Extracellular vesicles: a new frontier in diagnosing and treating graft-versus-host disease after allogeneic hematopoietic cell transplantation.

Introduction

Graft-versus-host disease (GvHD) is a serious complication of allo-HSCT [ 1 ]. According to the latest survey statistics, the cumulative incidence of acute GvHD (aGvHD) reported by each center within 100 days was 62%. After verification by the endpoint review committee, the incidence rate has been revised to 49%. 40% of all patients after allo-HSCT experience aGvHD (grades II-IV), with 19% having severe aGvHD (grades III-IV) in patients who received a transplant during the period 1990–1995. The incidence of aGvHD is declining over time. Among patients who received transplants between 2011 and 2015, the incidence of aGvHD in grades II-IV was 28%, and the incidence of grades III-IV aGvHD was 11% [ 2 – 4 ]. In recent decades, the primary clinical treatment of GvHD still relies on inducing overall immunosuppression ( 5 – 6 ). However, due to the need for more accuracy of this method, it leads to a high non-recurrence mortality rate secondary to infection. Also, it increases the risk of resistance to treatment and relapse of blood cancer [ 6 ]. Therefore, exploring new prevention and treatment methods in the clinic is urgent.

Extracellular vesicles (EVs) constitute a group of membranous vesicles enclosed by a lipid bilayer ranging from the nano- to micrometer scale ( 7 – 8 ). Many recent studies have indicated that EVs are vital in the genesis, progression, diagnosis, treatment, and prognosis of immune disorders [ 9 – 12 ]. EVs can be discharged into the extracellular milieu via the active secretion of viable cells or the exfoliation of dead cells, thereby being ubiquitously present in diverse body fluids and cell supernatants [ 13 ]. Because of their contents, various proteins, nucleotides, lipids, and metabolites are often used as carriers for various diseases [ 14 ]. Furthermore, these components can precisely reflect the characteristics of the source cells, so EVs are also valuable biomarkers for disease diagnosis [ 13 , 15 ]. Studies have shown that the components of EVs in the serum of patients after allo-HSCT are unique. Hence, EVs have potential value in early diagnosis and monitoring of the prognosis of GvHD [ 16 – 18 ]. Furthermore, clinical and preclinical studies have shown that EVs, as an essential component of paracellular secretion, have biological functions similar to those of parental cells ( 19 – 20 ), such as immune regulation, proliferation promotion and inhibition of apoptosis, and anti-inflammatory and antioxidant functions. Mesenchymal Stromal/Stem Cells (MSCs) are pluripotent stem cells with self-renewal and multi-differentiation potential [ 21 – 23 ]. MSCs are a potential treatment for GvHD due to their multiple biological properties, such as immune regulation [ 24 ]. In recent years, it has been widely documented that EVs derived from mesenchymal stem cells (MSCs) are capable of evading numerous side effects and substituting for MSCs in exerting therapeutic functions, concurrently significantly enhancing the clinical manifestations and mortality of GvHD [ 25 – 27 ]. In addition, researchers have also found that EVs can serve as an excellent delivery carrier [ 28 ], and the surface and interior of its lipid bilayer can be personalized and loaded with some tissue-targeted molecules or enhanced therapeutic drugs to improve its efficacy in intervening GvHD [ 29 ].

Literature reviews on EVs and GvHD reported thus far have mainly emphasized the role and mechanism of EVs in the pathogenesis and progression of GvHD. In contrast, our review will concentrate primarily on the clinical diagnostic value of EVs in diagnosing GvHD and the translational application value of MSC-EVs in treating GvHD. More specifically, this review will discuss the occurrence and development of GvHD from multiple standpoints, including the pathogenesis, classification, and the latest therapeutic progress of GvHD, and review the progress of applying EV-based diagnosis and treatment in GvHD, as well as future clinical application prospects.

Classification and pathogenesis of GvHD

Allo-HSCT is an efficacious modality for managing diverse malignant hematological diseases, including leukemia, lymphoma, myelodysplastic syndrome, aplastic anemia, and multiple myeloma [ 30 – 32 ]. In 1990, the results of the first survey conducted by the European Bone Marrow Transplant Group indicated that a total of 143 centers reported 4,234 hematopoietic cell transplants (HCT). As of 2019, a total of 700 centers from 51 countries reported that the number of HCT in Europe and its partner countries reached 48,512, among which 19,798 cases (41%) were allogeneic transplants and 28,714 cases (59%) were autologous transplants ( 33 – 34 ). In 2018, over 22,000 cases of HCT were reported in the United States [ 35 ]. Currently, haploid HSCT (haplo-HSCT) is widely used worldwide, and with the successful application of haplo-HSCT, the number of hematopoietic HSCT cases has increased rapidly. Over the past three decades, the scale of haplo-HSCT in Europe has witnessed a remarkable increase. By 2019, the quantity of haplo-HSCT had nearly multiplied by eight [ 34 , 36 ]. Data from the Chinese Hematopoietic Stem Cell Transplantation Registry showed that in 2019, almost 10,000 cases of allo-HSCT were performed in 140 medical institutions in China, among which haplo-HSCT accounted for 60% [ 37 – 38 ]. Although the therapeutic efficacy of allo-HSCT in the treatment of malignant hematological diseases has been continuously improved, the frequent occurrence of complications such as GvHD, infections, toxic reactions, and relapses still constitutes significant factors restricting the success of transplantation.

GvHD is caused by the active attack of T lymphocytes on allogeneic donor grafts against alloantigens in recipients after allo-HSCT, primarily targeting the skin, gastrointestinal tract, and liver, manifesting as skin congestion, maculopapular rash, and intractable diarrhea ( 4 – 5 ). Based on the timing of the GvHD after allo-HSCT, GvHD is mainly divided into two forms: acute GvHD (aGvHD) that occurs within 100 days and chronic GvHD (cGvHD) within more than 100 days. In 2005, the National Institutes of Health (NIH) of the United States refined this classification and distinguished GvHD based on disease characteristics [ 39 ]. aGvHD mainly affects the skin, liver, and gastrointestinal tract, while cGvHD may affect any body organ, and its diagnosis is not time-limited [ 40 ]. The pathophysiological process of aGvHD is mainly divided into three stages: the initiation phase, the phase of T cell activation, and the effector phase [ 4 ]. At the initiation stage, the intervention of chemotherapy and/or radiotherapy induces the release of danger signals in the host tissues, such as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), which further activates the host antigen-presenting cells (APC). APC mainly includes professional APC (dendritic cells) and non-professional APC (epithelial and mesenchymal cells) [ 41 ]. APC, beyond merely presenting self-antigens and allogeneic antigens to invigorate pathogenic T cells, orchestrates the development of GvHD by penetrating tissues and organs, secreting a cascade of inflammatory cytokines, and generating both autoantibodies and alloantibodies via an array of intricate mechanisms [ 42 ]. In contrast to aGvHD, donor and host APC can initiate CD4-mediated cGvHD [ 43 ]. Therefore, blocking donor T cells from recognizing the recipient’s alloantigen is a promising strategy for preventing GvHD [ 44 ]. In the subsequent stage of T cell activation, the host APC activates the allogeneic donor CD4 + and CD8 + T cells. Subsequently, in the effector stage, effector T cells and pro-inflammatory cytokines lead to the damage or apoptosis of tissues such as the gastrointestinal tract, skin, and liver, thereby triggering the emergence of aGvHD symptoms ( 4 – 5 ). Compared with aGvHD, the pathogenesis of cGvHD is more complex. It is usually divided into three stages: acute inflammation, chronic inflammatory immune dysregulation, and abnormal tissue repair leading to continuous fibrosis ( 45 – 46 ). During the first stage, the inflammatory signals initiated by the injury of the host tissue stimulate the activation of donor alloreactive T cells, thereby causing damage or apoptosis of the host target cells. In the second stage, the peripheral tolerance of regulatory T cells and B cells is lost, concurrently, self-reactive and alloreactive T cell populations emerge. These factors contribute to the persistence of the T17 cell-mediated inflammatory response. In the third stage, activated macrophages stimulate the generation of transforming growth factor-β (TGF-β) and platelet-derived growth factor-α (PDGF-α), which further impairs the function of regulatory T cells and intensifies the inflammation [ 47 ]. The abnormal tissue repair process results in continuous fibrosis, and patients may present with scleroderma or obliterative bronchiolitis syndrome. Moreover, the donor B cell’s steady state and tolerance mechanism are disrupted, leading to decreased memory function. Donor T follicular helper cells (Tfh) have been verified to proliferate in secondary lymphoid organs and, by secreting cytokines such as IL-21 and B cell activating factors, facilitate the survival and proliferation of donor B cells and their differentiation into plasma cells that generate abnormal anti-host immunoglobulins [ 48 ]. B-cell lymphocytopenia and humoral immunodeficiency are prominent features of cGvHD, which may result in the body not producing sufficient antibodies or antibody deficiency, resulting in increased susceptibility to infection by pathogens [ 49 ]. Therefore, preventive strategies that block the development of pathogenic B cells or inhibit Tfh cells may better prevent cGvHD (Fig. 1 ) [ 46 , 50 ]. In addition to T and B cells, macrophages, NK cells, and other immune microenvironment cells also play a role in aGvHD and cGvHD [ 49 ].

Classification, biosynthesis, release, and uptake of EVs

EVs are membrane vesicles of nanoscale to microscale size that are actively released or shrunk by cells into the extracellular environment. According to EVs’ biological process, diameter size, and biophysical and chemical properties, the International Society for Extracellular Vesicles has further classified EVs [ 8 , 51 ]. According to the diameter, they are divided into apoptotic bodies (ABs), microparticles, microvesicles (MVs), nanovesicles, and nanoparticles; according to the different cellular and tissue sources, they are divided into prostasomes, dexosomes, oncosomes, synaptic vesicles, cardiosomes, and vexosomes; and according to function, they are divided into matrix vesicles. According to the extracellular secretion characteristics, they are divided into exosomes, exosome-like vesicles, and ectosomes. Exosomes are vesicles formed by the fusion of multivesicular bodies (MVBs) and the plasma membrane. Microvesicles are vesicles formed by the plasma membrane directly to the outgoing bud, and apoptotic bodies are vesicles released by apoptosis [ 52 ]. Current research has mainly focused on the following three categories: apoptotic bodies, microvesicles, and exosomes. After their initial discovery, EVs were considered to be cell fragments. EVs transfer bioactive proteins or genetic material carried by their internal or surface from donor cells to recipient cells through direct membrane fusion, ligand-mediated interaction, a variety of cell endocytic pathways, and other mechanisms, thus playing a variety of biological functions (Fig. 2 ) [ 7 , 52 ]. Tissues and cells in a diseased state specifically express specific ligand molecules. Based on this physiological and pathological process, researchers have designed various specific targeting short peptide molecules such as RVG (Rabies virus glycoprotein) peptide, RGD (Arg-Gly-Asp) peptide, and Kim-1 (Kidney injury molecule-1) peptide to modify the surface of EV vesicles, thereby achieving precise drug delivery [ 28 , 53 – 55 ]. In addition, the ligand of CXCR4 (C-X-C chemokine receptor type 4), namely SDF-1/CXCL12 (Stromal cell-derived factor-1), is highly expressed in the inflammatory site. Research indicates that EVs overexpressing CXCR4 can specifically be home to the inflammatory regions, which offers novel ideas for treating diseases such as ulcerative colitis, tumors, osteoporosis, and periodontitis [ 56 – 59 ]. Recently, Stanford et al. [ 60 ]. have developed bioengineered multifunctional EVs for targeted delivery of biological agents to T cells. These EVs can specifically target T cells and actively enrich target cargo molecules. Furthermore, this technology significantly improves the efficiency of uptake and fusion of engineered EVs by receptor cells. In contrast to traditional engineering approaches, this novel method does not require the isolation of EVs synthesized by parental cells in vitro. Instead, it enables in situ engineering of parental cells in vivo through a one-step process, allowing the biologically synthesized multifunctional EVs to be directionally transported via the blood circulation to target cells and directly exert their functions.

