Mixed lymphocyte reaction-conditioned MSC-derived extracellular vesicles enhance graft survival via miR-638-mediated immunoregulation

Abstract

Background

Mesenchymal stem cells (MSCs) require priming by proinflammatory stimuli for optimal immunosuppressive effects. Our previous work identified mixed lymphocyte reaction-conditioned medium (MLR-CdM) as a potent enhancer of MSC immunosuppressive properties. This study evaluates the immunomodulatory potential of MSC-derived extracellular vesicles preconditioned with MLR-CdM (MSC-EV^MLR) compared to IFN-γ (MSC-EV^IFN), focusing on key miRNAs and mechanisms involved.

Methods

We assessed the ability of MSC-EV^MLR and MSC-EV^IFN to modulate lymphocyte proliferation and cytokine expression in vitro. To identify potential effector molecules within MSC-EV^MLR, we performed miRNA array analysis combined with dose-response experiments using MLR-CdM under varying stimulation conditions. We used a murine allogeneic heterotopic heart transplantation model to investigate the impact of MSC-EV^MLR on graft survival and its immunomodulatory effects.

Results

MSC-EV^MLR outperformed MSC-EV^IFN in suppressing lymphocyte proliferation and steering cytokine expression toward an anti-inflammatory profile in vitro. Through miRNA array analysis and dose-response experiments with MLR-CdM, miR-638 was identified as a potential effector molecule in MSC-EV^MLR. In vivo study demonstrated that MSC-EV^MLR significantly prolonged graft survival, which was associated with a marked decreased proinflammatory cytokines IL6 and IFN-γ and increase in regulatory T cells (Tregs) and within the transplanted heart tissue. These effect was significantly reduced upon miR-638 knockdown. Additionally, the miR-638/Fosb axis was identified as a key pathway that promoted Treg differentiation and induced immune tolerance.

Conclusions

Preconditioning MSCs with MLR-CdM, a blend of inflammatory stimuli, potentiates the immunoregulatory capacity of MSC-EV beyond the effects of IFN-γ stimulation alone. This study advances the understanding of MSC-EV-based therapies in transplantation.

Graphical Abstract

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Introduction

In transplantation medicine, organ failure resulting from critical diseases poses a substantial challenge, with organ transplantation standing as the definitive therapeutic intervention for patients with end-stage organ failure.¹ The longevity and success of transplanted organs are heavily reliant on effective immunosuppressive management to counteract graft rejection. Current therapeutic strategies predominantly utilize broad-spectrum immunosuppressive agents such as corticosteroids, tacrolimus, mycophenolate mofetil, or cyclosporine A. Despite their efficacy, these treatments indiscriminately dampen the immune response, leading to a plethora of adverse outcomes, including increased susceptibility to infections, cancer, cardiovascular complications, metabolic disruptions, and direct toxicity to the graft, thereby jeopardizing its function.² This scenario underscores the urgent need for more precise, potent, and less harmful immunosuppressive modalities for clinical transplantation.

Among the emerging solutions to this challenge, mesenchymal stem cells (MSCs) have emerged as a beacon of hope, courtesy of their profound immunomodulatory potential.³ MSCs hailing from the embryonic mesoderm are characterized by their multipotency, ease of procurement, and expansibility. These attributes, coupled with their low immunogenic profile and ability to modulate various components of the immune system, including T and B cells, NK cells, monocytes, and regulatory T cells (Tregs), render them attractive candidates for mitigating transplant rejection and fostering immune tolerance.⁴

Despite the therapeutic promise of MSCs, their application in enhancing graft survival has been met with inconsistent outcomes, fueling debate over their efficacy and safety. Some studies report that despite MSCs’ ability to inhibit lymphocyte proliferation in vitro, their transplantation does not necessarily extend graft survival.⁵ In some cases, MSCs have been observed to accelerate rejection processes when used in conjunction with low doses of cyclosporine A.⁶ This discordance is attributed, in part, to the dual nature of MSC-mediated immunosuppression, which is not inherently active but requires priming by proinflammatory cytokines.⁷ This realization has steered research towards understanding the dynamics of MSC activation and the pursuit of optimal conditions that potentiate their immunoregulatory function.

The role of MSC-derived extracellular vesicles (MSC-EVs) in immune regulation presents a new frontier in the search for advanced immunosuppressive therapies.⁸ MSC-EVs are nanoscale vesicles laden with bioactive molecules, including proteins, mRNAs, DNAs, and microRNAs (miRNAs), capable of mimicking the biological functions of parent cells, including immune regulation. These vesicles offer several advantages over cell-based therapies, including enhanced safety, storage convenience, and ease of transport.⁹ Crucially, the function and yield of MSC-EVs are influenced by various factors including inflammatory stimuli. Exposure to inflammatory cytokines, such as IFN-γ, not only increases the production of MSC-EVs but also enhances their capacity to inhibit T-cell proliferation and induce Treg-cell generation.¹⁰ Interactions with peripheral blood mononuclear cells (PBMCs) in co-culture systems have shown that MSC-EVs can modulate the expression of proinflammatory and anti-inflammatory cytokines, facilitating a shift from Th1 to Th2 responses.¹¹ These findings underscore the potential of appropriately stimulated MSC-EVs as potent immunosuppressive agents. However, identifying the optimal stimulatory conditions for harnessing the immunosuppressive potential of MSC-EVs remains an area of active investigation. Although IFN-γ is a commonly used stimulant for eliciting MSCs’ immunosuppressive activity, emerging evidence suggests that combining multiple inflammatory factors may yield more potent immunoregulatory effects. Zhang et al demonstrated that co-stimulation of MSCs with IFN-γ and transforming growth factor-beta (TGF-β) significantly surpassed the effects of either cytokine alone in promoting the differentiation of mononuclear cells into Tregs.¹² Another group has proposed that a conditioning medium composed of tumor necrosis factor-alpha (TNF-α), IFN-γ and Interleukin 1β (IL1β) that mimics a pro-inflammatory environment effectively improves the immunomodulatory activity of MSC-EVs.^13,14 These findings suggest that the synergistic effect of multiple inflammatory cytokines on MSCs may optimize the immunoregulatory properties of MSC-EVs, enhancing their potency in suppressing immune responses.

