exosomes could restore kidney injury in pigs with metabolic syndrome and kidney artery stenoses possibly mediated by increased kidney blood supply, suppressed glomerular filtration, modified kidney inflammation, and abrogated kidney fibrosis and medullary oxygenation, supporting kidney normal function. It has been supposed that MSC exosomes elicited these protective effects upon IL-10 upregulation as an anti-inflammatory cytokine [110]. Another research indicated that BM-MSC exosomes could deliver exogenous microRNA let7c with strong anti-fibrotic activity via suppression of kidney inflammation in rodent models [111]. Respecting to the glial cell line-derived neurotrophic factor (GDNF) neurotrophic property, exosomes derived from GDNF-overexpressed AT-MSCs were found to stimulate angiogenesis and enhance peritubular capillary loss in tubulointerstitial fibrosis in vivo by activating the SIRT1 signaling pathway and inducing the production of phosphorylated endothelial nitric oxide synthase (p-eNOS) [146]. Moreover, in an experimental model of type I diabetes, intravenous administration of MSC exosomes significantly ameliorated diabetes-induced kidney injury by suppressing apoptosis of podocytes and tubular epithelial cells and inducing tubular vascular survival and regeneration. MSC exosomes contained angiogenesis-related factors such as transforming growth factor- (TGF-), angiogenin, and bone morphogenetic protein-7 (BMP-7) played central roles in these processes [147].

As well, another study in 30 participants with COPD indicated that administration of MSC exosomes was well tolerated because none of them experienced any unwanted events [152]. Moreover, investigation of the safety and efficacy of exosomes (ExoFlo) derived from allogeneic BM-MSCs in 24 patients with severe 2019 coronavirus disease (COVID-19) revealed that systemic injection of the ExoFlo caused restored patients clinical condition and oxygenation without any serious events during 2weeks follow-up, suggesting MSC exoxomes as a capable therapeutic candidate for severe COVID-19 [153].

Tumor progression and angiogenesis are related to a range of growth factor receptors and the activation of signaling pathways associated with these receptors. Stimulation or phosphorylation of the intracellular domain of these receptors leads to downstream signaling via Akt, PKC, and ERK kinase pathways which promote malignant cell proliferation and migratory phenotypes in tumor cells [179]. MSC exosomes are recognized to deliver their content to adjacent tumor cells and to affect tumor development with tumor-supporting or tumor-inhibiting mechanisms.

Neovascularization is known to be a critical biochemical step in tumor development and metastases. Both tumor and stromal cells in the tumor microenvironment participate a pivotal role in the regulation of tumor angiogenesis through the secretion of diverse signaling molecules and factors [184, 185]. Studies in different tumors have shown that exosomes produced from different cell types, in particular MSCs, could affect angiogenic response by targeting diverse molecules of angiogenetic signaling pathways. Latest experiments have shown that MSC-derived exosomes release components that act as angiogenic stimuli or angiogenic repressors. Thus, conflicting effects of MSC-derived exosomes on angiogenesis in different tumors may arise due to several elements, for example, the variance in the form of MSCs (i.e., BM-MSCs versus tissue-derived MSCs), the variability of MSC growth conditions, and the different dosage or interval of MSC infusion. Exosomes derived from MSCs have been shown to dramatically increase the expression of VEGF in tumor cells by activation of ERK1/2 and p38 MAPK pathways, sustaining tumor growth, and angiogenesis [182]. Also, AT-MSC-derived exosomes can deliver miR-125a as a proangiogenic factor to endothelial cells and consequently facilitate the development of endothelial tip cells and angiogenesis by suppressing the expression of repressing angiogenic inhibitor delta-like 4 (DLL4) expression [111]. Conversely, exosomal miR-16 has been reported to significantly reduce VEGF expression in breast cancer cells leading to the inhibition of angiogenesis in vitro and in vivo [169]. Additionally, Pakravan et al. revealed that the transfer of miR-100 from human BM-MSC-derived exosomes to breast cancer cells decreased VEGF expression and secretion by modulating the mTOR/HIF-1/VEGF signaling axis [173]. MSC-derived exosomes can also be used as an efficient modulating angiogenetic factor for tumor therapy.

