Extracellular vesicles (EVs) are small vesicular structures released by cells, and contain a variety of bioactive molecules, including proteins, nucleic acids and lipids, which facilitate the transfer of information between cells. EVs play critical roles in a plethora of physiological processes and pathological mechanisms. These roles include transporting bioactive molecules, facilitating intercellular communication and modulating the functions of target cells via receptor interaction. Moreover, EVs have emerged as valuable tools in the fields of disease diagnosis and therapeutic intervention, particularly in the context of conditions like cardiovascular disorders and neurological ailments (1,2). Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal properties that serve a role in cell therapy due to their potent immunomodulatory and regenerative effects (2). Recent studies have highlighted the therapeutic potential of MSCs through their paracrine products. MSCs can regulate the immune response, promote angiogenesis and exhibit anti-inflammatory effects by secreting a series of bioactive factors and MSC-derived EVs (MSC-EVs), thus potentially offering therapeutic benefits for a variety of diseases (3–5). Isolation and purification techniques for MSC-EVs continue to be refined, emphasizing the importance of determining their identity. Although EVs have broad application prospects in the medical field, there are still some challenges in their separation and purification techniques, such as improving the separation efficiency and avoiding contamination. A number of methods, including electron microscopy, nanotrack analysis, western blotting and flow cytometry, have been employed to identify MSC-EVs (2). Studies have shown that MSC-EVs exert similar biological effects as MSCs, including modulation of inflammatory responses, promotion of tissue repair and antioxidant properties (6–9). These features enable them to serve important roles in the regulation and treatment of numerous diseases. In the context of infectious diseases, such as renal tuberculosis (TB), urinary TB and TB, MSC-EVs have demonstrated marked efficacy in improving renal function and quality of life through mechanisms such as reduction of renal injury, stimulation of renal cell regeneration and repair, suppression of inflammatory responses, and antiviral/antibacterial pathways (10–13). Furthermore, comprehensive research and clinical trials are key in fully unlocking the therapeutic potential of MSC-EVs in disease treatment (10,14,15).

Based on the established understanding of Mycobacterium tuberculosis (Mtb) infection mechanisms and the unique properties of MSCs, therapeutic strategies utilizing MSCs for the treatment of TB and multidrug-resistant TB (MDR-TB) have garnered interest (11–13,15,16). Previous studies have further elucidated the therapeutic potential of MSCs (6–9,17,18). Yang et al (19) conducted a study in which human umbilical cord-derived MSCs (HUC-MSC-EVs) were co-cultured with Mtb-infected THP-1 macrophages. The findings indicated that MSCs were able to augment the immune response of macrophages to Mtb by activating immune receptors and inducing the production of inflammatory mediators. The present review provides an important platform for advancing TB diagnostics and therapeutic interventions, representing a notable step toward understanding and controlling Mtb pathogenesis in human hosts. Results from studies utilizing MSC-EVs have demonstrated the potential of MSC-EVs in therapeutic applications across diverse animal models, including cardiac, brain, kidney and lung injury animal models (Table I). Furthermore, MSC-EVs exhibit remarkable efficacy in mitigating the increased bacterial load associated with Mtb infection, while effectively suppressing pro-inflammatory cytokine production (10,14,20,21). However, research on the specific application of MSC-EVs in TB therapy remains relatively limited. This review aims to provide a comprehensive overview of various themes, encompassing an analysis of isolation methodologies for MSC-EVs and the therapeutic potential of MSC-EVs in a range of application areas, with a particular focus on their potential in the treatment of TB, current research trends and future directions in the field.

A core theory behind the enhanced biological effects of MSC-EVs is their ability to transfer bioactive molecules to target cells, thereby modulating numerous cellular processes. MSC-EVs can promote tissue repair, reduce inflammation and regulate immune responses, making them a promising therapeutic option for a range of diseases (6). The present research is primarily concerned with addressing challenges pertaining to the separation efficiency, stability and safety of MSC-EVs. Moving forward, additional enhancements in research methodologies and the accumulation of clinical trial data will be necessary to facilitate the progression of MSC-EVs therapy towards clinical implementation.