In light of the quest for optimal stimulatory conditions for MSCs, our previous research has provided insightful findings that underscore the potential of mixed lymphocyte reaction-conditioned medium (MLR-CdM) as a potent stimulant.¹⁵ We discovered that treating MSCs with MLR-CdM not only significantly increased the secretion of proteins by MSCs but also markedly enhanced their capacity to suppress mixed lymphocyte reactions (MLR). This increase in protein yield and the improved ability to modulate immune responses suggest that MLR-CdM, as a consequence of various inflammatory cytokines, could be instrumental in augmenting the immunosuppressive functions of MSC-EVs. Building on these data, our current study aimed to delve deeper into the mechanisms underlying the enhanced immunosuppressive capacity of MSC-EVs following preconditioning with MLR-CdM. We hypothesized that MLR-CdM preconditioning not only primes MSC-EVs for improved immunomodulatory function, but also alters the composition of bioactive molecules within MSC-EVs, potentially enriching them with key immunoregulatory components. To test this hypothesis, we employed miRNA microarray analysis to identify the critical components within MSC-EVs that are modulated by MLR-CdM preconditioning. Subsequently, we validated the roles and mechanisms of the key components in mediating the immunosuppressive effects of MSC-EVs.

Materials and methods

Preparation of MSC-conditioned medium and MSC-EV extraction

Human umbilical cord mesenchymal stem cells (MSCs) were purchased from Saliai (http://www.saliai.com/;). The derivation and characterization of these MSCs have been documented in previous studies by the source lab.^16,17 MSCs were cultured in minimum essential medium plus 5% UltraGRO-Advanced GMP cell culture supplement (Helios, HPCFDCGL50). Cells from passages 4 to 8 were used for the subsequent experiments. MSCs were cultured to reach a confluence of 60%-70%. The cells were then subjected to different treatments to obtain 3 variants of MSC-conditioned medium (MSC-CdM), which were used for subsequent MSC-extracellular vesicle (MSC-EV) extraction and characterization. The groups were as follows: (1) Control Group (MSC-CdM^Ctr): the complete culture medium was replaced with serum-free DMEM. After 24 hours, the supernatant was collected. (2) IFN-γ-primed group (MSC-CdM^IFN): MSCs were stimulated with IFN-γ (50 ng/mL, MedChemExpress, #HY-P7025) for 24 hours. The medium was then replaced with serum-free DMEM, and the supernatant was collected after an additional 24 hours. (3) MLR-CdM Primed Group (MSC-CdM^MLR): MSC-CdM^MLR was prepared as describe previously.¹⁵ Briefly, spleen cell suspensions from C57/BL6 and Balb/C mice were prepared. Balb/C spleen cells were treated with mitomycin C (40 μg/mL, MedChemExpress, #HY-13316) for 30 minutes and seeded at a density of 0.5 × 10^5 cells per well in 96 well plate to serve as stimulating cells. C57/BL6 spleen cells, serving as responder cells, were mixed with stimulating cells at a 10:1 ratio and co-cultured for 96 hours. The resulting MLR-conditioned medium (MLR-CdM) was collected, centrifuged to remove cellular debris, quantified using the BCA method (Thermo Fisher, #23225), and then used to stimulate MSCs at a concentration of 50 ng/mL. After 24 hours, the medium was replaced with serum-free DMEM and the supernatant was collected after an additional 24 hours. Following the above treatments, the conditioned media from the three groups (MSC-CdM^Ctr, MSC-CdM^IFN, and MSC-CdM^MLR) were utilized for MSC-EV extraction. MSC-EVs were isolated using ultracentrifugation, as described previously,¹⁸ resulting in 3 distinct MSC-EV groups: MSC-EV^Ctr, MSC-EV^IFN, and MSC-EV^MLR, respectively. A schematic diagram of the MSC-EV^MLR preparation is shown in Figure 1A. The morphology and size of MSC-EVs were analyzed by electron microscopy (TEM). The presence and expression levels of extracellular vesicle-specific markers CD63 (Thermo Fisher, #12-0639-42 for flow cytometry; Thermo Fisher, #10628D for western blotting) and CD9 (Thermo Fisher, #12-0098-42 for flow cytometry; Cell Signaling, #13174 for western blotting) were confirmed by flow cytometry and western blotting analysis, respectively.

Quantification of Cytokines in MLR-CdM

The cytokine composition of the MLR-CdM was analyzed using the Cytometric Bead Array (CBA) Kit (BD Biosciences, #552364) according to the manufacturer’s protocol. Briefly, 50 μL of MLR-CdM was incubated with cytokine capture beads and phycoerythrin (PE)-conjugated detection antibodies in a 96-well plate at room temperature for 2 hours, protected from light. The beads were then washed twice with the provided wash buffer and resuspended in 200 μL of wash buffer. The levels of six key inflammatory cytokines, including Interleukin 10 (IL10), Interleukin 12p70 (IL12p70), monocyte chemoattractant protein-1 (MCP-1), TNF-α, IFN-γ, and Interleukin 6 (IL6), were quantified using a flow cytometer (Beckman Cytoflex). Cytokine concentrations were calculated by generating a standard curve using known concentrations of cytokine standards provided in the kit. Data acquisition and analysis were performed using BD FCAP Array Software (BD Biosciences).

Immunomodulatory effects of MSC-EV on lymphocyte proliferation

To elucidate the immunomodulatory potential of MSC-EVs in lymphocyte proliferation, a comprehensive experimental setup was designed using murine models. Spleen cells from C57/BL6 and Balb/C mice were harvested to establish a mixed lymphocyte reaction (MLR) system, providing an in vitro model for assessing lymphocyte proliferation in response to MSC-EV treatment. Briefly, spleen cells from Balb/C mice were isolated and treated with mitomycin C (40 μg/mL, MedChemExpress, #HY-13316) for 30 minutes to serve as stimulating cells. These cells were seeded in 96-well plates at a density of 0.5 × 10^5 cells per well. Concurrently, spleen cells from C57/BL6 mice were prepared as responder cells. The stimulator cells (Balb/C splenocytes) were mixed with responder cells (C57/BL6 splenocytes) at a ratio of 1:10 (0.5 × 10^5: 5 × 10^5 cells per well) in MLR culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin; 200 μL per well). Three groups of MSC-EV—MSC-EV^Ctr, MSC-EV^IFN or MSC-EV^MLR were added to co-cultures of C57/BL6 and Balb/C spleen cells. The cultures were incubated for 72 hours under optimal conditions (37°C, 5% CO₂). Following the incubation period, the proliferation of lymphocytes was quantified using a bromodeoxyuridine (BrdU) incorporation assay (Abcam, #ab126556), according to the manufacturer’s instructions.