Tumor metastases initiate with the separation of the tumor cells from the primary neoplasm and moving to the distant organ site, which includes a series of distinct multistep biological events. Any of these events are influenced by the biological and molecular alteration of cancer cells and the collaboration of stromal cells in the microenvironment of the tumor [186, 187]. Emerging results have confirmed that MSC-derived exosomes contribute to tumor metastases and the development of a pre-metastatic niche [188]. Studies in MCF-7 breast cancer cells revealed that exosomes secreted by AT-MSCs could successfully upregulate Wnt/-catenin signaling pathways and increase expressions of Wnt target genes such as Axin2 and Dickkopf-1 (Dkk1), thereby facilitating the progression and migration of cancer cells [161]. Similarly, MSC-derived exosomes promote the migration and invasion of HGC-27gastric cancer cells via enhancing the development of epithelial-mesenchymal transition (EMT) and boosting the expression of mesenchymal markers, and reducing the expression of epithelial markers in HGC-27 cells. It was further demonstrated that the expression of OCT4 and Lin28B increased in HGC-27 cells after treatment with MSC exosomes. Besides, these exosomes promoted the development and metastatic potential of HGC-27 cells by activating the Akt signaling pathway [163].

On the other hand, several reports are demonstrating that treatment with MSC-derived exosomes could prevent tumor migration and invasion. Shimbo et al. found that the transfer of miR-143 enriched exosomes into osteosarcoma cells may substantially alter the physiological features of osteosarcoma cells and suppress the migration ability of these cells [189]. Moreover, Ono et al. have demonstrated that the exosomal transfer of miR-23b from the BM-MSCs to breast cancer cells suppressed the target gene, myristoylated alanine-rich C-kinase substrate (MARCKS), which is responsible for promoting cell cycling and motility. Therefore, these findings suggest that exosomes maintained breast cancer cells in dormancy and prevents their recurrence [172]. In conclusion, these observations show that MSC-derived exosomes affect cell-to-cell communication from different pathways which may be essential for the modulation of tumor metastases.

Exosomes are recognized as promising natural nanovesicles for clinical therapeutic and diagnostic applications due to their effective biocompatibility and biodistribution properties. Further, engineered/modified exosomes can be used as effective therapeutic options for cancer because of their properties as natural important intercellular contact mediators and their innate ability in packaging and transition of therapeutic agents such as medicines, certain unique mRNAs, miRNAs, regulatory miRNAs, lipids, and proteins [190]. Over the past decades, the use of MSCs and their unlimited capacity in the development of exosomes as a nanocarrier in anticancer therapy have been extensively studied. It has been shown that the induction of MSCs to secrete exosomes loaded with antagomyR-222/223 and systemic administration of these exosomes could sensitize breast cancer cells to carboplatin-based therapy and enhance the cell dormancy of cancer cells to increase host survival in breast cancer [160]. Furthermore, it has been reported that MSC-derived exosomes loaded with anticancer protein, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), elicited considerable apoptosis in a series of cell lines [191]. Besides, the use of exosomes as a natural drug delivery vehicle has several benefits over artificial delivery systems as exosomes are derived from natural cell supplies with low immunogenicity and reduced toxicity following delivery to the recipient cells. On the other hand, exosomes have a phospholipid bilayer structure that expresses ligands and receptors that can bind with specific ligands and receptors or target cell membranes and internalize their encapsulated contents to target cells. The small size of exosomes increases their ability for long circulation and thus inhibits deterioration or elimination of reticuloendothelial (RES) organs, enhances their extravasation from the blood vessels, and ultimately penetrates deep tissue. The capacity of exosomes in the sustained release of a specific amount of drugs increases their therapeutic potency due to extended circulation of drugs and their accumulation in the recipient cells [192, 193]. Meanwhile, MSC-released exosomes loaded with paclitaxel (PTX) demonstrate the potent anticancer activity by inhibiting the proliferative activity of the CFPAC-1 pancreatic cell line [194]. Also, it has been shown that PTX-loaded MSC-derived exosomes hindered the development of different tumor cell populations in vitro and obstructed the appearance of metastasis in primary tumors in vivo [195]. Despite the promising future of exosomes as a drug vehicle for the treatment of different forms of the tumor, the lack of consistent protocols for the production, isolation, and purification of exosomes leads to contradictory findings that limit their usage. Variable conditions in the MSC culture medium and the harvesting of exosomes, as well as the type of tumor, may affect the pro- or anti-tumor attributes of the MSC exosomes. These discrepancies in the features of MSC exosomes can vary the microenvironment of the tumor and the systemic environment of the host from tumor inhibition to tumor promotion, thereby affecting the outcome of treatment [37, 196]. Therefore, the achievement of favorable clinical effects with limited undesired toxicity requires further exploration in the field of engineering exosomes accompanied by advancement in the production and isolation of exosome techniques.