After in-depth studies over the past decades, various biological effects of EVs have gradually emerged, including mediating intercellular signaling, antigen presentation, immune regulation, and promotion of angiogenesis ( 61 – 62 ). EVs, particularly those derived from MSCs, exert a favorable therapeutic impact in various refractory diseases, including diabetes, systemic lupus erythematosus, spinal cord injury, malignant hematological disorders, and the associated GvHD [ 27 ]. In addition, EVs also have good biocompatibility, low immunogenicity and toxicity, biodegradability, and other advantages, and have gradually become crucial in nanomedicine research [ 28 , 55 ]. Engineered EVs also bring new hope for personalized and accurate treatment of various diseases [ 7 , 55 , 63 ]. The lipid bilayer of EVs is wrapped with many proteins, nucleotides, lipids, and metabolites, which are used as biomarkers for diagnosing and treating disease. Various cells secrete and release EVs in different disease conditions, carrying proteins, nucleic acids, and lipids. The content carried by EVs serves as a reflection of the cellular state of the parent cell at the time of secretion, and these components undergo dynamic adjustments in response to changes in the physiological state and environment of the parent cell during EV secretion. Consequently, by analyzing these EVs, we can accurately and precisely reflect alterations in the pathophysiological state of their originating parent cells. Furthermore, given that EVs have specificity and stability of cell types and can be obtained from body fluids, various components within EVs can become suitable biomarkers for early diagnosis, treatment, prognosis, and other diseases. EVs as biomarkers are essential for individualized disease diagnosis and monitoring [ 64 ].

The diagnostic value of EVs in aGvHD and cGvHD

aGvHD and cGvHD represent some of the most vital long-term complications ensuing from allo-HSCT and constitute the prime cause of non-relapse mortality [ 47 ]. aGvHD and cGvHD not only significantly reduce long-term survival and quality of life for patients post-transplantation but have also been associated with a variety of other comorbidities. Although current research has improved our understanding of the pathogenesis of aGvHD and cGvHD, reliable biomarkers for predicting the occurrence of aGvHD and cGvHD have yet to be determined. Since 30–70% of patients develop GvHD after allo-HSCT [ 40 ], it is urgent to identify appropriate early diagnosis and prognostic markers for the early detection and intervention of this post-transplant complication. A diverse array of biomarkers for GvHD, encompassing genetic, plasma, cellular, and microbiome-related markers, is being progressively explored [ 65 ]. Within the clinical realm, dependable serum biomarkers for the diagnosis and differential diagnosis of aGvHD and cGvHD remain scarce. Endoscopy, other supplementary examinations, and histopathological assessment still constitute the predominant methods for the diagnosis and differential diagnosis of aGvHD. Liquid biopsy is a non-invasive blood testing approach that analyzes and diagnoses diseases like cancer by monitoring biomarkers such as free circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), exosomes, and circulating RNA that are released into the blood by tumors or metastatic foci [ 66 ]. With the rapid evolution of liquid biopsy technology, the screening of biomarkers for the diagnosis and prognosis of GvHD in blood, urine, and other biological fluids holds considerable value. As an essential component of liquid biopsy, EVs are expected to be an attractive biomarker for the diagnosis and prognosis of GvHD due to their high number and easy capture.

The source or quantity of circulating EVs and their diagnostic application in GvHD

The incidence and mortality of cardiovascular events in allo-HSCT survivors are significantly increased, with endothelial dysfunction and thrombosis risk being the main contributors. Therefore, early monitoring of vascular injury and prothrombotic activity in allo-HSCT survivors may be an essential strategy for preventing and treating cardiovascular diseases. In establishing and developing plant resistance to viral host, the number, membrane antigen subgroups, and contents carried by EVs undergo significant changes. Studies have shown that circulating EVs derived from allo-HSCT survivors may be an evaluation index of endothelial injury and procoagulant activity [ 67 ]. The study recruited 45 survivors post-allo-HSCT and 45 controls. Most patients who underwent allo-HSCT experienced aGvHD (44%) and/or cGvHD (66%). The authors found that allo-HSCT survivors had significantly increased levels of platelet and erythrocyte-derived EVs compared with the control group [ 67 ]. Furthermore, among allo-HSCT survivors with a history of thrombotic microangiopathy, erythrocyte-derived MVs are markedly elevated. Several studies have shown that the number of EVs in the circulating blood of patients with GvHD is significantly increased, and the increase in the secretion of EVs is significantly associated with the risk of aGvHD ( 16 – 17 ). Carneiro et al. [ 16 ]. found that in patients with circulating EVs in plasma, high levels of total EVs (TEVs) and red blood cell-derived EVs (EryEVs) counts strongly correlated with the occurrence of aGvHD. In contrast, platelet-derived EVs (PEVs) were not associated with the risk of aGvHD. Some studies have shown that EVs derived from red blood cells, platelets, or vascular endothelial cells may be a new class of biomarkers. The proportion and functional alterations of T cell subsets are paramount for the occurrence and progression of GvHD. Moreover, T cells released EVs at a constant low level, which suddenly increased after activation. Hence, detecting the variations of EV subpopulations derived from T cells might offer a more direct and efficient means for monitoring the occurrence of GvHD. Nagasawa et al. [ 17 ]. discovered that T cell-specific CD3 + CD8 + EVs and CD3 + HLA-DR + EVs levels were strongly correlated with the risk of aGvHD. By examining the EVs derived from the peripheral blood of 20 patients who underwent HSCT, they ascertained that the levels of CD3 + CD4 + EVs and CD3 + CD8 + EVs were correlated explicitly with the counts of CD4 + and CD8 + T lymphocytes, respectively. Meanwhile, CD3 + CD8 + EVs and CD3 + HLA-DR + EVs escalated in patients with GvHD and were more specifically competent in reproducing the persistence of GvHD than soluble interleukin 2 receptor (sIL-2R). Engraftment syndrome (ES) is a non-infectious fever syndrome that typically occurs during the recovery of neutrophils after HSCT, and its clinical manifestations include fever, rash, and pulmonary infiltration ( 68 – 69 ). This syndrome can occur in patients undergoing both autologous and allo-HSCT. Because the symptoms of ES are similar to those of aGvHD, it was once called super-acute GvHD [ 17 ]. In engraftment syndrome, sIL-2R was significantly elevated, whereas CD3 + HLA-DR + EVs were not. Furthermore, ferritin and sIL-2R were conspicuously elevated in pretransplant hemophagocytic syndrome; However, CD3 + HLA-DR + EVs showed little change. CD3 + CD4 +− , CD3 + CD8 +− , and CD3 + HLA-DR +− EVs can effectively stimulate cell-mediated immune responses. The proportion and functional alterations of T cell subsets exert significant roles in the occurrence and development of GvHD. Thus, detecting the variations of EV subsets derived from T cells might constitute a more direct and practical approach for monitoring the occurrence of GvHD. In contrast to other conventional biomarkers, CD3 + CD8 + and CD3 + HLA-DR + may exhibit superior performance in in monitoring and assessing aGvHD. The researchers independently developed a novel EV technology platform for automatic acoustic capture and separation to overcome the technical bottleneck of separating EVs from small plasma volumes. They found that the amount of EV captured in plasma increased more than two-fold in most patients after allo-HSCT [ 18 ]. The expression of platelet-specific miR-142-3p in EVs after transplantation is closely correlated with the expected platelet count dynamics. More importantly, the authors also found that some miRNAs in plasma EVs were strongly correlated with the occurrence and development of GvHD. These research results imply that the monitoring of circulating EVs is expected to become a valuable biomarker for the occurrence and development of GvHD and the prognosis of treatment (Fig. 3 ).

The membrane antigen components of circulating EVs and the diagnostic application of GvHD

Studies have demonstrated that specific membrane components of EVs, such as lipids and membrane proteins, as well as their contents, including nucleotides and metabolites, are closely associated with the occurrence and progression of diseases. Recently, studies have shown that surface antigens of EVs have a potential correlation with the occurrence of aGvHD ( 70 – 71 ). Baur et al. [ 72 ]. discovered that circulating EVs in the plasma of patients after allo-HSCT, expressed PD-L1 and PD-L1 + EVs, attained the peak of PD-L1 expression 6 weeks after allo-HSCT. The expression of PD-L1 in these EVs was associated with their capacity to inhibit T cells and the emergence of GvHD. The development of aGvHD is closely related to the appearance of EVs with high PD-L1 expression in allo-HSCT post-transplantation. Moreover, the expression level of PD-L1 in patients is positively correlated with the severity of GvHD. And PD-L1 expression levels will significantly decrease only after successful treatment of GvHD. The above results suggest that PDL1 EVs may be used to monitor the clinical efficacy of GvHD treatment. In another study, Lia et al. [ 70 ]. detected serum EVs of 41 patients with multiple myeloma who received allo-HSCT using 13 antibodies against specific membrane proteins and found that the membrane antigens CD146, CD31, and CD140-α + EVs had a strong correlation with the appearance of aGvHD. Among them, CD146 (MCAM-1) increased the risk of aGvHD by nearly 60%, whereas CD31 (PECAM-1) and CD140 (PDGFR) decreased the risk of almost 40% and 60%, respectively. The emergence of EVs with differential expression of membrane antigens indicates that the risk of aGvHD significantly increases for patients after transplantation. The variations of EV subpopulations may precede the clinical manifestations of GvHD, offering crucial early warning information for the early prevention and clinical intervention of GvHD. In a follow-up study, Lia et al. [ 71 ]. further observed and monitored the expression profiles of CD146, CD31, CD140a, CD120a, CD26, CD144, CD30, and their miRNA loading on EVs in 32 consecutive patients, which showed a significant correlation with the pathogenesis of aGvHD. They also found that the expression profile of EVs and plasma biomarkers (such as ST2, sTNFR1, and REG3a) potentially correlated with aGvHD. The combination of CD146, sTNFR1, and miR-100 or miR-194 in EVs was closely related to the pathogenesis of aGvHD. Its diagnostic efficacy, indicated by an AUROC (area under the receiver operating characteristic curve) > 0.975, suggested that the expression profile of EVs was expected to be a promising biomarker for the early diagnosis of aGvHD. The above studies have demonstrated that the abnormal alterations in the expression profiles of membrane antigen components of EVs and their carried contents, such as nucleotides or proteins, are closely associated with the disease occurrence and progression of GvHD. The individual or combined detection of EV components is anticipated to become an excellent biomarker for the early diagnosis or prognosis monitoring of aGvHD.

The nucleotide components of circulating EVs and their diagnostic application in GvHD

Recent studies have shown that miRNAs encapsulated by circulating EVs in patients are expected to be promising biomarkers for the early monitoring of aGvHD and cGvHD. Lim et al. [ 18 ]. showed that miRNAs in plasma EVs can predict GvHD and correlate with disease severity and patient survival. The authors utilized acoustic fluidic capture technology to capture EVs in plasma samples from 20 consecutive patients with high-risk/relapsed hematological malignancies 12 weeks pre- and post-allo-HSCT. They ascertained that the number of EVs in most patients increased by more than 2-fold after transplantation. Through the analysis of the miRNA expression profiling of EVs, the expression of miR-15b-3p, miR-30a-5p, miR-130a-3p, miR-145-5p, and miR-342-3p in plasma EVs was conspicuously augmented after transplantation; in contrast, the expression of miR-18a-5p, miR-92b-3p, miR-93-5p, miR-141-3p, and miR-486-5p was significantly diminished. Importantly, they found that upregulated miRNAs in plasma EVs were positively correlated with infection and GvHD, respectively, while the downregulated miRNAs were negatively correlated with these complications. Furthermore, platelet-specific miR-142-3p expression was also associated with expected platelet count dynamics in graft EVs. This study reveals that it is crucial to construct a rapid separation technology platform for plasma EVs based on acoustic principles and elucidate EV miRNA’s important significance as a potential biomarker for early prevention and diagnosis of infection or GvHD after allo-HSCT. In another study, Lacina et al. [ 73 ]. performed the detection of miRNA expression profiles in 3 patients with cGvHD and 4 patients without cGvHD and no disease symptoms and identified that there were three differentially expressed miRNAs in patients with cGvHD, namely hsa-miR-29c-3p, hsa-miR-374b-5p, and hsa-miR-630. Among them, the latter two levels were significantly decreased by 2.7-fold and 4.1-fold, respectively, while the expression of hsa-miR-29c-3p was elevated considerably by 5.8-fold. These results suggest that the miRNA profiles of plasma EVs might be used as markers for the pathogenesis of cGvHD.