Effects of MSC-EV on lymphocyte cytokine profiles

To assess how MSC-EVs influence the cytokine secretion profiles of lymphocytes, MSC-EV^Ctr, MSC-EV^IFN or MSC-EV^MLR were introduced into co-cultures of C57/BL6 and Balb/C spleen cells as previously described. After 72 hours under standard culture conditions (37°C and 5% CO₂), the supernatants were collected from each well, carefully centrifuged to eliminate any suspended cells, and subjected to cytokine analysis. Cytokine profiles were assessed using a Cytometric Bead Array (CBA) kit (BD Biosciences, #564085), according to the manufacturer’s instructions. This technique allowed for the simultaneous detection and quantification of multiple inflammatory cytokines present in cell culture supernatants, including IFN-γ, TNF-α, IL6, IL10, Interleukin 2 (IL2), Interleukin 4 (IL4), and Interleukin 17A (IL17A). Data acquisition and analysis were performed using BD FCAP Array Software (BD Biosciences).

miRNA profiling of MSC-EV

To investigate the differential expression of microRNAs (miRNAs) in MSC-EVs following stimulation with IFN-γ and MLR-CdM, we performed miRNA microarray analysis. Total RNA was isolated from MSC-EV^IFN and MSC-EV^MLR using the miRNeasy® Mini Kit (Qiagen, #217004). miRNA profiling was performed using Illumina HiSeqTM 2500 (Aksomics). Following normalization, miRNA expression levels were analyzed to identify significant differences between the 2 datasets. Differentially expressed miRNAs were determined by a fold-change of >1.5. Heatmaps illustrating these differentially expressed miRNAs were generated using the omicshare cloud platform.

Dose-response analysis of differentially expressed miRNAs in MSC-EV^MLR

To explore the potential dose-dependent response of differentially expressed miRNAs in MSC-EV^MLR, MSCs were stimulated with various concentrations of MLR-CdM. Following stimulation, MSC-EVs were collected, and the expression levels of miR-634, miR-638, miR-371b-3p, and miR-3156-3p were quantified using q-PCR. q-PCR reactions were performed using miRNA-specific primers and probes, with normalization to an endogenous small nucleolar RNA control, and fold changes in expression were calculated using the 2^(-ΔΔCt) method.

Immunomodulatory impact of miR-638 knockdown in MSC-EV^MLR

To investigate the role of miR-638 in mediating the immunosuppressive effects of MSC-EV^MLR, MSCs were transfected with a miR-638 inhibitor synthesized by GenePharma Co., Ltd. Lipofectamine 2000 (Thermo Fisher, #11668019) was used as the transfection reagent, according to the manufacturer’s protocol. Six hours post-transfection, the medium was replaced with MLR-CdM to stimulate the MSCs. After 24 hours of stimulation, the medium was changed to serum-free medium and the cells were incubated for an additional 24 hours. Subsequently, the cells were harvested for RNA extraction to assess the knockdown efficiency of miR-638, and the culture supernatants were collected for the extraction of extracellular vesicles (miR-638^KD-MSC-EV^MLR). The expression levels of miR-638 in these vesicles were analyzed using q-PCR. MSC-EV^MLR and miR-638^KD-MSC-EV^MLR were co-cultured with MLR comprising C57/BL6 and Balb/C mouse spleen cells, as described in an earlier section “Immunomodulatory Effects of MSC-EV on Lymphocyte Proliferation.” After a 72-hour incubation period, lymphocyte proliferation was evaluated using the BrdU incorporation assay, as outlined in earlier sections of this study. Concurrently, cytokine profiling was conducted to elucidate the impact of MSC-EVs on cytokine secretion in MLR cultures. The levels of cytokines were quantified by flow cytometry using a CBA kit (BD Biosciences, #564085) as described in the earlier section.

Establishment of an allogeneic heterotopic heart transplantation model in mice

Balb/C (male, 8-10 weeks) and C57/BL6 mice (male, 8-10 weeks) were purchased from Vitalriver. The handling of the animals and experimental protocols applied in this study were in compliance with ARRIVE guidelines 2.0, and approved by the Committee on Ethics of Medicine at Naval Medical University (approval number: 2022SLYS7). Experimental animals were assigned unique identification numbers and subsequently assigned to groups using a randomization process based on these numbers. To explore the immunomodulatory function of MSC-EVs and the role of miR-638 in this context, we established an allogeneic heterotopic heart transplantation model using a previously described method.¹⁵ Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg). The neck area of recipient C57/BL6 mice was prepared by shaving and making a transverse incision along the midline, two-thirds of the way from the jaw to the sternum. The external jugular vein was isolated and cauterized over a 3-5 mm segment. The proximal and distal ends were clamped and cut, and a segment of the vein was everted over the inserted cannula to expose the endothelium. This segment was then ligated with a 6-0 silk suture and the cannula was removed. A similar procedure was used to prepare the carotid artery for anastomosis. Balb/c mice served as donors. Following anesthesia with pentobarbital sodium (50 mg/kg), a midline abdominal incision was made and the intestines were displaced to expose the inferior vena cava. Heparinized saline (0.5 mL) was injected downstream to perfuse the heart tissue. The vena cava, aorta, and pulmonary artery were ligated and cut, and the heart was excised. The donor heart aorta was anastomosed to the recipient’s carotid artery and the pulmonary artery was anastomosed to the external jugular vein. Blood flow was restored by releasing the clamps, allowing the transplanted heart to re-perfuse. Postoperatively, recipients were administered rapamycin orally (1 mg/kg/day) for the first 10 days. Additionally, MSC-EV (10 ug in 100uL PBS) were injected intraperitoneally on days 1, 3, and 5 post-transplantation. The animals were divided into 6 groups (85 mice in total): rapamycin (Rapa) (n = 14), MSC-EV^Ctr (n = 14), MSC-EV^IFN (n = 14), MSC-EV^MLR (n = 14), miR-638^KD-MSC-EV^MLR (n = 15), and a PBS control group (n = 14) that received intraperitoneal PBS injection (100 µL) on days 1, 3, and 5 post-transplantation. The primary endpoint was graft survival, monitored daily by palpation (presence or absence of regular contractions) until cessation of heartbeat, as previously described.¹⁹

Histological evaluation of rejection

Upon cessation of the cardiac allograft beating, the grafts were promptly harvested and fixed in 10% PBS–formalin. The fixed tissues were then embedded in paraffin, and transventricular sections of 5 μm thickness were prepared. These sections were subsequently processed for hematoxylin and eosin (H&E) staining to evaluate histological changes and rejection. The stained sections were examined under a light microscope, and the degree of rejection was assessed based on established histopathological criteria. The analysis focused on identifying key indicators of rejection, including inflammatory cell infiltration, myocardial damage, and tissue disorganization. All histological evaluations were performed by blinded pathologists to ensure objectivity and accuracy in the assessment.