The isolation of EVs is crucial for scientific research (98) and forms the basis for maintaining their purity, yield and accuracy in subsequent functional studies. The source of MSCs used in EV research varies, with the majority being isolated from the bone marrow (51%), followed by umbilical cord/placental tissue (23%) and adipose tissue (13%). In addition, MSCs derived from embryonic or induced pluripotent stem cells account for 8% of MSCs, while other sources account for 5% of the total (26). Currently, a number of methods are commonly used for EV separation, including differential centrifugation, size exclusion chromatography, immunoaffinity capture, ultracentrifugation and ultrafiltration, each with its own unique attributes (99).

Although research on MSC-EVs in the treatment of intrapulmonary or extrapulmonary TB is still limited, preliminary findings have shown promising results (10,20). This necessitates further investigation and summarization of the current research in this area.

According to statistics from the World Health Organization, the global TB incidence reached 10.8 million in 2023, which was a 0.9% increase from 10.7 million in 2022 (109). Among extrapulmonary TB cases, genitourinary TB, particularly renal TB, is the most prevalent form (110). Previous studies have increasingly suggested that MSCs hold therapeutic promise for inflammatory and sclerotic diseases, including renal TB (11,12). Supporting this potential, one study has evaluated BM-MSCs as a combination therapy for experimental renal TB model in rabbits. Researchers induced renal TB by injecting TB H37Rv into the renal cortex under ultrasound guidance. All animals developed renal failure within 2.5 months. Anti-TB drugs reduced the levels of albumin, copper protein and elastase, improved the balance of mediators/inhibitors, and enhanced the inflammatory response, while BM-MSC transplantation exhibited different benefits. At 1 month post-transplant, inflammatory protein levels decreased, kidney-specific destructive inflammation subsided and mature connective tissue was formed, indicating activated repair mechanisms (86). Researchers compared the cellular reactions of MSCs to the virulent H37Rv strain and the attenuated H37Ra strain. The results showed that early infection (≤24 h) with H37Rv led to a marked increase in the phosphorylation of toll-like receptor (TLR)-2, protein kinase AMP-activated catalytic subunit-α (PRKAA)-1 and PRKAA2 in MSCs compared with attenuated H37Ra infection. Activation of the TLR2-AMPK pathway enhanced autophagic induction in H37Rv-infected MSCs, as demonstrated by elevated Atg9b expression and an increased LC3-II/LC3-I ratio. In addition, metabolic analysis revealed that, compared with H37Ra infection, H37Rv infection led to increased expression of SLC2A3, PFKFB3, HK1 and ABCA1 in MSCs. Despite the differences in autophagy induction, the bacterial loads between the two strains remained similar, indicating a potential role of apoptosis and immune inflammation in the control of mycobacterial growth (111).

EVs exhibit therapeutic relevance in TB. Host- or pathogen-derived EVs carry pathogenic antigens, regulating immune responses, metabolism and cell death (112). Specifically, EVs from Mtb-infected cells control inflammatory cytokines through antigen presentation, transmit immunoregulatory molecules to T helper cells and promote adaptive immune responses (113,114). Studies have shown that infection with Mtb promotes the release of MSC-EVs without affecting MSC proliferation; these vesicles can be internalized by macrophages and induce time-dependent pro-inflammatory responses through upregulation of TNF-α, RANTES and iNOS. This process is primarily mediated via the TLR2/4-MyD88 signaling pathway, and in vivo experiments further demonstrate that EVs derived from Mtb-infected MSCs (Mtb-MSC-EVs) can trigger pro-inflammatory responses in mice even during anti-TB treatment (87). A rabbit model of renal TB was established by injecting Mtb H37Rv into the renal cortical parenchyma, followed by intravenous administration of MSC-EVs in combination with standard anti-TB therapy (ATT). Compared with ATT alone, the combination treatment significantly increased serum anti-inflammatory cytokine levels while reducing pro-inflammatory cytokines and improving alkaline phosphatase, adenosine deaminase and body weight. Further analyses indicated that MSC-EVs are enriched with antimicrobial peptides such as lactotransferrin, lysozyme and cystatin B, as well as proteins with anti-inflammatory and immunomodulatory properties, suggesting a potential role in alleviating renal injury (15).

Beyond therapy, Mtb-derived membrane vesicles show potential as next-generation vaccines, eliciting stronger adjuvant-free immunity compared with the traditional Bacillus Calmette-Guérin (BCG) (115). EV-associated genes may also serve as biomarkers or therapeutic targets (114). Given their efficacy in preclinical models of acute/chronic kidney inflammation and functional impairment, MSC-EVs represent a promising strategy for renal TB treatment (10,91,116).