In 2005, the new consensus standard classification established by the National Institutes of Health (NIH) defined aGvHD that occurs more than 100 days after transplantation as late-onset aGvHD (LA GvHD) [ 39 ]. According to this NIH definition, approximately 20–40% of patients with cGvHD may be reconsidered as LA GvHD. LA GvHD is also associated with higher non-relapsed mortality after allo-HSCT compared to non-GvHD [ 74 ]. The underlying mechanism of LA GvHD remains poorly understood, and the identification of differentially expressed miRNAs in EVs is of significant importance in revealing the role of miRNAs in this disease. Yoshizawa et al. [ 75 ]. collected EVs from the plasma of 5 patients with LA GvHD, 5 patients with non-GvHD, and 8 healthy controls and performed miRNA expression profiling. They identified 55 miRNAs with differential expression between LA GvHD and non-GvHD. Among them, through conducting further verification on the 10 miRNAs (such as miR-19a, miR-30c, miR-125b, miR-128, miR-142-3p, miR-191, miR-193a, miR-193b, miR-423-5p, and miR-574-3p) that were most differentially expressed, miR-128 was upregulated to a significantly greater extent in the pathogenesis of LA GvHD, with an area under the curve (AUC) of 0.975. Previous studies have indicated that miR-128 shows abnormal expression in the tissues and blood samples of some patients with malignant tumors and is associated with tumor progression. Moreover, MirTarBase analysis showed that miR-128 target genes were involved in the immune system and inflammatory response. These results further imply that miR-128 present within EVs is expected to act as a novel and non-invasive biomarker for the early diagnosis of LA GvHD (Table 1 ). Overall, the attribute of circulating EV miRNA constitutes a non-invasive and highly promising biomarker for the early diagnosis of acute, chronic, and late-onset GvHD.

Circulating EVs and the medication and prognosis monitoring of GvHD

Treatment with rabbit anti-T lymphocyte globulin (ATLG) is an effective strategy for the prevention of GvHD, and its effectiveness in preventing GvHD has been demonstrated in randomized studies. However, its impact on relapse remains a significant concern for broader application. Therefore, the early adjustment of the ATLG dosage in line with the host characteristics is expected to minimize its side effects (such as immune reconstitution, recurrence, and infection), representing an effective means to improve the clinical intervention of ATLG. Storci et al. [ 76 ]. found that the serum ATLG activity level was significantly decreased in 23 patients with GvHD. Meanwhile, the concentration of CD69 + EVs in patients with GvHD increased at the time before infusion. The researchers obtained consistent results in 12 other validation cohorts of HSCT patients who received ATLG treatment. These results suggest that high levels of serum CD69 + EVs are significantly correlated with an augmented risk of GvHD, and are anticipated to be an effective means for customizing ATLG dose monitoring for individualized prevention of GvHD.

Therapeutic effects of EVs in GvHD and its related complications

Long-term suppression of the host immune system and methods to improve transplant tolerance remain the main challenges in organ transplantation and GvHD [ 77 ]. MSC-EVs have been used in the treatment of a variety of immune diseases. Allogeneic human MSC products can be used in the clinical treatment of GvHD, but clinical application data indicate that these MSC products have certain limitations in treatment practice. Many patients with aGvHD respond to cell therapy of human bone marrow/umbilical cord MSCs/stem cells. However, the mechanism of these cells to improve complications related to aGvHD remains to be clarified. MSC-EVs are due to their unique biological distribution and significant dose-dependent therapeutic effects. The unique immunomodulatory properties of MSC-EVs make them particularly important in grafts in which the immune system is overactive, such as allo-HSCT. Recent studies have revealed that MSC-EVs have promising therapeutic effects in preclinical and clinical studies. Some specific miRNAs, membrane proteins, membrane lipids, and soluble factor binding components carried by MSC-EVs can treat GvHD and its related complications through various mechanisms, such as regulation of T cell-mediated adaptive immunity and APC-mediated innate immunity and direct regeneration of injured target organs.

Therapeutic effects of EVs in aGvHD

MSCs have been utilized in treating aGvHD, cGvHD, and their associated complications, as they discharge a wide range of mediators, including immunosuppressive molecules, growth factors, chemokines, and EVs (Tables 2 and 3 ) [ 89 ]. Studies have investigated the effects of MSC administration on aGvHD, with differing results. Increasing evidence suggests that MSC-derived cytokines, EVs, and other components are critical in alleviating GvHD. Kordelas et al. [ 25 ]. were the first to report a clinical case of mucosal GvHD after PBSCT for myelodysplastic syndrome. A female patient developed moderate mucosal GvHD after her first allo-HSCT using PBSC from an HLA-identical female donor for myelodysplastic syndrome at age 13. At age 20, she was diagnosed with secondary AML and underwent a second allogeneic stem cell transplant with PBSC from a male donor of the same HLA, resulting in hyperacute skin GvHD (Grade 4). The patient experienced severe intestinal and cutaneous GvHD, treated with steroids and MSC-EVs. Shortly after starting MSC-EV therapy, the pro-inflammatory cytokine response in peripheral blood mononuclear cells significantly decreased, leading to a marked improvement in GvHD symptoms: diarrhea severity was notably reduced post-treatment. Skin and mucosal GvHD also showed significant improvement within 2 weeks and remained stable even after 4 months of MSC-EV treatment. Consequently, steroid dosage was reduced from 125 mg to 30 mg per day. The patient’s condition stayed stable for several months. Although the patient ultimately passed away from pneumonia seven months after receiving MSC-EVs, these promising results suggest that MSC-EVs may offer a novel and safe approach for treating refractory GvHD and other inflammation-related disorders.

EVs improve GvHD by regulating the function of CD4 + and CD8 + T cells

Cord blood-derived multipotent stem cells (CB-SCs) were found to improve the development of the GvHD response in xenohuman PBMC-induced NSG mouse models in vivo. Mechanistically, CB-SCs improve allogeneic response in vitro, an effect associated with CB-SC-derived EVs that suppress the proliferation and activation of T cells in allogeneic mixed lymphocyte cultures [ 90 ]. The dysfunction of CD4 + T cells in GvHD may be an essential factor aggravating GvHD. A recent study by Li et al. [ 91 ]. revealed that hucMSC-EVs can efficaciously reduce damage from GvHD by alleviating disorders of redox metabolism and inflammatory cytokine bursts in CD4 + T cells. Moreover, the authors discovered that miR-16-5p, which is abundantly present in hucMSC-EVs, can alleviate endoplasmic reticulum stress and decrease the apoptosis of CD4 + T cells through targeted inhibition of the ATF6/CHOP signaling axis, facilitate the development of CD4 + IL-10 + T cells, and restore immune homeostasis during the pathogenesis of GvHD. Wang et al. [ 87 ]. found that hucMSC-EV treatment could regulate the immune response to prevent life-threatening aGvHD. Further investigations disclosed that the infusion of EVs conspicuously mitigated the clinical score and pathological impairment in mice afflicted with aGvHD and diminished their mortality rate. After EV treatment, the frequency and absolute quantity of CD3 + CD8 + T cells were significantly decreased, whereas the proportion of CD3 + CD4 + and CD3 + CD8 + T cells escalated; the concentrations of IL-2, IFN-γ, and TNF-α in serum dropped, while the level of IL-10 in serum ascended. In vitro experiments showed that EVs could dose-dependently inhibit mitogen-induced splenocyte proliferation, inhibit the expression of pro-inflammatory cytokines levels of IL-2, IFN-γ, and TNF-α, and promote the expression of IL-10. The results of this study also suggested that hucMSC-EVs are an ideal alternative for the prevention of aGvHD after allo-HSCT.

The miRNAs within EVs derived from bone marrow MSCs play a crucial role in regulating immune responses; however, the function of miRNAs in treating aGvHD remains undefined. Liu et al. [ 86 ] uncovered through high-throughput sequencing that miR-223 was strikingly highly expressed in both hucMSC-EVs and mBMSC-EVs and disclosed that miR-223 derived from MSC-EVs could diminish donor T cell migration by targeting ICAM-1, thereby mitigating the symptoms of aGvHD. Mechanistic studies showed that miR-223 could inhibit ICAM-1 expression in mouse lymphatic endothelial cells to inhibit T-cell adhesion and migration, and miR-223 AgomiR infusion could alleviate the clinical symptoms of aGvHD, reduce donor T cell infiltration into the spleen, liver, and intestinal tract, and reduce the expression of the inflammatory cytokines IFN-γ, TNF-α, and IL-17. Fujii et al. [ 79 ]. systematically administered human bone marrow MSC-EVs not only prolonged the survival of aGvHD mice but also reduced the damage of various target organs of GvHD. In GvHD mice treated with EVs, CD4 + and CD8 + T cells were inhibited, and the proportion of CD62L − CD44 + and CD62L + CD44 − T cells was reduced, suggesting that EVs can inhibit the functional differentiation of T cells from the naïve phenotype to the expression of the effector phenotype. In addition, EVs also preserved the CD4 + CD25 + Foxp3 + regulatory T-cell population. Amarnath et al. [ 83 ]. found that CD73-expressing BMMSC-EVs mediated adenosine-based immunosuppression and could improve x-GvHD. The authors first established an x-GvHD model of human CD4 + Th1 cell death and explored immunomodulation using clinical-grade BMMSC products. BMMSCs could reverse the established, lethal x-GvHD by significantly inhibiting the Th1 cell effector function. The implantation of BMMSCs was limited to the lungs without affecting regulatory T cells, suggesting that the paracrine mechanism of BMMSCs was the key mechanism mediating this effect. Further studies found that serum CD73-expressing MSC-EVs in BMMSCs recipients increased significantly and promoted adenosine accumulation in vitro. BMMSC-mediated immunomodulatory effects were wholly abolished by drug therapy with adenosine A2A receptor antagonists. To investigate the underlying mechanisms and reveal their potential clinical relevance, we examined the content of MSC-EVs in serum samples collected from patients before and after treatment with BMMSCs. We found that MSC-EVs expressing CD73 promoted adenosine accumulation in serum samples from patients after treatment with BMMSCs. BMMSCs effectively modulate experimental GvHD by promoting adenosine-based immunosuppression through paracrine activation.

EVs improve GvHD by regulating the function of regulatory T cells

Zhang et al. [ 85 ]. found that EVs derived from immortalized human embryonic stem cell-derived MSCs increased T-reg production in mice with allogeneic skin grafts but not in engrafted mice. MSC-EVs were jointly utilized in combination with mouse splenic CD4 + T cells activated by anti-CD3/CD28 monoclonal antibodies or splenic CD11c + cells that are rich in allogeneic antigen-presenting cells (APCs) to determine whether mouse CD4 + CD25 + T cells or CD4 + CD25 + Foxp3 + cells could be induced. MSC-EVs have also been utilized in a lethal human-SCID chimeric mouse GvHD model. In this model, human PBMC was infused into irradiated NSG mice to induce GvHD. MSC-EVs can induce CD4 + CD25 + T cells or CD4 + CD25 + Foxp3 + Tregs activated by allogeneic CD11c + cells rather than those activated by anti-CD3/CD28 monoclonal antibodies. This induction effect demonstrates a dose-dependent relationship with EVs. MSC-EVs attenuated symptoms of GvHD and increased survival in a murine model of a GvHD induced by a human antimouse CD4 + T cell effector stimulated by transplanted human APC. The levels of human CD4 + CD25 + CD127 low/− Tregs in surviving MSC-EVs-treated mice were significantly higher than those in surviving mice treated with the TNF inhibitor etanercept. MSC-EVs facilitate the generation of regulatory T cells in vivo and in vitro via the APC-mediated pathway.

EVs improve GvHD by regulating the function of other immune cells and the inflammatory response

MSC-EVs have been found to alleviate GvHD and preserve the graft-versus-leukemia (GVL) effect by inhibiting the immunomodulatory functions of DCs, macrophages, and T lymphocytes in allo-HSCT animal models [ 92 ]. Treatment with MSC-EVs can effectively reduce the clinical symptoms of GvHD patients after HSCT and significantly improve the survival rate of HSCT recipients. Studies in GvHD patients have shown that MSC-EVs inhibit the release of IFN-γ and TNF-α through activated NK cells, thus reducing the lethal function and inflammatory response of NK cells [ 93 ]. MiRNA microarray screening showed that miR-125a-3p was the most significantly upregulated in EV, and its downstream target genes were closely related to the inhibition of T-cell proliferation. Recently, Li et al. [ 88 ]. found that hBMMSC-EVs could reduce pathological damage caused by aGvHD and promote the survival of mice by regulating the function of DCs and T cell subsets and inhibiting the inflammatory response of mice. The intervention of EV resulted in a decrease in the expression of IL-2, IFN-γ, and TNF-α, an increase in the expression of IL-10, reduced body weight loss, clinical scores, and mortality in aGvHD mice. Further studies found that EV treatment resulted in a 7- to 8-fold increase in CD8α + and CD11b + conventional DCs and changed the subsets of helper T cells and Tregs, reducing the proliferation of cytotoxic T cells, which was conducive to inflammation suppression in aGvHD mice. Because human experimentation is strictly restricted by moral, ethical, legal, and other factors, the human-mouse GvHD model (xenogeneic GvHD, x-GvHD) has become an ideal experimental tool for GvHD research because of its reliability and stability.