Evaluating the immunomodulatory effects of miR-638 mimic in MSC-EVs

To further investigate the role of miR-638 in mediating the immunosuppressive effects of MSC-EVs, MSCs were transfected with a miR-638 mimic synthesized by GenePharma Co., Ltd. Lipofectamine 2000 (Thermo Fisher, #11668019) was used as the transfection reagent, according to the manufacturer’s protocol. Six hours post-transfection, the medium was changed to serum-free medium and the cells were incubated for an additional 24 hours. Subsequently, the cells were harvested for RNA extraction to assess the expression levels of miR-638, and the culture supernatants were collected for the extraction of extracellular vesicles (miR-638-MSC-EV^Ctr). The expression levels of miR-638 in these vesicles were analyzed using q-PCR to compare MSC-EV^Ctr and miR-638-MSC-EV^Ctr. MSC-EV^Ctr and miR-638-MSC-EV^Ctr were co-cultured with MLR comprising C57/BL6 and Balb/C mouse spleen cells, as described in an earlier section “Immunomodulatory Effects of MSC-EV on Lymphocyte Proliferation.” After a 72-hour incubation period, lymphocyte proliferation was evaluated using the BrdU incorporation assay, as outlined in earlier sections of this study. In vivo experiments were conducted to assess the effects of MSC-EV^Ctr and miR-638-MSC-EV^Ctr in an allogeneic heterotopic heart transplantation model, following the method described above. Mice were divided into 2 groups: Rapa + MSC-EV^Ctr (n = 8) and Rapa + miR-638-MSC-EV^Ctr (n = 8). Graft survival was monitored daily by palpation, and histological evaluation of the grafts was performed as previously described. H&E staining was used to assess inflammatory infiltration and myocardial fiber organization in the transplanted hearts.

Cytokine and regulatory T-cell analysis post-transplantation

At 48 hours post-transplantation, the mice (n = 3 per group) were euthanized with an overdose of pentobarbital sodium (150 mg/kg) administered intraperitoneally. Samples from the serum, spleen, and transplanted hearts were collected for further analysis. Regulatory T cell (Treg) percentages in the peripheral blood, spleen, and heart transplant were assessed by flow cytometry (Mouse Regulatory T Cell Staining Kit, eBioscience, #88-8118). For cytokine analysis, graft tissues were homogenized, and proteins were extracted using RIPA buffer. Protein concentrations were determined using a BCA assay (Thermo Fisher, #23225). Cytokine levels (IL6, TNF-α, IFN-γ, IL10, IL2, IL4, and IL17A) in the serum and graft tissue were quantified using the CBA kit (BD Biosciences, #564085) and analyzed by flow cytometry.

Immunostaining

At 7 days post-transplantation, the mice were euthanized with an overdose of pentobarbital sodium (150 mg/kg, n = 3 per group). The grafts were promptly harvested, fixed, embedded in paraffin, and sectioned for immunostaining using anti-FoxP3 antibody (Abcam, #ab215206). DAPI was used to counterstain the cell nuclei. The percentage of FoxP3 + cells (per field) was then calculated by dividing the number of FoxP3 + nuclei by the total number of nuclei and multiplying by 100%.

Prediction and verification of Fosb as target gene of miR-638

A bioinformatics tool (TargetScan, http://www.targetscan.org/) was used to analyze the potential target genes of miR-638, identifying Fosb as a candidate gene. The association between miR-638 and Fosb expression was assessed using a luciferase reporter assay. Constructs containing the 3′ untranslated region (3′UTR) of Fosb, either wild-type or mutated in the predicted miR-638 binding sites, were cloned into luciferase reporter vectors. These constructs were co-transfected into HEK293T cells along with a miR-638 mimic or inhibitor using Lipofectamine 2000 (Thermo Fisher, #11668019) following the manufacturer’s guidelines. Luciferase activity was measured 48 hours post-transfection using a dual-luciferase reporter assay system and the results were normalized to Renilla luciferase activity. miR-638 mimics, negative control mimics (NC), negative control inhibitor (anti-NC), miR-638 inhibitor (anti-miR-638) were synthesized by GenePharma Co, Ltd. Transfections were performed using Lipofectamine 2000 (Thermo Fisher, #11668019), following the manufacturer’s protocol. After 48 hours of transfection, total RNA and protein were extracted for subsequent reverse transcription q-PCR and Western blot analysis.

Treg differentiation assay

Naive CD4+ T cells were isolated from C57/BL6 mouse spleens using magnetic-activated cell sorting (Miltenyi Biotec, #130-104-453). CD4+ T cells were cultured under Treg-polarizing conditions with low-dose IL2 (PeproTech, #200-02), TGF-β (PeproTech, #100-21), and anti-CD3 antibodies (BD Biosciences, Clone 145-2C11, #550275) as previously described.²⁰ MSC-EV^MLR, miR-638^KD-MSC-EV^MLR, and the Fosb inhibitor T-5224 (40 μM, MedChemExpress, #HY-12270) were added to the cultures to assess their impact on Treg differentiation. After a specified incubation period, the cells were stained with antibodies against FoxP3 (Mouse Regulatory T Cell Staining Kit, eBioscience, #88-8118), and the proportion of FoxP3+ Tregs was determined by flow cytometry.

Western blotting

Protein samples were lysed (Thermo Fisher, # 89901) and quantified using the BCA assay (Thermo Fisher, #23225). Proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 hour in a 5% non-fat milk solution to prevent non-specific binding. After blocking, the membranes were incubated with primary antibodies specific to the target proteins overnight at 4°C. The primary antibodies used in this experiment were anti-CD9 (Cell Signaling, #13174), anti-CD63 (Thermo Fisher, #10628D), and anti-Fosb (Cell Signaling, #2251). After primary antibody incubation, the membranes were washed three times with TBST (Tris-buffered saline, 0.1% Tween 20) and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The membranes were washed 3 times with TBST before detection using enhanced chemiluminescence (ECL). Signals were visualized and quantified using a chemiluminescence imaging system. Protein loading was normalized to that of GAPDH detected using an anti-GAPDH antibody as loading control.

