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MSC-sEVs exacerbate senescence by transferring bisecting GlcNAcylated GPNMB

Stem Cell Research & Therapy volume 16, Article number: 23 (2025) Cite this article

Abstract

Background

The senescence of bone marrow mesenchymal stem cells (BMMSCs) is increasingly recognized as a critical factor contributing to the pathophysiology of age-related diseases. Recent studies suggest that small extracellular vesicles (sEVs) derived from the serum of elderly individuals may play a pivotal role in promoting BMMSC senescence. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane glycoprotein, is upregulated during cellular senescence and can regulate stem cell ageing. However, the precise mechanisms by which GPNMB influences BMMSCs senescence remain poorly understood. Understanding this relationship could provide valuable insights into therapeutic strategies for enhancing BMMSCs function and mitigating age-related degeneration.

Methods

In this study, we conducted comprehensive in vitro experiments to elucidate the effects of sEVs isolated from the serum of elderly donors on the senescence of BMMSCs. We employed advanced proteomic analysis to quantify the expression levels of GPNMB in both BMMSCs and sEVs. Statistical methods were utilized to investigate the correlations between GPNMB expression, glycosylation modifications, and established senescence markers.

Results

Our findings demonstrate a robust positive correlation between the expression of GPNMB in BMMSCs and sEVs and the induction of cellular senescence. Notably, we observed that elevated levels of GPNMB, particularly those bearing bisecting N-acetylglucosamine (GlcNAc) modifications, significantly enhance the senescent phenotype of BMMSCs. Furthermore, we identified the bisecting GlcNAc modification at the Asn 249 residue of GPNMB as a critical determinant for its senescence-promoting function.

Conclusions

This study elucidates the substantial role of sEVs derived from mesenchymal stem cells in exacerbating BMMSC senescence through mechanisms that are critically dependent on the presence of bisecting GlcNAcylated GPNMB. These insights emphasize the necessity of targeting glycosylation modifications of GPNMB in the design of novel senolytic therapies aimed at mitigating cellular ageing and its associated pathologies.

Introduction

Aging is an inevitable, time-dependent process that eventually limits the capacity of cells to maintain efficient homeostasis and repair mechanisms, resulting in a general decline in physiological functions throughout the body. Small extracellular vesicles (sEVs), containing a variety of bioactive proteins, lipids, or nucleic acids, are important for mediating cell-to-cell communication [1, 2], and are now of great interest in the context of ageing and age-related diseases [3,4,5]. Specifically, sEVs derived from aged mesenchymal stem cells (MSCs) are particularly noteworthy, as these cells are central to tissue repair and homeostasis [6, 7]. In contrast to their youthful counterparts, MSCs from aged individuals exhibit a decline in their regenerative potential, a process that is further exacerbated by the senescence-inducing effects of sEVs they secrete. Several studies have now demonstrated that sEVs from aged BMMSCs promote senescence and inflammation in neighboring cells. For example, studies have shown that sEVs from aged BMMSCs carry pro-senescent factors, leading to the accelerated senescence of young MSCs and other target cells [8]. These sEVs can also modulate immune responses, potentially exacerbating chronic inflammation commonly observed in aging tissues. The cargo of these vesicles, including microRNAs and proteins, influences a range of signaling pathways that contribute to cellular dysfunction. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane protein that was originally identified in melanoma cell lines [9]. It was highly upregulated in Parkinson’s disease (PD) and Alzheimer’s disease (AD) [10, 11]. Recently, studies have identified GPNMB as a molecular target for ageing treatment [8]. It has been reported that GPNMB promotes cancer cell aggressiveness and inhibits CD8 + T cell activation by being packaged into sEVs during the progression of hepatocellular carcinoma (HCC) [12]. However, as a highly N-linked glycosylated protein [13, 14], whether glycosylation modifications can alter GPNMB function in sEVs during MSC ageing process need to be fully elucidated.

In the present study, we found the expression of GPNMB protein were significantly elevated in senescent MSCs and its sEVs. Further investigations have demonstrated that one type N-linked glycosylation, bisecting GlcNAc, on GPNMB is pivotal to its function. It was elucidated that bisecting GlcNAcylated-modified GPNMB protein plays a pivotal role in the sEVs-mediated ageing process of MSCs. These findings offer a novel direction for future research into the mechanism of GPNMB protein in the ageing process of MSCs.

Materials and methods

The work has been reported in line with the ARRIVE guidelines 2.0.