Therapeutic effects of EVs in cGvHD and GvHD-related complications

cGvHD is a systemic, multi-organ syndrome; the main affected target organs include the skin, eyes, liver, and gastrointestinal tract. Norooznezhad et al. [ 84 ]. reported a second case of EV treatment in a patient with cutaneous cGVHD, showing clinically acceptable results for both the team and the patient. The results remained stable for 4 months without recurrence. This study only investigated the treatment of hPMSCs with EVs. The authors suggest that studies on BMMSC from other sources, such as bone marrow, should be carried out to treat cGvHD. In addition, changes in the environment of BMMSCs could also be considered another variable in future studies. The incidence and severity of skin ulcers, wounds, keratinization, and atrophic lesions were reduced, and wound healing was observed. Additionally, there was a significant improvement in the degree of sclerosis and dryness of the skin after treatment. Dry eye syndrome (DES) associated with GvHD is characterized by extensive inflammatory destruction of the ocular surface, leading to unbearable pain and visual impairment. Current clinical treatments have limited efficacy. Zhou et al. [ 78 ]. reported that MSC-EVs can relieve GvHD-related DES by inhibiting inflammation and improving epithelial cell recovery in mice and humans. In a prospective clinical trial, the authors found that hucMSC-EVs significantly improved the symptoms of 28 patients with refractory GvHD-related DES. After intervention with hucMSC-EVs, the fluorescein score was reduced, the tear film break-up time was prolonged, tear secretion was increased, and the OSDI score was reduced. Similarly, in a mouse model of GvHD-related DES-induced by benzalkonium chloride (BAC), the symptoms of DES were effectively alleviated after treatment with mouse bone marrow MSC-EVs (mBMMSC-EVs). Mechanistic studies found that a miR-204 molecule enriched in MSC-EVs can target the IL-6/IL-6R/STAT3 signaling axis to reprogram pro-inflammatory M1 macrophages into immunosuppressive M2 macrophages to attenuate DES. To evaluate the safety and efficacy of MSC-EVs in a transplant patient population, Lightner et al. [ 94 ]. used MSC-EVs to treat isolated intestinal transplant patients ( n = 2), liver allograft patients ( n = 2), modified multi-visceral transplantation patients ( n = 3), and GvHD patients ( n = 2). The results showed that the clinical symptoms of all patients improved within 24 h after EVs treatment, the serological laboratory evaluation indicators, the lung symptoms and skin manifestations of GvHD improved, and the graft inflammation/rejection was entirely resolved histologically within 7 days after administration.

CD4 + T cells and Th17 cells play a crucial role in the pathogenesis of cGvHD [ 47 , 49 ]. Lai et al. [ 80 ]. found that hBMMSC-EVs could effectively prolong the survival of cGvHD mice, reduce the clinical and pathological scores of mice, and inhibit the skin, lung, and liver fibrosis process by exerting powerful immunomodulatory effects on GvHD effector T cells. In the process of EVs intervention in cGvHD, CD4 + T cell activation and lung infiltration were reduced, the expression of Th17 cell-related transcription factors and pro-inflammatory cytokines was significantly reduced, and the pathogenic T cells expressing IL-17 and regulatory cells inducing IL-10 expression were inhibited. EVs blocked Th17 cell differentiation of Th17 cells from PBMCs of healthy donors and patients with cGvHD in vitro and improved the Treg phenotype. hBMMSC-EVs could enhance the survival of patients with cGvHD and their pathological injury by inhibiting Th17 cell proliferation and activity of Th17 cells and inducing Treg expression. Early administration of MSC-EVs to inhibit persistent Th17 cell-mediated inflammation may block the progression of the fibrotic stage by exacerbating overactive inflammation levels. Therefore, MSC-EVs are expected to be effective reagents for the prevention or treatment of cGvHD.

Engineering EVs for the treatment of GvHD and its related complications

Owing to its remarkable biocompatibility and physical and chemical stability, EVs are regarded as outstanding means of transportation by many researchers. Both hucMSC-EVs and arsenic trioxides (ATOs) have been shown to treat aGvHD through immunomodulation. In addition to immunomodulation, hucMSC-EVs have a unique drug delivery function, which is believed to improve their activity (Fig. 4 ). ATOs are effective drugs for the treatment of acute promyelocytic leukemia. In addition to the direct intervention of ATOs in acute promyelocytic leukemia, recent studies have also found that it can play an immunomodulatory role in inhibiting the development of autoimmune disorders such as GvHD. Kavian et al. [ 95 ]. found that applying ATOs can improve the clinical symptoms of scleroderma GvHD mice. At the same time, Liu et al. [ 96 ]. also found that ATOs can transfer macrophages to the M2 state to prolong the survival of aGVHD mice. Other research reports also show that ATOs are expected to be a new method for the treatment of immune disorders, such as experimental colitis and autoimmune encephalomyelitis, rheumatoid arthritis, and systemic lupus erythematosus [ 97 – 100 ], indicating that ATOs may be expected to be a new method for the treatment of GvHD. However, their wide clinical use is limited due to significant toxicity. Therefore, the use of hucMSC-EVs to deliver ATOs cannot only reduce their toxicity but also improve the therapeutic effect of GvHD. Su et al. [ 29 ]. prepared ATOs-loaded hucMSC-EVs to intervene in aGvHD. In vitro and in vivo results indicated that ATOs-hucMSC-EVs mainly reduced the severity of aGvHD by regulating the mTOR autophagy signal to reset inflammatory macrophages to the M2 phenotype without weakening the activity of GVL response. In another study, Zhou et al. [ 78 ]. found that miR-204 rich in MSC-EVs could effectively relieve the clinical symptoms of GvHD-related DES in both preclinical and prospective clinical trials. Subsequently, to achieve more efficient therapeutic efficacy, the authors constructed an L929 cell line with miR-204 precursor overexpression by gene manipulation and isolated L929-EVs overexpressing miR-204, significantly weakening dry eye symptoms. Establishing this engineered EV therapeutic platform also brought new ideas and hope for treating complex GvHD-related DES.

BMMSCs have been found to regulate T cell homeostasis in vivo, thus protecting patients after allo-HSCT from potentially fatal events of GvHD. Epidermal growth factor (EGF) has been reported to induce the expression of miR-21 and AKT, which improves cell survival in vitro. Additionally, the level of miR-21 is positively correlated with Foxp3 expression of Foxp3 (forkhead box P3), which affects the regulation of the natural function of Tregs. Overexpression of miR-21 has been reported to enhance proliferation, invasion, and differentiation damage to BMMSCs. Zhu et al. ( 81 – 82 ) utilized EGF to facilitate the phosphorylation of c-Jun in BMMSCs, thereby promoting the expression miR-21 in BMMSC-EVs. This led to an enhancement of the expression of Foxp3 in regulatory T cells (Tregs), thereby improving the therapeutic effect of BMSCs on aGvHD. In the aGvHD mouse model, EGF significantly increased the therapeutic efficacy of BMMSCs by reducing the expression of IFN-γ and organ damage. The skin is the site most frequently affected by acute and chronic GvHD [ 101 ]. Systemic sclerosis (SSc) and sclerodermatous-GvHD (Scl-GvHD) are chronic autoimmune diseases of connective tissue. Recent studies have shown that MSCs are a promising method for treating autoimmune fibrotic skin diseases SSc and Scl-GvHD. MSCs and their EVs play a crucial role in rebalancing immune and inflammatory disorders, resisting oxidative stress, and inhibiting overly activated fibrosis [ 102 ].

Regulatory T cells (Tregs) are a subset of CD4 + T cells, which can play a decisive immunosuppressive role in autoimmune response diseases, including aGvHD, and some miRNAs are involved in the pathophysiological process of GvHD. EVs secreted by MSCs stimulated by TGF-β combined with IFN-γ have stronger immunosuppressive activity. They can promote monocyte transformation into Tregs more effectively, among which IDO may be a key mediator [ 103 ]. In addition, Wang et al. [ 104 ]. found that apoptotic bodies released by IFN-γ/TGF-β1-stimulated apoptotic MSCs significantly inhibited the proliferation of T cells, increased the number of Tregs, maintained the activity of immunoregulatory T cells, and reduced the expression of pro-inflammatory T cells. These research discoveries suggest that the constituents derived from MSCs induced by various stimulating molecules (such as molecules like EVs and apoptotic bodies) likewise offer novel strategies for treating of GvHD and other immune-related disorders.

Studies have shown that the therapeutic MSCs used for therapeutic purposes, as well as EVs secreted by host vascular cells, and these EVs themselves, express varying levels of high procoagulant tissue factor (TF/CD142) on their surfaces [ 23 , 105 – 107 ]. TF/CD142 associated with EVs is a crucial endothelial injury and coagulopathy biomarker. Intravenous infusion of MSC may affect the cell’s hematological compatibility and safety [ 108 – 110 ]. Therefore, clinicians must be cautious when handling MSC or EV therapy to prevent potentially fatal thromboembolic events during infusion or blood contact, especially in patients with potential coagulopathy, such as GvHD and COVID-19. The immunogenicity of EVs constitutes a significant factor restricting their future clinical transformation and application [ 111 ]. At present, basic research has yet to concentrate entirely on this challenge. Hence, it is particularly urgent to gain an in-depth understanding of the various factors influencing the potential immunogenicity of EVs and the strategies for reducing immune recognition to enhance therapeutic efficacy. Hence, eliminating or reducing the expression of TF/CD142 and other immunogenic genes on the surface of MSCs and their derived EVs through CRISPR (Clustered regularly interspaced short palindromic repeats) technology and different approaches can enhance the biocompatibility of MSC-related products after intravenous infusion. These strategies might serve as effective measures to augment the therapeutic efficacy of MSCs or their derived EVs in treating GvHD.

Discussion and perspectives

In the past few decades, studies have shown that EVs have the potential for clinical translation and application in diagnosing and treating various diseases, including GvHD, which has attracted widespread attention from the medical research and clinical application fields. EVs can be secreted and released by almost all cells and are widely present in cell culture supernatant and various body fluids, such as blood, urine, cerebrospinal fluid, and saliva. The existence of the natural lipid bilayer structure of EVs can protect the internal cargo from the degradation of cytoplasmic enzymes to allow the carrying of various functional molecules such as nucleic acid, protein, lipid, metabolites, and other components stably in the extracellular environment; this unique advantage makes EVs a circulating marker of clinical diagnosis and transformation. Many studies have shown that the number of circulating EVs, membrane protein, and internal nucleic acid molecules are significantly different in the occurrence and development of aGvHD and cGvHD, which further suggests that EVs may be used for the prevention, diagnosis, and prognosis monitoring of GvHD as well as guidance of clinical medication. By revealing the expression profiles of EV proteins and nucleic acids and the secretion levels of EVs in different cell subsets during the occurrence and development of GvHD, it is expected to become a promising biomarker for the prevention, diagnosis, and monitoring of disease progression and patient survival. However, challenges persist with insufficient sample sizes, lag isolation methods, and low EV capture yields in EV-based clinical biomarker studies. To overcome the technical bottleneck of EV separation from small plasma volume and improve the yield of EV capture, the researchers independently developed a novel EV technology platform for automatic acoustic capture and separation [ 18 ]. The improvement of this technique holds great promise for the early diagnosis of EV-based clinical biomarkers of GvHD. In recent years, the EV separation technology has continuously improved, especially with the emergence of microfluidic and other technologies, so that the cost of EV separation has reduced, the required sample size has reduced, and the capture yield of EV has continually improved [ 112 ]. The translational clinical application of plasma-derived EVs in transplant-related diseases will have a broader scope for application.

EVs have biological functions similar to mother cells and advantages such as low immunogenicity, good biocompatibility, and a greater tendency to reach the injured site. Therefore, in recent years, scientists are gradually trying to use natural EVs, especially MSC-EVs, to treat complex and refractory diseases, including diabetes [ 113 ], systemic lupus erythematosus [ 114 ], rheumatoid arthritis [ 115 ], malignant hematological tumors [ 116 ], GvHD [ 12 , 27 ], and other immune disorders [ 117 ]. Studies have shown that fibrosis caused by collective barrier surface regeneration failure and maladaptive repair plays a vital role in the pathogenesis and progression of GvHD. Furthermore, median survival, GvHD clinical score, and histological score improved in mice treated with MSC-EV compared to untreated GvHD mice, and prophylactic treatment with MSC-EV reduced the severity of GvHD and improved survival [ 118 ]. In addition to MSCs, EVs derived from various immunosuppressive cells can also inhibit the onset and progression of GvHD. Myeloid-derivative-suppressor cells (MDSCs) suppress the immune response. Some studies have shown that MDSCs can inhibit GvHD by promoting Tregs activity, suggesting that MDSC-EVs may be a key component in alleviating GvHD [ 26 ]. Although immunosuppressive therapy could control inflammation associated with aGvHD and cGvHD, damage to organ tissue homeostasis will lead to long-term organ damage and affect patient quality of life [ 6 ].