Statistical analysis

All statistical analyses were performed using Graphpad Prism Version 10.2.1. Comparisons between 2 groups were conducted using Student’s t-test, while one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test was used for multiple comparisons. Survival rates were analyzed using the Kaplan-Meier method, and differences between survival curves were assessed with the log-rank test. Statistical significance was set at P < .05.

Results

Characterization of MSC-EV

A schematic diagram illustrates the preparation of MSC-EV^MLR (Figure 1A). Electron microscopy (TEM) was used to examine the morphology of MSC-EVs. The results revealed that all examined MSC-EV samples displayed a characteristic double-membrane structure typical of extracellular vesicles (Figure 1B). To confirm the exosomal nature of the isolated vesicles, flow cytometry, and Western blotting analyses were conducted to detect the presence of the exosomal markers CD9 and CD63. Both analyses demonstrated positive expression of CD9 and CD63 in MSC-EV^ctr, MSC-EV^IFN, and MSC-EV^MLR (Figure 1C-D).

Differential immunomodulatory impact of MSC-EVs on mixed lymphocyte reactions and cytokine dynamics

We introduced MSC-EV^ctr, MSC-EV^IFN, and MSC-EV^MLR into the MLR system. This system consisted of co-cultures of C57/BL6 and Balb/C spleen cells. After 72 hours of co-culture, lymphocyte proliferation was evaluated using a BrdU assay. The supernatants were collected for cytokine expression analysis. Our findings indicated that all variants of MSC-EVs were capable of suppressing MLR to varying extents. Among these, MSC-EV^MLR exhibited the most significant suppressive effect on MLR, outperforming MSC-EV^IFN (P < 0.01, Figure 2A). Treatment with MSC-EV^Ctr, MSC-EV^IFN, and MSC-EV^MLR significantly modulated cytokine profiles within the MLR system. Specifically, there was a marked downregulation in the expression of pro-inflammatory cytokines (IL6, TNF-α, and IFN-γ) and upregulation of the anti-inflammatory cytokine (IL10) across all MSC-EV-treated groups. Notably, the MSC-EV^MLR group exhibited a more pronounced effect in decreasing IL6 and IFN-γ levels, and increasing IL10 expression compared to the MSC-EV^IFN group (Figure 2B). These results suggest that MSC-EV^MLR derived from MLR-CdM stimulation possesses superior immunosuppressive capabilities compared to MSC-EV^IFN and MSC-EV^Ctr. To further investigate the underlying factors contributing to this enhanced immunosuppressive effect, we analyzed the cytokine composition within the MLR-CdM. The CBA analysis revealed elevated levels of multiple cytokines, including IL10, IL12p70, MCP-1, TNF-α, IFN-γ, and IL6, in the MLR-CdM. Notably, MCP-1 and IL12p70 exhibited the most significant upregulation among the detected cytokines (Supplementary Figure 1).

Identification of miR-638 as potential effector in MSC-EV^MLR mediated Immunomodulation

To elucidate the molecular basis underlying the distinct immunosuppressive capacities of MSC-EVs following IFN-γ and MLR-CdM stimulation, we performed a miRNA microarray analysis. A comparative study revealed a unique set of miRNAs enriched in MSC-EV^MLR (Figure 3A, Supplementary Table 1).

Among the significantly altered miRNAs, a literature search revealed that miR-638 is closely associated with immune regulation. In patients with diabetic nephropathy, serum miR-638 levels were significantly lower compared than in healthy controls. The use of miR-638 mimics significantly decreased the levels of inflammatory factors in human mesangial cells induced by high glucose.²¹ miR-638 exhibits a distinct expression pattern in lupus nephritis, with decreased glomerular and increased tubulointerstitial expression, suggesting its critical role in modulating local immune responses.²² However, its role in MSC-EV^MLR-mediated immunosuppression remains unclear. Therefore, we focused on miR-638 to understand its specific role in the immunomodulatory effects observed in MSC-EV^MLR. A further investigation of the expression dynamics of the miR-638 under varying concentrations of MLR-CdM stimulation revealed a particularly compelling pattern for miR-638. Its expression in MSCs demonstrated a clear dose-response relationship, markedly adjusting in proportion to the concentration of MLR-CdM (Figure 3B). Additionally, we examined other miRNAs enriched in MSC-EV^MLR identified through miRNA array analysis, including miR-634, miR-371b-3p, and miR-3156-3p. While these miRNAs responded to MLR-CdM stimulation, they did not exhibit a dose-dependent expression pattern as prominently as miR-638 (Supplementary Figure 2), further underscoring the unique regulatory potential of miR-638 in this context.

The role of miR-638 in MSC-EV^MLR-induced immunomodulation and cytokine regulation

Following the identification of miR-638 dose-dependent expression in MSC-EV^MLR, which suggested its pivotal role in enhancing the immunosuppressive function of these extracellular vesicles, we further explored the mechanistic aspects through q-PCR analysis. This confirmed that miR-638 levels were significantly higher in MSC-EV^MLR than in MSC-EV^IFN and MSC-EV^ctr (Figure 4A), aligning with the miRNA chip data that indicated a five-fold increase. To better understand the temporal dynamics of miR-638 expression, we stimulated MSCs with MLR-CdM for 24 and 48 hours, followed by a 24-hour incubation in serum-free medium before collecting the conditioned medium for EV extraction, resulting in MSC-EV^MLR-24 and MSC-EV^MLR-48, respectively. q-PCR analysis revealed that miR-638 expression in MSC-EV^MLR-48 tended to be lower than in MSC-EV^MLR-24 (though not statistically significant, P = .06, Supplementary Figure 3). Therefore, in subsequent experiments, we adhered to the initial experimental design, using MSC-EV stimulated with MLR-CdM for 24 hours. To assess the direct impact of miR-638 on immunomodulatory capabilities of MSC-EV^MLR, we employed a miR-638 inhibitor, leading to the successful knockdown of miR-638 in both MSCs (termed miR-638^KD-MSC^MLR, Figure 4B) and MSC-EV^MLR (termed miR-638^KD-MSC-EV^MLR, Figure 4C). This modification notably impaired the ability of MSC-EV^MLR to suppress mixed lymphocyte reactions (P < .05, Figure 4D), highlighting the critical role of miR-638 in mediating this process. Additionally, the knockdown of miR-638 altered cytokine profiles, with a significant reversal observed in the expression of key pro-inflammatory cytokine IFN-γ and anti-inflammatory cytokine IL10 (Figure 4E), suggesting the indispensable role of miR-638 in the immunosuppressive function of MSC-EV^MLR.