The lungs are particularly susceptible to Mtb infection (117). A clinical trial has investigated the systemic transplantation of autologous MSCs in patients with MDR-TB or extensively drug-resistant TB refractory to conventional therapy. A group of patients who did not respond well to standard TB treatment showed positive outcomes after receiving stem cell therapy with MSCs. At 36 months postoperatively, all 27 patients exhibited clinical improvement. Within 3–4 months after surgery, 20 patients cleared bacteria from their lungs and 11 had reduced lung tissue cavities. A total of 16 patients received stem cell therapy along with standard treatment, 9 patients experienced sustained remission of TB for up to 2 years, and 6 patients showed marked improvements in both bacterial load and structural changes in their lungs. The study suggested that combining autologous MSCs with conventional TB therapy may improve outcomes in patients with drug-resistant TB (118).

In a Cornell model of Mtb persistence in mice (119), it was observed that the highly virulent green fluorescent protein-labeled Mtb strain H37Rv persisted in CD271⁺ BM-MSCs 90 days after treatment with isoniazid and pyrazinamide. Infected BM-MSCs were identified using pyronidazole, an in vivo marker of hypoxia. Notably, the treatment regimen successfully eradicated Mtb infection in the lungs of the mice. Subsequent experiments involved the isolation of CD271⁺ BM-MSCs from post-treatment mice, which were then transplanted into healthy mice. Notably, the recipients developed active TB infections in their lungs, determining the presence of viable Mtb within BM-MSCs. In addition, this study revealed that Mtb infection induced a notable increase in the hypoxic phenotype of CD271⁺ BM-MSCs. In a parallel investigation involving individuals previously treated for TB, Mtb-containing CD271⁺ BM-MSCs exhibited heightened expression of HIF-1α and reduced levels of CD146, a cell surface marker typically downregulated in hypoxic human BM-MSCs. These findings suggested that the Mtb harbored by CD271⁺ BM-MSCs may be situated within an anoxic microenvironment, which serves a key role in promoting the quiescent state of Mtb (119). Chenari et al (120) conducted a study in which MSCs were isolated from mouse adipose tissue, and their phenotype and differentiation properties were evaluated. Cell-conditioned medium (CM) collected from these MSCs was administered intranasally to mice. This study showed that CM helped achieve an improved balance in the inflammatory response to BCG infection, thereby reducing lung tissue damage. Previous studies have identified two types of scavenger receptors (macrophage receptor with collagenous structure and scavenger receptor class B member 1) that are involved in the ability of MSCs to phagocytize Mtb (121,122). Blocking the receptor with antibodies or inhibiting its expression through siRNA technology has been proven to reduce the uptake of Mtb. Additionally, research has indicated that infusion of autologous MSCs into patients with TB is safe and can enhance lung immunity (121). Furthermore, a study explored the potential of MSC-EVs in combatting Mtb infection in alveolar epithelial type II cells. Treatment with MSC-EVs markedly inhibited the increase in bacterial load induced by Mtb infection and prevented the production of pro-inflammatory cytokines (20).

Genitourinary TB commonly results in bladder contracture and diminished bladder capacity, and in severe cases, a notably reduced bladder size until the bladder becomes completely atretic (59). Conservative treatment of stage IV bladder TB is typically ineffective, warranting consideration of surgical intervention. Although numerous surgical strategies have proven to be effective, there is a substantial risk of complications (110). Electrolyte imbalances, metabolic abnormalities, excessive mucus production, stone formation, recurrent urinary infections and altered drug metabolism should be considered when addressing potential serious complications and surgical techniques in the context of the urinary system (123). In a study utilizing a New Zealand rabbit model of bladder TB, the efficacy of interstitial injection of MSCs combined with standard anti-TB treatment in restoring bladder function was determined. To track the localization of MSCs in tissues, cells were tagged with superparamagnetic iron oxide nanoparticles. The findings indicated that the nanoparticles were readily absorbed by the cells without compromising their proliferation ability. A single administration of MSCs directly into the bladder mucosa proved to be effective in reducing bladder wall deformation and inflammation (54). In a treatment trial involving 20 female ‘Totoro’ rabbits with experimental genital TB, the administration of MSCs markedly enhanced the repair process of the body and effectively promoted re-epithelialization of the fallopian tubes 2 months after inoculation. MSCs also exhibit a bactericidal mechanism similar to that of macrophages, involving the production of ROS and the transfer of hydrolases from lysosomes to phagosomes. In addition, MSCs exhibit the potential to enhance the bactericidal capacity of macrophages by regulating their phenotypes. These findings serve as an important theoretical foundation for the development of novel approaches for TB treatment using EVs (55). A number of studies regarding the use of MSC-EVs for TB treatment are ongoing and summarized in Fig. 2 (15,54,55,87,111,119,120,123). In addition, a summary of research on EVs specifically targeting TB is provided in Table III. This includes studies involving EVs derived from Mtb-infected cells and patients, EVs administered to mice for immunization purposes, EVs isolated from serum samples of TB patients, EVs investigated in TB animal models and EVs discovered in pleural fluid samples (10,20,124–133).