MSCs and their derived EVs improve aGvHD and, as well as GvHD-related complications, by mediating tissue regeneration and repair to reduce disease severity of disease and mortality. Unlike traditional nanomaterials, EVs are gradually regarded as natural drug and gene delivery carriers due to their better biocompatibility, safety, and physical and chemical stability [ 119 ]. By loading a variety of bioactive molecules into EVs or performing specific targeted modifications on the surface of EVs, these functional EV modification strategies can not only improve the internal circulation time of drugs, reduce drug clearance rate, and reduce drug toxicities but also achieve accurate targeted delivery of drugs, thereby improving the efficacy of treatment. In recent years, some studies have confirmed that engineered EVs can achieve the purpose of improving the treatment of aGvHD and cGvHD and related complications by delivering nucleic acid or drugs and other therapeutic molecules ( 29 , 78 , 81 – 82 ), which also brings novel ideas and strategies for the treatment of diseases. Previous studies have shown that human BMMSC-EVs could treat acute steroid-refractory GvHD [ 25 ]. However, Madel et al. [ 120 ]. found that the efficacy of hBMMSC-EV preparations in alleviating symptoms in advanced mouse GvHD models needs to be more consistent between batches. Therefore, standardized MSC-EV production strategies may not guarantee the production of MSC-EV products with repeatable quality and effective mitigation of GvHD. Before administration, every MSC-EV preparation intended for clinical application must thoroughly evaluate its therapeutic efficacy both in vitro and in vivo.

Based on EVs, both therapy and drug delivery have been proven to possess favorable safety and efficacy in preclinical models and early clinical trials. Consequently, EVs are anticipated to become the next-generation therapeutic and drug delivery systems. However, factors such as the origin and biogenesis of EVs, size, endogenous/exogenous contents, production and storage approaches, dosage, infusion rate, and biomolecular corona significantly influence their immunogenicity [ 121 ]. Consequently, it is imperative to formulate methodologies for minimizing the immunogenicity of EVs to expedite clinical progress. In addition, there are still some differences and controversies in the purification and identification, bioactivity determination, and quality control of EVs derived from stem cells, including embryonic stem cells and MSCs, and there still needs to be unified standards. To address these challenges and actively promote the research and application of stem cell-derived EVs in the disease treatment field, the Extracellular Vesicle Research and Application Branch of the Chinese Research Hospital Association enlisted domestic experts and scholars specializing in stem cell-derived EVs. In 2022, for the first time, two national group standards, “Small Extracellular Vesicles Derived from Human Pluripotent Stem Cells” (T/CRHA 002-2021) [ 122 ] and “Small Extracellular Vesicles Derived from Human Mesenchymal Stem Cells” (T/CRHA 001-2021) [ 123 ], were formulated and published.

Conclusions

Although EVs have broad application potential and advantages in the diagnosis and treatment of diseases, there are still many bottlenecks and challenges in clinical translation and application, including purity and standard extraction, large-scale production and preparation, and long-term in vivo application safety evaluation, still have a long way to go. It is postulated that via the concerted endeavors of scholars specializing in EV research both nationally and internationally, in conjunction with the progressive enhancement of EV separation and purification technology, certain impediments that EVs confront in diagnosis and application will be successively surmounted. Additionally, the application and transformation of stem cell EVs in the therapeutic management of multiple intricate and recalcitrant clinical disorders such as GvHD will also exhibit more promising prospects and application values.

Acknowledgements

Not applicable.

Author contributions

P.W. and Z.W. designed, drafted, wrote, and revised this manuscript. Y.S. participated in editing and revising the paper. P.W., Z.C., M.W., and B.W. participated in the conception and design, financial support, and final approval of the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 82303775); the Natural Science Foundation of Anhui Province (2308085QH273); China Postdoctoral Science Foundation (2023M733398); Natural Science Research Project of Anhui Educational Committee (No. 2022AH050739); Research Fund of Anhui Institute of translational medicine (No. 2022zhyx-C65); The Youth Project of the Research Fund of Anhui Medical University (No. 2023xkj018).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors consent to publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peipei Wu, Zhangfei Wang, and Yongping Sun contributed equally to this work.

Contributor Information

Zhixiang Cheng, Email: chengzhixiang@sina.com.

Min Wang, Email: minwang@ahmu.edu.cn.

Baolong Wang, Email: wbl196555@163.com.

References

1. Zeiser R, Blazar BR. Acute Graft-versus-Host Disease - Biologic process, prevention, and therapy. N Engl J Med. 2017;377(22):2167–79.

2. Reshef R, Saber W, Bolaños-Meade J, Chen G, Chen YB, Ho VT, Ponce DM, Nakamura R, Martens MJ, Hansen JA, Levine JE, Acute GVHD. Diagnosis and adjudication in a multicenter trial: A report from the BMT CTN 1202 biorepository study. J Clin Oncology: Official J Am Soc Clin Oncol. 2021;39(17):1878–87.

3. Greinix HT, Eikema DJ, Koster L, Penack O, Yakoub-Agha I, Montoto S, Chabannon C, Styczynski J, Nagler A, Robin M, Robinson S, Chalandon Y, Mikulska M, Schönland S, Peric Z, Ruggeri A, Lanza F, De Wreede LC, Mohty M, Basak GW, Kröger N. Improved outcome of patients with graft-versus-host disease after allogeneic hematopoietic cell transplantation for hematologic malignancies over time: an EBMT mega-file study. Haematologica. 2022;107(5):1054–63.

4. Malard F, Holler E, Sandmaier BM, Huang H, Mohty M. Acute graft-versus-host disease. Nat Reviews Disease Primers. 2023;9(1):27.

5. Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet (London England). 2009;373(9674):1550–61.

6. Chakraverty R, Teshima T. Graft-versus-host disease: a disorder of tissue regeneration and repair. Blood. 2021;138(18):1657–65.

7. S ELA, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12(5):347–57.

8. Théry, C.; Witwer, K. W.; Aikawa, E.; Alcaraz, M. J.; Anderson, J. D.; Andriantsitohaina,R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G. K.; Ayre, D. C.; Bach, J.M.; Bachurski, D.; Baharvand, H.; Balaj, L.; Baldacchino, S.; Bauer, N. N.; Baxter,A. A.; Bebawy, M.; Beckham, C.; Bedina Zavec, A.; Benmoussa, A.; Berardi, A. C.; Bergese,P.; Bielska, E.; Blenkiron, C.; Bobis-Wozowicz, S.; Boilard, E.; Boireau, W.; Bongiovanni,A.; Borràs, F. E.; Bosch, S.; Boulanger, C. M.; Breakefield, X.; Breglio, A. M.; Brennan,M.; Brigstock, D. R.; Brisson, A.; Broekman, M. L.; Bromberg, J. F.; Bryl-Górecka,P.; Buch, S.; Buck, A. H.; Burger, D.; Busatto, S.; Buschmann, D.; Bussolati, B.;Buzás, E. I.; Byrd, J. B.; Camussi, G.; Carter, D. R.; Caruso, S.; Chamley, L. W.;Chang, Y. T.; Chen, C.; Chen, S.; Cheng, L.; Chin, A. R.; Clayton, A.; Clerici, S.P.; Cocks, A.; Cocucci, E.; Coffey, R. J.; Cordeiro-da-Silva, A.; Couch, Y.; Coumans,F. A.; Coyle, B.; Crescitelli, R.; Criado, M. F.; D’Souza-Schorey, C.; Das, S.; Datta Chaudhuri, A.; de Candia, P.; De Santana, E. F.; De Wever, O.; Del Portillo, H. A.;Demaret, T.; Deville, S.; Devitt, A.; Dhondt, B.; Di Vizio, D.; Dieterich, L. C.;Dolo, V.; Dominguez Rubio, A. P.; Dominici, M.; Dourado, M. R.; Driedonks, T. A.;Duarte, F. V.; Duncan, H. M.; Eichenberger, R. M.; Ekström, K.; El Andaloussi, S.;Elie-Caille, C.; Erdbrügger, U.; Falcón-Pérez, J. M.; Fatima, F.; Fish, J. E.; Flores-Bellver,M.; Försönits, A.; Frelet-Barrand, A.; Fricke, F.; Fuhrmann, G.; Gabrielsson, S.;Gámez-Valero, A.; Gardiner, C.; Gärtner, K.; Gaudin, R.; Gho, Y. S.; Giebel, B.; Gilbert,C.; Gimona, M.; Giusti, I.; Goberdhan, D. C.; Görgens, A.; Gorski, S. M.; Greening,D. W.; Gross, J. C.; Gualerzi, A.; Gupta, G. N.; Gustafson, D.; Handberg, A.; Haraszti,R. A.; Harrison, P.; Hegyesi, H.; Hendrix, A.; Hill, A. F.; Hochberg, F. H.; Hoffmann,K. F.; Holder, B.; Holthofer, H.; Hosseinkhani, B.; Hu, G.; Huang, Y.; Huber, V.;Hunt, S.; Ibrahim, A. G.; Ikezu, T.; Inal, J. M.; Isin, M.; Ivanova, A.; Jackson,H. K.; Jacobsen, S.; Jay, S. M.; Jayachandran, M.; Jenster, G.; Jiang, L.; Johnson,S. M.; Jones, J. C.; Jong, A.; Jovanovic-Talisman, T.; Jung, S.; Kalluri, R.; Kano,S. I.; Kaur, S.; Kawamura, Y.; Keller, E. T.; Khamari, D.; Khomyakova, E.; Khvorova,A.; Kierulf, P.; Kim, K. P.; Kislinger, T.; Klingeborn, M.; Klinke, D. J., 2nd; Kornek,M.; Kosanović, M. M.; Kovács Á, F.; Krämer-Albers, E. M.; Krasemann, S.; Krause, M.;Kurochkin, I. V.; Kusuma, G. D.; Kuypers, S.; Laitinen, S.; Langevin, S. M.; Languino,L. R.; Lannigan, J.; Lässer, C.; Laurent, L. C.; Lavieu, G.; Lázaro-Ibáñez, E.; Le Lay, S.; Lee, M. S.; Lee, Y. X. F.; Lemos, D. S.; Lenassi, M.; Leszczynska, A.; Li,I. T.; Liao, K.; Libregts, S. F.; Ligeti, E.; Lim, R.; Lim, S. K.; Linē, A.; Linnemannstöns,K.; Llorente, A.; Lombard, C. A.; Lorenowicz, M. J.; Lörincz Á, M.; Lötvall, J.; Lovett,J.; Lowry, M. C.; Loyer, X.; Lu, Q.; Lukomska, B.; Lunavat, T. R.; Maas, S. L.; Malhi,H.; Marcilla, A.; Mariani, J.; Mariscal, J.; Martens-Uzunova, E. S.; Martin-Jaular,L.; Martinez, M. C.; Martins, V. R.; Mathieu, M.; Mathivanan, S.; Maugeri, M.; McGinnis,L. K.; McVey, M. J.; Meckes, D. G., Jr.; Meehan, K. L.; Mertens, I.; Minciacchi, V.R.; Möller, A.; Møller Jørgensen, M.; Morales-Kastresana, A.; Morhayim, J.; Mullier,F.; Muraca, M.; Musante, L.; Mussack, V.; Muth, D. C.; Myburgh, K. H.; Najrana, T.;Nawaz, M.; Nazarenko, I.; Nejsum, P.; Neri, C.; Neri, T.; Nieuwland, R.; Nimrichter,L.; Nolan, J. P.; Nolte-‘t Hoen, E. N.; Noren Hooten, N.; O’Driscoll, L.; O’Grady,T.; O’Loghlen, A.; Ochiya, T.; Olivier, M.; Ortiz, A.; Ortiz, L. A.; Osteikoetxea,X.; Østergaard, O.; Ostrowski, M.; Park, J.; Pegtel, D. M.; Peinado, H.; Perut, F.;Pfaffl, M. W.; Phinney, D. G.; Pieters, B. C.; Pink, R. C.; Pisetsky, D. S.; Pogge von Strandmann, E.; Polakovicova, I.; Poon, I. K.; Powell, B. H.; Prada, I.; Pulliam,L.; Quesenberry, P.; Radeghieri, A.; Raffai, R. L.; Raimondo, S.; Rak, J.; Ramirez,M. I.; Raposo, G.; Rayyan, M. S.; Regev-Rudzki, N.; Ricklefs, F. L.; Robbins, P. D.;Roberts, D. D.; Rodrigues, S. C.; Rohde, E.; Rome, S.; Rouschop, K. M.; Rughetti,A.; Russell, A. E.; Saá, P.; Sahoo, S.; Salas-Huenuleo, E.; Sánchez, C.; Saugstad,J. A.; Saul, M. J.; Schiffelers, R. M.; Schneider, R.; Schøyen, T. H.; Scott, A.;Shahaj, E.; Sharma, S.; Shatnyeva, O.; Shekari, F.; Shelke, G. V.; Shetty, A. K.;Shiba, K.; Siljander, P. R.; Silva, A. M.; Skowronek, A.; Snyder, O. L., 2nd; Soares,R. P.; Sódar, B. W.; Soekmadji, C.; Sotillo, J.; Stahl, P. D.; Stoorvogel, W.; Stott,S. L.; Strasser, E. F.; Swift, S.; Tahara, H.; Tewari, M.; Timms, K.; Tiwari, S.;Tixeira, R.; Tkach, M.; Toh, W. S.; Tomasini, R.; Torrecilhas, A. C.; Tosar, J. P.;Toxavidis, V.; Urbanelli, L.; Vader, P.; van Balkom, B. W.; van der Grein, S. G.;Van Deun, J.; van Herwijnen, M. J.; Van Keuren-Jensen, K.; van Niel, G.; van Royen,M. E.; van Wijnen, A. J.; Vasconcelos, M. H.; Vechetti, I. J., Jr.; Veit, T. D.; Vella,L. J.; Velot, É.; Verweij, F. J.; Vestad, B.; Viñas, J. L.; Visnovitz, T.; Vukman,K. V.; Wahlgren, J.; Watson, D. C.; Wauben, M. H.; Weaver, A.; Webber, J. P.; Weber,V.; Wehman, A. M.; Weiss, D. J.; Welsh, J. A.; Wendt, S.; Wheelock, A. M.; Wiener,Z.; Witte, L.; Wolfram, J.; Xagorari, A.; Xander, P.; Xu, J.; Yan, X.; Yáñez-Mó, M.;Yin, H.; Yuana, Y.; Zappulli, V.; Zarubova, J.; Žėkas, V.; Zhang, J. Y.; Zhao, Z.;Zheng, L.; Zheutlin, A. R.; Zickler, A. M.; Zimmermann, P.; Zivkovic, A. M.; Zocco,D.; Zuba-Surma, E. K., Minimal information for studies of extracellular vesicles 2018(MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of extracellular vesicles 2018, 7 (1), 1535750.