The role of miR-638 in MSC-EV^MLR mediated immunoregulation and inflammatory response in heart transplantation

We evaluated the immunomodulatory effects of MSC-EV^MLR and the role of miR-638 in prolonging graft survival in an allogeneic heart transplant model. Figure 5A shows a schematic diagram of the animal experimental design and grouping. Daily monitoring of transplanted heart function through palpation revealed that all MSC-EV treatments extended the survival of heart transplants compared to the PBS control and Rapa group (Figure 5B, Supplementary Table 2). Importantly, the effectiveness in extending transplant survival demonstrated a gradation, with MSC-EV^MLR exhibiting the highest capability (medium survival 22 days), followed by MSC-EV^IFN (medium survival 15 days), and MSC-EV^Ctr (medium survival 11.5 days), showing the least effect. This gradation underscores the superior immunomodulatory potential of MSC-EV^MLR. Knockdown of miR-638 in MSC-EV^MLR weakened its ability to extend transplant survival (medium survival 12 days), indicating a pivotal role of miR-638 in enhancing protective effects of MSC-EV^MLR (Figure 5B, Supplementary Table 2). Histopathological analysis revealed marked inflammatory cell infiltration, disorganization, diminished cardiomyocytes, and severe myocardial damage in the PBS control group (Figure 5C). The Rapa group exhibited similar severe outcomes. In contrast, the Rapa + MSC-EV intervention groups showed significant improvements, with noticeable reductions in inflammatory cell infiltration, better organization of cardiomyocytes, and less myocardial damage, particularly in the MSC-EV^MLR group, which demonstrated the most pronounced benefits (Figure 5C). However, in the miR-638^KD-MSC-EV^MLR group, where miR-638 expression was knocked down, these pathological changes, including immune infiltration and myocardial fiber loss, reappeared (Figure 5C). We further explored the modulation of Tregs and expression of inflammatory cytokines to understand the comprehensive immunomodulatory effects of MSC-EVs in a heart transplant context. Given that 48 hours post-transplantation is a critical period for cytokine and inflammatory cell responses, making this a crucial time point for capturing the acute immune response,²³ mice were sacrificed to collect serum, spleen, and heart transplant tissues for analysis. The proportion of CD4 + CD25 + FoxP3 + Tregs in peripheral blood and transplanted heart was not significantly different across all groups (data not shown). However, MSC-EV treatment notably increased the percentage of CD4 + CD25 + FoxP3 + Tregs within the spleen, with MSC-EV^MLR demonstrating the most significant increase, followed by MSC-EV^IFN and MSC-EV^Ctr (Figure 6A-B). This augmentation was markedly attenuated in the miR-638^KD-MSC-EV^MLR group, indicating a reduction in Treg upregulation (Figure 6A-B). Cytokine profiling revealed that while peripheral blood cytokine levels remained unchanged across groups (data not shown), MSC-EV administration significantly reduced the levels of the pro-inflammatory cytokines IL6 and IFN-γ within the transplant heart, with MSC-EV^MLR showing the greatest decrease (Figure 6C). This effect was notably impaired in the miR-638^KD-MSC-EV^MLR group, suggesting a crucial role of miR-638 in modulating the cytokine environment (Figure 6C). Since no changes in Treg proportions were observed in the heart grafts at 48 hours post-transplantation, we examined the Treg changes in the transplanted hearts at 7 days post-transplantation. Immunofluorescence staining showed that MSC-EV^MLR treatment significantly increased the number of Tregs within the heart grafts, while this effect was substantially reduced in the miR-638^KD-MSC-EV^MLR group (Figure 6D-E). To provide a more comprehensive understanding the role of miR-638 in MSC-EVs, we transfected MSCs with a miR-638 mimic (termed miR-638-MSC^Ctr) and subsequently extracted EVs from these MSCs (termed miR-638-MSC-EV^Ctr). q-PCR analysis showed that miR-638 mimic transfection effectively increased miR-638 expression levels in both MSCs and MSC-EVs by over 2.5-fold (Supplementary Figure 4A-B). In vitro experiments revealed that miR-638-MSC-EV^Ctr had a significantly stronger inhibitory effect on MLR compared to MSC-EV^Ctr (Supplementary Figure 4C). In vivo experiments showed a trend towards improved graft survival in the Rapa + miR-638-MSC-EV^Ctr group (medium survival 14 days) compared to the Rapa + MSC-EV^Ctr group (medium survival 11 days), although the difference was not statistically significant (Supplementary Figure 4D, Supplementary Table 3). H&E staining indicated prominent immune infiltration and disorganized myocardial fibers in both groups (Supplementary Figure 4E). These findings underscore the pivotal function of miR-638 in MSC-EV^MLR in extending heart transplant survival, likely through the regulation of Treg populations and the suppression of pro-inflammatory cytokines.

miR-638/Fosb axis promotes Treg differentiation and immune tolerance in MSC-EV^MLR

Building on the established role of miR-638 in enhancing the immunosuppressive effects of MSC-EV^MLR, further bioinformatics analysis suggested Fosb as a potential target gene of miR-638 (Figure 7A). The interaction of miR-638 and Fosb was subsequently validated by a luciferase reporter assay, demonstrating that miR-638 directly downregulated Fosb expression (Figure 7B). HEK293T cells were treated with miR-638 mimics or inhibitors, which significantly affected Fosb expression (Figure 7C). Given the known involvement of Fosb in post-activation T-cell regulation,²⁴ we explored whether the miR-638/Fosb pathway influences the differentiation of Tregs, thereby inducing immune tolerance. Naïve CD4+ T cells were exposed to low doses of IL2, TGF-β, and anti-CD3 to simulate an environment for Treg differentiation. The addition of MSC-EV^MLR to this culture system markedly promoted FoxP3+ Treg differentiation, which was attenuated by the miR-638 knockdown (Figure 7D). Notably, addition of a Fosb inhibitor (T-5224) to the miR-638^KD-MSC-EV^MLR group partially restored the proportion of Tregs (Figure 7D), underscoring the intricate interplay between miR-638/Fosb axis and Treg differentiation.