MSC-EVs show marked promise in TB treatment, offering potent immunomodulation, direct antibacterial effects and tissue repair capabilities. MSC-EVs improve pathological outcomes, reduce inflammation and combat Mtb through numerous mechanisms. Furthermore, MSC-EVs improve kidney function, promote weight gain and restore serum adenosine deaminase activity. Targeted elimination of HIF-1α-high/CD146-low MSCs within hypoxic niches could potentially eradicate latent TB infections. MSCs can eliminate Mtb through delivery of ROS and lysosomal enzymes as aforementioned. Technologically, methods such as ultrafiltration combined with liquid chromatography enhance the purity and functional capacity of MSC-EVs. Notably, their therapeutic efficacy is dependent on the tissue source of the parent MSCs. Key challenges requiring resolution include establishing standardized protocols for mass production and determining long-term safety (134–137). In summary, the primary focus of current research encompasses enhancing the preparation techniques of MSC-EVs, elucidating the mechanisms underlying the biological action of MSC-EVs and utilizing MSC-EVs for the treatment of infectious diseases such as TB.

Future research directions include engineering EVs (such as MSC-EVs with high PD-L1 expression, conducting multi-omics studies and accelerating clinical translation. From a technological standpoint, there is a need to continue optimizing existing technologies and to investigate novel isolation strategies in order to improve the purity, yield and efficiency of MSC-EVs. This will provide strong support for the utilization of MSC-EVs in disease diagnosis, treatment and biomarker research, thereby advancing the field of EV-related research towards clinical applications. A key aspect in MSC-EVs clinical translation involves understanding how these conditions, such as temperature, storage duration and freeze-drying affect key properties such as morphology, functionality and therapeutic payload. Research has shown that MSC-EVs maintain their core biological activities, including anti-inflammatory effects and enhanced angiogenesis promotion, for up to 4–6 weeks when stored at temperatures of −20°C and −80°C. In addition, freeze-drying successfully preserved these functional properties (138). A combination of in vitro experiments and in vivo wound healing models has demonstrated that optimal storage conditions not only preserve stability but also ensure the functionality of miRNA and long non-coding RNA cargos within EVs (138). While there is currently a dearth of research specifically investigating the effects of MSC-EVs on Mtb infection utilizing integrated omics methodologies, findings from related studies offer encouraging prospects for utilizing MSC-EVs as therapeutic agents for TB. For example, a recent study took a comprehensive approach by integrating proteomics, metabolomics and transcriptomics analyses to elucidate the inhibitory mechanism of MSC-EVs on the HepG2 cell line of hepatocellular carcinoma, thereby contributing to advancements in this field (139). Despite their potential, current production platforms for MSC-EVs face a number of limitations during clinical application, including variability arising from donor sources, scalability challenges and inconsistent therapeutic outcomes. To effectively address these shortcomings, it is imperative to establish a scalable, standardized production platform that integrates high-quality MSC-EVs manufacturing with artificial intelligence-integrated, fully automated, Good Manufacturing Practice of Medical Products-compliant manufacturing of therapeutic EVs suitable for clinical translation (140). Collectively, MSC-EVs represent a promising novel therapeutic strategy with the potential to address drug-resistant TB and other challenging infections (125,141–143). However, prior to clinical translation, it is important to perform a thorough risk assessment, including analysis of factors such as immunogenicity, potential pro-inflammatory effects and the possibility of promoting TB dormancy.