9. Bai L, Shao H, Wang H, Zhang Z, Su C, Dong L, Yu B, Chen X, Li X, Zhang X. Effects of mesenchymal stem Cell-Derived exosomes on experimental autoimmune uveitis. Sci Rep. 2017;7(1):4323.

10. Wu X, Liu Y, Wei W, Liu ML. Extracellular vesicles in autoimmune vasculitis - Little Dirts light the fire in blood vessels. Autoimmun Rev. 2019;18(6):593–606.

11. Greening DW, Xu R, Ale A, Hagemeyer CE, Chen W. Extracellular vesicles as next generation immunotherapeutics. Sem Cancer Biol. 2023;90:73–100.

12. Fujii S, Miura Y. Immunomodulatory and regenerative effects of MSC-Derived extracellular vesicles to treat acute GVHD. Stem Cells. 2022;40(11):977–90.

13. Yu D, Li Y, Wang M, Gu J, Xu W, Cai H, Fang X, Zhang X. Exosomes as a new frontier of cancer liquid biopsy. Mol Cancer. 2022;21(1):56.

14. Pegtel DM, Gould SJ, Exosomes. Annu Rev Biochem. 2019;88:487–514.

15. Buzas EI. The roles of extracellular vesicles in the immune system. Nat Rev Immunol. 2023;23(4):236–50.

16. Carneiro TX, Marrese DG, Santos MGD, Gonçalves MV, Novis YAS, Rizzatti EG, Rocha V, Sandes AF, de Lacerda MP, Arrais-Rodrigues C. Circulating Extracellular Vesicles as a Predictive Biomarker for Acute Graft-Versus-Host Disease. Experimental hematology 2022.

17. Nagasawa M, Mitsuiki N, Yanagimachi M, Yamamoto M, Fukuda T, Miura O, Oba R, Igarashi A, Nagata K, Morio T. Utility of novel T-cell-specific extracellular vesicles in monitoring and evaluation of acute GVHD. Int J Hematol. 2021;113(6):910–20.

18. Lim HC, Soneji S, Pálmason R, Lenhoff S, Laurell T, Scheding S. Development of acoustically isolated extracellular plasma vesicles for biomarker discovery in allogeneic hematopoietic stem cell transplantation. Biomark Res. 2021;9(1):6.

19. Kooijmans SAA, de Jong OG, Schiffelers RM. Exploring interactions between extracellular vesicles and cells for innovative drug delivery system design. Adv Drug Deliv Rev. 2021;173:252–78.

20. Kim HY, Kwon S, Um W, Shin S, Kim CH, Park JH, Kim BS. Functional extracellular vesicles for regenerative medicine. Small 2022, 18 (36), e2106569.

21. Viswanathan S, Shi Y, Galipeau J, Krampera M, Leblanc K, Martin I, Nolta J, Phinney DG, Sensebe L. Mesenchymal stem versus stromal cells: international society for cell & gene therapy (ISCT ® ) mesenchymal stromal cell committee position statement on nomenclature. Cytotherapy. 2019;21(10):1019–24.

22. Bianco P, Cao X, Frenette PS, Mao JJ, Robey PG, Simmons PJ, Wang CY. The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med. 2013;19(1):35–42.

23. Moll G, Ankrum JA, Kamhieh-Milz J, Bieback K, Ringdén O, Volk HD, Geissler S, Reinke P. Intravascular mesenchymal stromal/stem cell therapy product diversification: time for new clinical guidelines. Trends Mol Med. 2019;25(2):149–63.

24. Zhao L, Chen S, Yang P, Cao H, Li L. The role of mesenchymal stem cells in hematopoietic stem cell transplantation: prevention and treatment of graft-versus-host disease. Stem Cell Res Ther. 2019;10(1):182.

25. Kordelas L, Rebmann V, Ludwig AK, Radtke S, Ruesing J, Doeppner TR, Epple M, Horn PA, Beelen DW, Giebel B. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia. 2014;28(4):970–3.

26. Hess NJ, Kink JA, Hematti P. Exosomes, MDSCs and Tregs: A new frontier for GVHD prevention and treatment. Front Immunol. 2023;14:1143381.

27. Lai P, Weng J, Guo L, Chen X, Du X. Novel insights into MSC-EVs therapy for immune diseases. Biomark Res. 2019;7:6.

28. Liang Y, Duan L, Lu J, Xia J. Engineering exosomes for targeted drug delivery. Theranostics. 2021;11(7):3183–95.

29. Su Y, Sun X, Liu X, Qu Q, Yang L, Chen Q, Liu F, Li Y, Wang Q, Huang B, Huang XJ, Zhang XH. hUC-EVs-ATO reduce the severity of acute GVHD by resetting inflammatory macrophages toward the M2 phenotype. J Hematol Oncol. 2022;15(1):99.

30. Saad A, de Lima M, Anand S, Bhatt VR, Bookout R, Chen G, Couriel D, Di Stasi A, El-Jawahri A, Giralt S, Gutman J, Ho V, Horwitz M, Hsu J, Juckett M, Kharfan-Dabaja MA, Loren A, Meade J, Mielcarek M, Moreira J, Nakamura R, Nieto Y, Roddy J, Satyanarayana G, Schroeder M, Tan CR, Tzachanis D, Burn J, Pluchino L. Hematopoietic cell transplantation, version 2.2020, NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Network: JNCCN. 2020;18(5):599–634.

31. Iftikhar R, Chaudhry QUN, Anwer F, Neupane K, Rafae A, Mahmood SK, Ghafoor T, Shahbaz N, Khan MA, Khattak TA, Shamshad GU, Rehman J, Farhan M, Khan M, Ansar I, Ashraf R, Marsh J, Satti TM, Ahmed P. Allogeneic hematopoietic stem cell transplantation in aplastic anemia: current indications and transplant strategies. Blood Rev. 2021;47:100772.

32. Chang YJ, Pei XY, Huang XJ. Haematopoietic stem-cell transplantation in China in the era of targeted therapies: current advances, challenges, and future directions. Lancet Haematol. 2022;9(12):e919–29.

33. Gratwohl A. Bone marrow transplantation activity in Europe 1990. European group for bone marrow transplantation (EBMT). Bone Marrow Transplant. 1991;8(3):197–201.

34. Passweg JR, Baldomero H, Chabannon C, Basak GW, de la Cámara R, Corbacioglu S, Dolstra H, Duarte R, Glass B, Greco R, Lankester AC, Mohty M, Peffault de Latour R, Snowden JA, Yakoub-Agha I, Kröger N. Hematopoietic cell transplantation and cellular therapy survey of the EBMT: monitoring of activities and trends over 30 years. Bone Marrow Transplant. 2021;56(7):1651–64.

35. Kanate AS, Majhail NS, Savani BN, Bredeson C, Champlin RE, Crawford S, Giralt SA, LeMaistre CF, Marks DI, Omel JL, Orchard PJ, Palmer J, Saber W, Veys PA, Carpenter PA, Hamadani M. Indications for hematopoietic cell transplantation and immune effector cell therapy: guidelines from the American society for transplantation and cellular therapy. Biology Blood Marrow Transplantation: J Am Soc Blood Marrow Transplantation. 2020;26(7):1247–56.

36. Passweg JR, Baldomero H, Ciceri F, Corbacioglu S, de la Cámara R, Dolstra H, Glass B, Greco R, McLornan DP, Neven B, de Latour RP, Perić Z, Ruggeri A, Snowden JA, Sureda A. Hematopoietic cell transplantation and cellular therapies in Europe 2021. The second year of the SARS-CoV-2 pandemic. A report from the EBMT activity survey. Bone Marrow Transplant. 2023;58(6):647–58.

37. [Chinese consensus. Of allogeneic hematopoietic stem cell transplantation for hematological disease (III) -acute graft-versus-host disease (2020)]. Zhonghua Xue ye Xue Za zhi = zhonghua Xueyexue Zazhi 2020, 41 (7), 529–36.

38. Xu LP, Lu PH, Wu DP, Sun ZM, Liu QF, Han MZ, Zhang X, Song YP, Song XM, Hu JD, Huang H, Lai YR, Wan DM, Chen J, Li CF, Xia LH, Wang JB, Liu DH, Huang XJ. Hematopoietic stem cell transplantation activity in China 2019: a report from the Chinese blood and marrow transplantation registry group. Bone Marrow Transplant. 2021;56(12):2940–7.

39. Filipovich AH, Weisdorf D, Pavletic S, Socie G, Wingard JR, Lee SJ, Martin P, Chien J, Przepiorka D, Couriel D, Cowen EW, Dinndorf P, Farrell A, Hartzman R, Henslee-Downey J, Jacobsohn D, McDonald G, Mittleman B, Rizzo JD, Robinson M, Schubert M, Schultz K, Shulman H, Turner M, Vogelsang G, Flowers ME. National institutes of health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biology Blood Marrow Transplantation: J Am Soc Blood Marrow Transplantation. 2005;11(12):945–56.

40. Jagasia MH, Greinix HT, Arora M, Williams KM, Wolff D, Cowen EW, Palmer J, Weisdorf D, Treister NS, Cheng GS, Kerr H, Stratton P, Duarte RF, McDonald GB, Inamoto Y, Vigorito A, Arai S, Datiles MB, Jacobsohn D, Heller T, Kitko CL, Mitchell SA, Martin PJ, Shulman H, Wu RS, Cutler CS, Vogelsang GB, Lee SJ, Pavletic SZ, Flowers ME. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: I. The 2014 Diagnosis and Staging Working Group report. Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 2015, 21 (3), 389–401.e1.

41. Koyama M, Hill GR. The primacy of Gastrointestinal tract antigen-presenting cells in lethal graft-versus-host disease. Blood. 2019;134(24):2139–48.