Discussion

In this study, we investigated the immunomodulatory effects of MSC-EVs, with a particular focus on MSC-EV^MLR, and identified the pivotal role of miR-638 in mediating these effects. Our findings demonstrate that MSC-EV^MLR exerts a profound immunosuppressive effect in an MLR system, surpassing those of MSC-EV^IFN and MSC-EV^Ctr. This observation is consistent with previous studies highlighting the potential of MSC-EVs to modulate immune responses, where the cargo within these vesicles, particularly non-coding RNAs, plays a crucial role in their functionality.^25,26 The superior performance of MSC-EV^MLR in suppressing lymphocyte proliferation and modulating cytokine expression highlights the critical influence of MLR-CdM preconditioning on enhancing the therapeutic efficacy of MSC-derived EVs. Further analysis of the cytokine composition within the MLR-CdM revealed significant upregulation of MCP-1 and IL12p70, suggesting that these cytokines may play a pivotal role in priming MSCs to secrete EVs with enhanced immunosuppressive properties.

Through miRNA array analysis, we identified several miRNAs enriched in MSC-EV^MLR. Combined with a review of existing literature, miR-638 emerged as a potential key factor contributing to the immunosuppressive properties of MSC-EV^MLR. The dose-dependent expression of miR-638, coupled with the diminished immunomodulatory effect upon its knockdown, emphasizes the specific contribution of this miRNA to the MSC-EV^MLR phenotype. The attenuation of pro-inflammatory cytokines and enhancement of anti- inflammatory IL10 expression in our MLR system further support miR-638’s role in steering MSC-EV^MLR towards an anti-inflammatory and immunosuppressive profile. This aligns with the emerging literature that recognizes the nuanced roles of specific miRNAs in dictating the functional outcomes of MSC-EVs in the immune system.^27,28

The in vivo validation of MSC-EV^MLR’s capacity to prolong heart transplant survival and its dependency on miR-638 to achieve maximum efficacy highlight the translational potential of MSC-EV^MLR in pre-clinical settings. Moreover, the increase in Treg proportions in spleen and modulation of cytokine profiles within the transplant tissue delineate a comprehensive mechanism through which MSC-EV^MLR, facilitated by miR-638, promotes transplant tolerance at the early phase of acute immune rejection. It is noteworthy that Treg levels in the grafts did not show significant changes during the initial 48 hours post-transplantation. However, by day 7 post-transplantation, the number of FoxP3 + Treg cells was significantly increased in the grafts of the MSC-EV^MLR group. Additionally, miR-638 knockdown inhibited the upregulation of Tregs by MSC-EV^MLR, indicating the critical role of miR-638 in this process. One possible explanation for the initial lack of significant Treg changes in the grafts is that MSC-EVs were administered intraperitoneally with only one intervention at 48 hours post-surgery. While MSC-EV^MLR effectively downregulated local pro-inflammatory cytokines IL6 and IFN-γ in the grafts, their impact on Treg populations may require a longer period to become evident. It is also worth mentioning that overexpressing miR-638 in MSC-EV^Ctr enhanced the inhibition of MLR in vitro, but this did not translate into a significant extension of graft survival in vivo. This suggests that other components within MSC-EVs, including non-coding RNAs or proteins, may also play a crucial role in the immunosuppressive effects mediated by MSC-EV^MLR. Inspired by these findings, future research should focus on elucidating the contributions of other non-coding RNAs and bioactive factors within MSC-EVs.

Our exploration of the miR-638/Fosb axis as a novel pathway influencing Treg differentiation and immune tolerance opens new avenues for therapeutic interventions. Fosb, as a member of the AP-1 transcription factor family, plays a role in immune regulation, including T-cell activation and differentiation.^24,29 Specific studies directly linking Fosb to Treg regulation are limited. However, Fosb’s involvement in the regulation of IL2,³⁰ a critical cytokine for Treg function, provides an indirect pathway through which it can influence Treg differentiation and function. Our study supported that miR-638 targeted Fosb to induce Treg differentiation, thereby contributing to the immunosuppressive effects of MSC-EV^MLR. By elucidating the miR-638/Fosb axis, we provide a potential target for enhancing the efficacy of MSC-EV-based therapies in transplantation and other immune-mediated conditions.

Recent studies have emphasized the importance of licensing MSCs to fully unleash their immunosuppressive capabilities.^31,32 While some studies suggest that combined stimulation with multiple inflammatory cytokines, such as IL1β and IFN-γ, enhances the immunosuppressive effects of MSCs more effectively than single cytokine stimulation,³³ this is not universally true. In fact, certain combinations, such as IFN-γ and Poly (I:C), may increase the expression of pro-inflammatory factors rather than enhancing immunosuppression.³⁴ Therefore, to fully activate the immunosuppressive abilities of MSCs, it is crucial to identify an appropriate stimulation strategy. In our study, we demonstrated that preconditioning MSCs with MLR-CdM significantly boosted their immunosuppressive potential compared with stimulation with IFN-γ alone. This finding underscores the superior efficacy of MLR-CdM as a stimulant, providing a more complex and physiologically relevant inflammatory milieu that better permits MSCs for therapeutic use. The enhanced performance of MSC-EV^MLR in suppressing lymphocyte proliferation and modulating cytokine profiles further supports the notion that a combination of inflammatory stimuli is more effective than individual cytokines in priming MSCs. Through CBA analysis we revealed that MCP-1 and IL12p70 were significantly upregulated in the MLR-CdM, suggesting that these cytokines may act as key effector molecules mediating the immunosuppressive properties of MSC-EV^MLR. However, the precise interplay between MCP-1, IL12p70, and other inflammatory mediators in the context of MSC-mediated immunomodulation remains incompletely understood. Future studies could focus on dissecting the signaling pathways and cellular interactions driven by these cytokines to optimize the therapeutic potential of MLR-CdM. Moreover, the cytokines detected using the CBA kit represent only a subset of the proteins present in the MLR-CdM. It is likely that MLR-CdM provides a broader range of stimulatory signals and mimics the complex in vivo inflammatory environment more closely than single cytokine stimulation, thereby enhancing the immunosuppressive function of MSCs more effectively. To further identify and understand the functional role of additional key mediators, high-throughput methods such as proteomic analysis are necessary. Nontherless, our study provides valuable insights into optimizing MSC-based therapies and highlights the potential of MLR-CdM as a potent preconditioning strategy to maximize the immunoregulatory function of MSC-EVs.