42. Hong C, Jin R, Dai X, Gao X. Functional contributions of antigen presenting cells in chronic Graft-Versus-Host disease. Front Immunol. 2021;12:614183.

43. Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD, Shlomchik MJ. Distinct roles for donor- and host-derived antigen-presenting cells and costimulatory molecules in murine chronic graft-versus-host disease: requirements depend on target organ. Blood. 2005;105(5):2227–34.

44. Koyama M, Hill GR. Alloantigen presentation and graft-versus-host disease: fuel for the fire. Blood. 2016;127(24):2963–70.

45. Flowers ME, Martin PJ. How we treat chronic graft-versus-host disease. Blood. 2015;125(4):606–15.

46. Cutler CS, Koreth J, Ritz J. Mechanistic approaches for the prevention and treatment of chronic GVHD. Blood. 2017;129(1):22–9.

47. Malard F, Mohty M. Updates in chronic graft-versus-host disease management. Am J Hematol. 2023;98(10):1637–44.

48. Hamilton BK. Updates in chronic graft-versus-host disease. Hematol Am Soc Hematol Educ Program. 2021;2021(1):648–54.

49. Cooke KR, Luznik L, Sarantopoulos S, Hakim FT, Jagasia M, Fowler DH, van den Brink MRM, Hansen JA, Parkman R, Miklos DB, Martin PJ, Paczesny S, Vogelsang G, Pavletic S, Ritz J, Schultz KR, Blazar BR. The biology of chronic Graft-versus-Host disease: A task force report from the National institutes of health consensus development project on criteria for clinical trials in chronic Graft-versus-Host disease. Biology Blood Marrow Transplantation: J Am Soc Blood Marrow Transplantation. 2017;23(2):211–34.

50. Greinix HT, Loddenkemper C, Pavletic SZ, Holler E, Socié G, Lawitschka A, Halter J, Wolff D. Diagnosis and staging of chronic graft-versus-host disease in the clinical practice. Biology Blood Marrow Transplantation: J Am Soc Blood Marrow Transplantation. 2011;17(2):167–75.

51. Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B, Cai H, Di Vizio D, Driedonks TAP, Erdbrügger U, Falcon-Perez JM, Fu QL, Hill AF, Lenassi M, Lim SK, Mahoney MG, Mohanty S, Möller A, Nieuwland R, Ochiya T, Sahoo S, Torrecilhas AC, Zheng L, Zijlstra A, Abuelreich S, Bagabas R, Bergese P, Bridges EM, Brucale M, Burger D, Carney RP, Cocucci E, Crescitelli R, Hanser E, Harris AL, Haughey NJ, Hendrix A, Ivanov AR, Jovanovic-Talisman T, Kruh-Garcia NA; Ku’ulei-Lyn, Faustino V, Kyburz D, Lässer C, Lennon KM, Lötvall J, Maddox AL, Martens-Uzunova ES, Mizenko RR, Newman LA, Ridolfi A, Rohde E, Rojalin T, Rowland A, Saftics A, Sandau US, Saugstad JA, Shekari F, Swift S, Ter-Ovanesyan D, Tosar JP, Useckaite Z, Valle F, Varga Z, van der Pol E, van Herwijnen MJC, Wauben MH. M.;, Wehman AM, Williams S, Zendrini A, Zimmerman AJ, Théry C, Witwer. K. W., Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. Journal of extracellular vesicles 2024, 13 (2), e12404.

52. Cocozza F, Grisard E, Martin-Jaular L, Mathieu M, Théry C. SnapShot: extracellular vesicles. Cell. 2020;182(1):262–e2621.

53. Zhang M, Hu S, Liu L, Dang P, Liu Y, Sun Z, Qiao B, Wang C. Engineered exosomes from different sources for cancer-targeted therapy. Signal Transduct Target Therapy. 2023;8(1):124.

54. Tang TT, Wang B, Li ZL, Wen Y, Feng ST, Wu M, Liu D, Cao JY, Yin Q, Yin D, Fu YQ, Gao YM, Ding ZY, Qian JY, Wu QL, Lv LL, Liu BC. Kim-1 targeted extracellular vesicles: A new therapeutic platform for RNAi to treat AKI. J Am Soc Nephrology: JASN. 2021;32(10):2467–83.

55. Wu P, Zhang B, Ocansey DKW, Xu W, Qian H. Extracellular vesicles: A bright star of nanomedicine. Biomaterials. 2021;269:120467.

56. Wang D, Jiang S, Zhang F, Ma S, C Heng B, Wang Y, Zhu J, Xu M, He Y, Wei Y, Zhang X, Xia X, Deng B. Cell membrane vesicles with enriched CXCR4 display enhances their targeted delivery as drug carriers to inflammatory sites. Adv Sci (Weinheim Baden-Wurttemberg Germany). 2021;8(23):e2101562.

57. Xu S, Liu B, Fan J, Xue C, Lu Y, Li C, Cui D. Engineered mesenchymal stem cell-derived exosomes with high CXCR4 levels for targeted SiRNA gene therapy against cancer. Nanoscale. 2022;14(11):4098–113.

58. Hu Y, Li X, Zhang Q, Gu Z, Luo Y, Guo J, Wang X, Jing Y, Chen X, Su J. Exosome-guided bone targeted delivery of Antagomir-188 as an anabolic therapy for bone loss. Bioactive Mater. 2021;6(9):2905–13.

59. Luo H, Chen D, Li R, Li R, Teng Y, Cao Y, Zou X, Wang W, Zhou C. Genetically engineered CXCR4-modified exosomes for delivery of miR-126 mimics to macrophages alleviate periodontitis. J Nanobiotechnol. 2023;21(1):116.

60. Stranford DM, Simons LM, Berman KE, Cheng L, DiBiase BN, Hung ME, Lucks JB, Hultquist JF, Leonard JN. Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells. Nat Biomedical Eng. 2024;8(4):397–414.

61. van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213–28.

62. Wiklander OPB, Brennan M, Lötvall J, Breakefield XO, El Andaloussi S. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med. 2019;11:492.

63. Sterzenbach U, Putz U, Low LH, Silke J, Tan SS, Howitt J. Engineered exosomes as vehicles for biologically active proteins. Mol Therapy: J Am Soc Gene Therapy. 2017;25(6):1269–78.

64. Escudé M, de Castilla P, Tong L, Huang C, Sofias AM, Pastorin G, Chen X, Storm G, Schiffelers RM, Wang JW. Extracellular vesicles as a drug delivery system: A systematic review of preclinical studies. Adv Drug Deliv Rev. 2021;175:113801.

65. Giaccone L, Faraci DG, Butera S, Lia G, Di Vito C, Gabrielli G, Cerrano M, Mariotti J, Dellacasa C, Felicetti F, Brignardello E, Mavilio D, Bruno B. Biomarkers for acute and chronic graft versus host disease: state of the Art. Expert Rev Hematol. 2021;14(1):79–96.

66. Nikanjam M, Kato S, Kurzrock R. Liquid biopsy: current technology and clinical applications. J Hematol Oncol. 2022;15(1):131.

67. Gavriilaki E, Sakellari I, Anyfanti P, Batsis I, Vardi A, Bousiou Z, Lazaridis A, Nikolaidou B, Zarifis I, Masmanidou M, Yiannaki E, Markala D, Anagnostopoulos A, Douma S, Gkaliagkousi E. Assessment of endothelial injury and Pro-Coagulant activity using Circulating microvesicles in survivors of allogeneic hematopoietic cell transplantation. Int J Mol Sci. 2020;21:24.

68. ElGohary G, Toor AA, Gergis U. Engraftment syndrome after allogeneic stem cell transplantation: a systematic review and meta-analysis. Bone Marrow Transplant. 2023;58(1):1–9.

69. Spitzer TR. Engraftment syndrome following hematopoietic stem cell transplantation. Bone Marrow Transplant. 2001;27(9):893–8.

70. Lia G, Brunello L, Bruno S, Carpanetto A, Omedè P, Festuccia M, Tosti L, Maffini E, Giaccone L, Arpinati M, Ciccone G, Boccadoro M, Evangelista A, Camussi G, Bruno B. Extracellular vesicles as potential biomarkers of acute graft-vs-host disease. Leukemia. 2018;32(3):765–73.

71. Lia G, Di Vito C, Bruno S, Tapparo M, Brunello L, Santoro A, Mariotti J, Bramanti S, Zaghi E, Calvi M, Comba L, Fascì M, Giaccone L, Camussi G, Boyle EM, Castagna L, Evangelista A, Mavilio D, Bruno B. Extracellular vesicles as biomarkers of acute Graft-vs.-Host disease after haploidentical stem cell transplantation and Post-Transplant cyclophosphamide. Front Immunol. 2021;12:816231.

72. Baur R, Karl F, Böttcher-Loschinski R, Stoll A, Völkl S, Gießl A, Flamann C, Bruns H, Schlötzer-Schrehardt U, Böttcher M, Schewe DM, Fischer T, Jitschin R, Mackensen A, Mougiakakos D. Accumulation of T-cell-suppressive PD-L1(high) extracellular vesicles is associated with GvHD and might impact GvL efficacy. J Immunother Cancer 2023, 11 (3).

73. Łacina P, Crossland RE, Wielińska J, Czyż A, Szeremet A, Ussowicz M, Wróbel T, Dickinson AM, Bogunia-Kubik K. Differential expression of MiRNAs from extracellular vesicles in chronic graft-versus-host disease: A preliminary study. Adv Clin Experimental Medicine: Official Organ Wroclaw Med Univ. 2023;32(5):539–44.

74. Omer AK, Weisdorf DJ, Lazaryan A, Shanley R, Blazar BR, MacMillan ML, Brunstein C, Bejanyan N, Arora M. Late acute Graft-versus-Host disease after allogeneic hematopoietic stem cell transplantation. Biology Blood Marrow Transplantation: J Am Soc Blood Marrow Transplantation. 2016;22(5):879–83.

75. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH, Ohyashiki K. Exosomal MiRNA signatures for Late-Onset acute Graft-Versus-Host disease in allogenic hematopoietic stem cell transplantation. Int J Mol Sci 2018, 19 (9).

76. Storci G, Barbato F, Ricci F, Tazzari PL, De Matteis S, Tomassini E, Dicataldo M, Laprovitera N, Arpinati M, Ursi M, Maffini E, Campanini E, Dan E, Manfroi S, Santi S, Ferracin M, Bonafe M, Bonifazi F. Pre-transplant CD69 + extracellular vesicles are negatively correlated with active ATLG serum levels and associate with the onset of GVHD in allogeneic HSCT patients. Front Immunol. 2022;13:1058739.

77. Liu H, Chen Y, Yin G, Xie Q. Therapeutic prospects of MicroRNAs carried by mesenchymal stem cells-derived extracellular vesicles in autoimmune diseases. Life Sci. 2021;277:119458.

78. Zhou T, He C, Lai P, Yang Z, Liu Y, Xu H, Lin X, Ni B, Ju R, Yi W, Liang L, Pei D, Egwuagu CE, Liu X. miR-204-containing exosomes ameliorate GVHD-associated dry eye disease. Sci Adv. 2022;8(2):eabj9617.

79. Fujii S, Miura Y, Fujishiro A, Shindo T, Shimazu Y, Hirai H, Tahara H, Takaori-Kondo A, Ichinohe T, Maekawa T. Graft-Versus-Host disease amelioration by human bone marrow mesenchymal stromal/stem cell-Derived extracellular vesicles is associated with peripheral preservation of Naive T cell populations. Stem Cells. 2018;36(3):434–45.

80. Lai P, Chen X, Guo L, Wang Y, Liu X, Liu Y, Zhou T, Huang T, Geng S, Luo C, Huang X, Wu S, Ling W, Du X, He C, Weng J. A potent Immunomodulatory role of exosomes derived from mesenchymal stromal cells in preventing cGVHD. J Hematol Oncol. 2018;11(1):135.

81. Erratum to. Epidermal growth factor stimulates Exosomal microRNA-21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells. FASEB Journal: Official Publication Federation Am Soc Experimental Biology 2023, 37 (7), e22990.

82. Zhu H, Lan L, Zhang Y, Chen Q, Zeng Y, Luo X, Ren J, Chen S, Xiao M, Lin K, Chen M, Li Q, Chen Y, Xu J, Zheng Z, Chen Z, Xie Y, Hu J, Yang T. Epidermal growth factor stimulates Exosomal microRNA-21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells. FASEB Journal: Official Publication Federation Am Soc Experimental Biology. 2020;34(6):7372–86.