Although our findings offer promising insights into the immunomodulatory potential of MSC-EV^MLR and the critical role of miR-638, several limitations warrant consideration. First, our study highlights the potential of MLR-CdM stimulation as a promising approach to enhance the immunosuppressive properties of MSC-EVs. While MCP-1 and IL12p70 were significantly upregulated in the MLR-CdM, it remains unclear whether they are the primary mediators of the observed effects or how they interact with other factors within the conditioned medium. Future studies should focus on elucidating these interactions and leveraging high-throughput analyses to identify additional contributors to MSC-EV-mediated immunosuppression. Second, while our study identified miR-638 as a key regulator in MSC-EV^MLR-mediated immunosuppression, its broader off-target effects and potential roles beyond Treg differentiation remain to be fully elucidated. Like many miRNAs, miR-638 likely exerts pleiotropic effects by targeting multiple genes and pathways. Although our study focused on the miR-638/Fosb axis and its impact on Treg differentiation, miR-638 may also influence other immune and non-immune cell types, highlighting the complexity of its regulatory functions. To this end, applying advanced techniques such as single-cell sequencing would be instrumental in uncovering the cellular heterogeneity and specific pathways involved. Third, while our study primarily focused on the early post-transplant phase (48 hours), extending the observation period could provide a more comprehensive understanding of the long-term immunomodulatory effects of MSC-EV^MLR. Future studies should incorporate additional time points to determine whether the immunosuppressive effects of MSC-EV^MLR persist or evolve over time. Additionally, optimizing the dosing frequency and quantity of MSC-EVs is critical for refining clinical protocols. In this study, we used multiple injections of MSC-EVs combined with rapamycin based on preliminary findings that either MSC-EVs alone or rapamycin combined with a single MSC-EV injection had limited efficacy in prolonging graft survival. However, the optimal dose, injection frequency, and delivery methods remain undefined and warrant further exploration. Fourth, other components within MSC-EVs, including the miRNAs with significant expression differences identified in our miRNA array, as well as other bioactive molecules and cellular sub-organelles like mitochondria, might also contribute to the immunosuppressive effects mediated by MSC-EVs. Further research is needed to elucidate these contributions. Finally, the clinical translation of MSC-EV^MLR faces significant challenges, including scalable production, standardization of MLR conditioning protocols, quality control of miR-638 content and rigorous safety assessment. Scalable production can be addressed by advanced bioreactor systems that ensure GMP-compliant, serum-free, and xeno-free conditions while maintaining EV quality and consistency.³⁵ Standardizing responder-to-stimulator cell ratios, incubation times, and potency assays is essential to ensure reproducibility and reliability across batches of MLR-CdM. For miR-638, rigorous quality control measures, such as digital droplet PCR (ddPCR) and next-generation sequencing (NGS), are necessary for precise quantification and profiling. These should be paired with standardized EV isolation techniques, such as size-exclusion chromatography, to minimize batch variability. Functional assays are also essential to validate the bioactivity of miR-638 and its therapeutic relevance. Safety remains a critical consideration, despite the low immunogenicity of MSC-EVs due to their lack of cell surface antigens.³⁶ However, the potential unintended effects of bioactive cargo and long-term immunomodulatory consequences must still be comprehensively evaluated through rigorous preclinical studies.

Conclusion

Our study revealed that the potent immunomodulatory effects of MSC-EV^MLR were significantly enhanced by miR-638 in suppressing lymphocyte proliferation and modulating cytokine profiles toward anti-inflammatory outcomes. The critical role of miR-638, particularly through the miR-638/Fosb axis, in promoting transplant tolerance and Treg differentiation underscores the therapeutic potential of MSC-EV^MLR in immune regulation. Despite these promising results, further research is essential to address the challenges of translating these findings into clinical applications, highlighting the need for comprehensive studies on the broader implications of MSC-EV-based therapies.

Acknowledgments

We would like to acknowledge that the schematic diagrams included in this article were created using Figdraw (https://www.figdraw.com).

Author contributions

Yue Ding Ding and Jiyuan Wang (Data curation, Investigation, Writing—original draft; Xiaoting Liang, Yue Ding, and Jiyuan Wang (Data curation and Writing—review & editing). ; Xueyang Zheng, Yu Chen, Fanyuan Zhu, Fang Lin, Kexin Ma (Resources); Xiaoting Liang and Shu Han (Investigation, Project administration, Supervision, Writing—review & editing)..

Funding

This research was supported in part by a Natural Science Foundation of Shanghai (24ZR1459300 to Xiaoting Liang), National Natural Science Grant of China (81500207 to Xiaoting Liang) and the Pyramid Talent Project (YQ677 to Yue Ding).

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

The miRNA array data has been uploaded to figshare (DOI: 10.6084/m9.figshare.26537251). The data will be made available upon request.

Consent for publication

All named authors consent to the publication of this manuscript.

Ethics approval

The human umbilical cord mesenchymal stem cells (MSCs) used in this study were purchased from Saliai, a supplier based in Guangzhou, China. The derivation and characterization of these MSCs have been thoroughly documented in previous studies conducted by the source lab, as referenced in (Life Sci, 2020. 246: p. 117401.; NPJ Regen Med, 2024. 9(1): p. 4.) Although a Material Transfer Agreement (MTA) was not provided within the purchase, the MSCs were obtained from a reputable source that adheres to strict ethical standards, as evidenced by the detailed characterization and ethical approvals reported in the published studies. Shanghai East Hospital, with its GMP-certified laboratory, is fully equipped to handle human-derived cells and adheres to all relevant regulatory standards. While our laboratory maintains compliance with GMP standards, specific institutional ethical approval for the use of commercially sourced human cells in this project was not obtained. The handling of the animals and experimental protocols used in this study were approved by the Committee on Ethics of Medicine at Naval Medical University (approval number: 2022SLYS7, approval date: May 22, 2022, titled “Study of Immunosuppression Induced by Enhancing miR-638 Expression in Mesenchymal Stem Cell Exosomes Stimulated by Mixed Lymphocyte Reaction Culture Supernatants”).

References

Author notes