83. Amarnath S, Foley JE, Farthing DE, Gress RE, Laurence A, Eckhaus MA, Métais JY, Rose JJ, Hakim FT, Felizardo TC, Cheng AV, Robey PG, Stroncek DE, Sabatino M, Battiwalla M, Ito S, Fowler DH, Barrett AJ. Bone marrow-derived mesenchymal stromal cells Harness purinergenic signaling to tolerize human Th1 cells in vivo. Stem Cells. 2015;33(4):1200–12.

84. Norooznezhad AH, Yarani R, Payandeh M, Hoseinkhani Z, Kiani S, Taghizadeh E, Thakor AS, Mansouri K. Human placental mesenchymal stromal cell-derived exosome-enriched extracellular vesicles for chronic cutaneous graft-versus-host disease: A case report. J Cell Mol Med. 2022;26(2):588–92.

85. Zhang B, Yeo RWY, Lai RC, Sim EWK, Chin KC, Lim SK. Mesenchymal stromal cell exosome-enhanced regulatory T-cell production through an antigen-presenting cell-mediated pathway. Cytotherapy. 2018;20(5):687–96.

86. Liu W, Zhou N, Liu Y, Zhang W, Li X, Wang Y, Zheng R, Zhang Y. Mesenchymal stem cell exosome-derived miR-223 alleviates acute graft-versus-host disease via reducing the migration of donor T cells. Stem Cell Res Ther. 2021;12(1):153.

87. Wang L, Gu Z, Zhao X, Yang N, Wang F, Deng A, Zhao S, Luo L, Wei H, Guan L, Gao Z, Li Y, Wang L, Liu D, Gao C. Extracellular vesicles released from human umbilical Cord-Derived mesenchymal stromal cells prevent Life-Threatening acute Graft-Versus-Host disease in a mouse model of allogeneic hematopoietic stem cell transplantation. Stem Cells Dev. 2016;25(24):1874–83.

88. Li KL, Li JY, Xie GL, Ma XY. Exosomes released from human bone Marrow-Derived mesenchymal stem cell attenuate acute Graft-Versus-Host disease after allogeneic hematopoietic stem cell transplantation in mice. Front Cell Dev Biology. 2021;9:617589.

89. Kelly K, Rasko JEJ. Mesenchymal stromal cells for the treatment of graft versus host disease. Front Immunol. 2021;12:761616.

90. Descalzi-Montoya DB, Yang Z, Fanning S, Hu W, LoMauro K, Zhao Y, Korngold R. Cord Blood-Derived multipotent stem cells ameliorate in vitro/in vivo alloreactive responses, and this effect is associated with Exosomal microvesicles in vitro. Transplantation Cell Therapy. 2024;30(4):e3961–39614.

91. Li W, Si Y, Wang Y, Chen J, Huo X, Xu P, Jiang B, Li Z, Shang K, Luo Q, Xiong Y. hUCMSC-derived exosomes protect against GVHD-induced Endoplasmic reticulum stress in CD4(+) T cells by targeting the miR-16-5p/ATF6/CHOP axis. Int Immunopharmacol. 2024;135:112315.

92. Jiang Y, Zhao J, Wang M, Huang F, Li J, Liu R, Wan J, Hao S. Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models. Front Immunol. 2023;14:1284936.

93. Shafeghat Z, Dorfaki M, Dehrouyeh S, Arab FL, Roozbehani M, Falak R, Faraji F, Jafari R. Mesenchymal stem cell-derived exosomes for managing graft-versus-host disease: an updated view. Transpl Immunol. 2023;81:101957.

94. Lightner AL, Fujiki M, Elshawy M, Dadgar N, Barnoski A, Osman M, Fulmer CG, Vaidya A. Mesenchymal stem cell extracellular vesicles as a new treatment paradigm in solid abdominal organ transplantation: A case series. Stem Cells Dev. 2024;33(5–6):107–16.

95. Kavian N, Marut W, Servettaz A, Laude H, Nicco C, Chéreau C, Weill B, Batteux F. Arsenic trioxide prevents murine sclerodermatous graft-versus-host disease. J Immunol (Baltimore Md: 1950). 2012;188(10):5142–9.

96. Liu X, Su Y, Sun X, Fu H, Huang Q, Chen Q, Mo X, Lv M, Kong Y, Xu L, Huang X, Zhang X. Arsenic trioxide alleviates acute graft-versus-host disease by modulating macrophage polarization. Sci China Life Sci. 2020;63(11):1744–54.

97. An K, Xue MJ, Zhong JY, Yu SN, Lan TS, Qi ZQ, Xia JJ. Arsenic trioxide ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice by inducing CD4(+) T cell apoptosis. J Neuroinflamm. 2020;17(1):147.

98. Basso PJ, Sales-Campos H, Nardini V, Duarte-Silva M, Alves VBF, Bonfá G, Rodrigues CC, Ghirotto B, Chica JEL, Nomizo A, Cardoso CRB. Peroxisome Proliferator-Activated receptor alpha mediates the beneficial effects of Atorvastatin in experimental colitis. Front Immunol. 2021;12:618365.

99. Li C, Zhang J, Wang W, Wang H, Zhang Y, Zhang Z. Arsenic trioxide improves Treg and Th17 balance by modulating STAT3 in treatment-naïve rheumatoid arthritis patients. Int Immunopharmacol. 2019;73:539–51.

100. Hamidou M, Néel A, Poupon J, Amoura Z, Ebbo M, Sibilia J, Viallard JF, Gaborit B, Volteau C, Hardouin JB, Hachulla E, Rieger F. Safety and efficacy of low-dose intravenous arsenic trioxide in systemic lupus erythematosus: an open-label phase IIa trial (Lupsenic). Arthritis Res Therapy. 2021;23(1):70.

101. Hong J, Fraebel J, Yang Y, Tkacyk E, Kitko C, Kim TK. Understanding and treatment of cutaneous graft-versus-host-disease. Bone Marrow Transplant. 2023;58(12):1298–313.

102. Yang H, Cheong S, He Y, Lu F. Mesenchymal stem cell-based therapy for autoimmune-related fibrotic skin diseases-systemic sclerosis and sclerodermatous graft-versus-host disease. Stem Cell Res Ther. 2023;14(1):372.

103. Zhang Q, Fu L, Liang Y, Guo Z, Wang L, Ma C, Wang H. Exosomes originating from MSCs stimulated with TGF-β and IFN-γ promote Treg differentiation. J Cell Physiol. 2018;233(9):6832–40.

104. Wang J, Donohoe E, Canning A, Moosavizadeh S, Buckley F, Brennan M, Ryan AE, Ritter T. Immunomodulatory function of licensed human bone marrow mesenchymal stromal cell-derived apoptotic bodies. Int Immunopharmacol. 2023;125:111096. (Pt A).

105. Moll G, Ankrum JA, Olson SD, Nolta JA. Improved MSC minimal criteria to maximize patient safety: A call to embrace tissue factor and hemocompatibility assessment of MSC products. Stem Cells Translational Med. 2022;11(1):2–13.

106. Moll G, Drzeniek N, Kamhieh-Milz J, Geissler S, Volk HD, Reinke P. MSC therapies for COVID-19: importance of patient coagulopathy, thromboprophylaxis, cell product quality and mode of delivery for treatment safety and efficacy. Front Immunol. 2020;11:1091.

107. Ringdén O, Moll G, Gustafsson B, Sadeghi B. Mesenchymal stromal cells for enhancing hematopoietic engraftment and treatment of Graft-Versus-Host disease, hemorrhages and acute respiratory distress syndrome. Front Immunol. 2022;13:839844.

108. Moll G, Rasmusson-Duprez I, von Bahr L, Connolly-Andersen AM, Elgue G, Funke L, Hamad OA, Lönnies H, Magnusson PU, Sanchez J, Teramura Y, Nilsson-Ekdahl K, Ringdén O, Korsgren O, Nilsson B, Le Blanc K. Are therapeutic human mesenchymal stromal cells compatible with human blood? Stem Cells. 2012;30(7):1565–74.

109. Moll G, Ignatowicz L, Catar R, Luecht C, Sadeghi B, Hamad O, Jungebluth P, Dragun D, Schmidtchen A, Ringdén O. Different procoagulant activity of therapeutic mesenchymal stromal cells derived from bone marrow and placental decidua. Stem Cells Dev. 2015;24(19):2269–79.

110. Burrello J, Caporali E, Gauthier LG, Pianezzi E, Balbi C, Rigamonti E, Bolis S, Lazzarini E, Biemmi V, Burrello A, Frigerio R, Martinetti G, Fusi-Schmidhauser T, Vassalli G, Ferrari E, Moccetti T, Gori A, Cretich M, Melli G, Monticone S, Barile L. Risk stratification of patients with SARS-CoV-2 by tissue factor expression in Circulating extracellular vesicles. Vascul Pharmacol. 2022;145:106999.

111. Xia Y, Zhang J, Liu G, Wolfram J. Immunogenicity of extracellular vesicles. Adv Mater (Deerfield Beach Fla) 2024, 36 (33), e2403199.

112. Chen J, Zheng M, Xiao Q, Wang H, Chi C, Lin T, Wang Y, Yi X, Zhu L. Recent advances in Microfluidic-Based extracellular vesicle analysis. Micromachines 2024, 15 (5).

113. Sun Y, Shi H, Yin S, Ji C, Zhang X, Zhang B, Wu P, Shi Y, Mao F, Yan Y, Xu W, Qian H. Human mesenchymal stem cell derived exosomes alleviate type 2 diabetes mellitus by reversing peripheral insulin resistance and relieving β-Cell destruction. ACS Nano. 2018;12(8):7613–28.

114. Yang C, Sun J, Tian Y, Li H, Zhang L, Yang J, Wang J, Zhang J, Yan S, Xu D. Immunomodulatory effect of MSCs and MSCs-Derived extracellular vesicles in systemic lupus erythematosus. Front Immunol. 2021;12:714832.

115. You DG, Lim GT, Kwon S, Um W, Oh BH, Song SH, Lee J, Jo DG, Cho YW, Park JH. Metabolically engineered stem cell-derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis. Sci Adv. 2021;7:23.

116. Weng Z, Zhang B, Wu C, Yu F, Han B, Li B, Li L. Therapeutic roles of mesenchymal stem cell-derived extracellular vesicles in cancer. J Hematol Oncol. 2021;14(1):136.

117. Riazifar M, Mohammadi MR, Pone EJ, Yeri A, Lässer C, Segaliny AI, McIntyre LL, Shelke GV, Hutchins E, Hamamoto A, Calle EN, Crescitelli R, Liao W, Pham V, Yin Y, Jayaraman J, Lakey JRT, Walsh CM, Van Keuren-Jensen K, Lotvall J, Zhao W. Stem Cell-Derived exosomes as nanotherapeutics for autoimmune and neurodegenerative disorders. ACS Nano. 2019;13(6):6670–88.

118. Gupta M, Tieu A, Slobodian M, Shorr R, Burger D, Lalu MM, Allan DS. Preclinical studies of MSC-Derived extracellular vesicles to treat or prevent graft versus host disease: a systematic review of the literature. Stem Cell Reviews Rep. 2021;17(2):332–40.

119. Li YJ, Wu JY, Liu J, Xu W, Qiu X, Huang S, Hu XB, Xiang DX. Artificial exosomes for translational nanomedicine. J Nanobiotechnol. 2021;19(1):242.

120. Madel RJ, Börger V, Dittrich R, Bremer M, Tertel T, Phuong NNT, Baba HA, Kordelas L, Staubach S, Stein F, Haberkant P, Hackl M, Grillari R, Grillari J, Buer J, Horn PA, Westendorf AM, Brandau S, Kirschning CJ, Giebel B. Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model. Cytotherapy. 2023;25(8):821–36.

121. Xia Y, Zhang J, Liu G, Wolfram J. Immunogenicity of extracellular vesicles. Adv Mater (Deerfield Beach Fla) 2024, e2403199.

122. Li Q, Li B, Chen Z, Xu W, Yin H, Deng Z, Li H, Yan X, Hao X, Li L, Tao Z, Liu B, Ye T, Luo L, Qian H, Fu Q-L, Wang Q, Zheng L, Wang Y. Requirements for human pluripotent stem cell derived small extracellular vesicles. Interdisciplinary Med 2023, 1 (2), e20220018.

123. Li Q, Li B, Ye T, Xu W, Yin H, Deng Z, Li H, Yan X, Hao X, Li L, Tao Z, Liu B, Chen Z, Luo L, Qian H, Fu Q-L, Wang Q, Zheng L, Wang Y. Requirements for human mesenchymal stem cell-derived small extracellular vesicles. Interdisciplinary Med 2023, 1 (1), e20220015.

Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.