Cell lines and cell culture

Mouse bone marrow mesenchymal stem cell line MSC-T was from Cell Bank at the Chinese Academy of Sciences (Shanghai, China) and cultured in minimal essential medium alpha (MEMα) added with 10% fetal bovine serum (FBS; Biological Industries, Beit HaEmek, Israel) and 1% penicillin/streptomycin (Beyotime Biotechnology; Haimen, Jiangsu, China) at 37 °C in 5% CO2 atmosphere.

Isolation and culture of mouse primary BM mesenchymal stem cells

Mouse primary MSCs (abbreviated as m-MSCs) were isolated from tibia and femoral marrow. Mice were anesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg body weight), administered to induce deep anesthesia. After confirming the absence of the righting reflex, the animals were placed on a heating pad to maintain body temperature during the procedure. After anesthesia, euthanasia was performed by cervical dislocation to ensure rapid and humane death. The bone marrow was flushed with PBS, and cells were filtered through a 70 μm strainer. After centrifugation, the cells were cultured for 24 h in MEMα medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 incubator. Culture medium was changed every 3 days. Cells positive for hematopoietic markers CD34 (#119309, BioLegend; San Diego, CA, USA), CD45 (#157605) were excluded by gating, and the expression of MSC markers CD44 (#103011), CD73 (#117203), and CD90 (#105201) were evaluated in the remaining adherent cells by flow cytometry (ACEA Biosciences; San Diego, CA, USA).

Isolation and culture of human primary mesenchymal stem cells

Written informed consent was obtained from all bone marrow donors in accordance with Declaration of Helsinki guidelines. All experimental designs using human tissues were reviewed and approved by the Research Ethics Committee of Northwest University. Human primary MSCs (abbreviated as h-MSCs) were isolated from the bone marrow of healthy subjects. First, mononuclear cells are isolated by Ficoll-Hypaque gradient centrifugation. Primary bone marrow mesenchymal stem cells (MSCs) are isolated from bone marrow monocytes by density gradient centrifugation, cultured in DMEM-hyaline medium, and expanded through selective adhesion, with MSCs characterized by surface markers. DMEM/F12 medium containing 10% FBS is added to the flask and incubated at 37 °C with 5% CO2.

Stable transfection of GPNMB

GPNMB gene was amplified via PCR and linked to lentiviral overexpression vector pLVX-AcGFP-N1 (Takara; Shiga, Japan). Lentiviral vector pLKO.1 (Sigma-Aldrich; MO, USA) encoding short hairpin RNAs (shRNAs) targeting GPNMB were generated using piLentisiRNA. Scrambled piLenti-siRNA was used as negative control. Lentiviral vectors were packed in HEK293T using Lipofectamine 2000 reagent (Thermo Fisher Scientific; San Jose, CA, USA), together with pMD2.G and psPAX2 (Addgene; Cambridge, MA, USA). Lentivirus particles were harvested 48 h after transfection, and MSCs were infected with the resultant lentivirus. Transfected cells were cultured in complete medium with puromycin (2 µg/ml) for 4 days. Stable transfectants were selected, and confirmed by western blotting analysis.

Clinical samples

Serum samples of healthy elderly and young people were collected from Xi’an First Hospital affiliated with Northwest University (Table S1). Written informed consent was obtained from all patients.

Western blotting

Cells were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl2, 5% glycerol) containing 1% protease inhibitor (Sigma-Aldrich; St. Louis, MO, USA). The lysate containing cellular proteins was then centrifuged (14000 × g, 15 min, 4 °C), after which the supernatant was collected and the protein concentration was determined by the Bicinchoninic acid (BCA) method (Beyotime). Proteins (30 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad; Hercules, CA, USA). The membranes were blocked using 3% BSA (# ST023,Beyotime), incubated with the primary antibody MMP9 (#A0289), p16 (#A23882), IL-6 (#A22222, ABclonal, Wuhan, China), and subsequently incubated with the corresponding enzyme-labelled secondary antibody conjugated with HRP (#A0208, Beyotime). Bands were displayed by enhanced chemiluminescence (ECL, Vazyme, Nanjing, China) and imaged using a bioluminescence imaging system (Tanon; Shanghai, China).

Lectin blotting

The proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membranes were soaked in 3% (w/v) BSA in TBST at 37 °C for 2 h. The membranes were incubated with biotin-coupled lectins PHA-E or PHA-L (Vector Labs; Burlingame, CA, USA) overnight at 4 °C. Subsequently, they were incubated with the appropriate enzyme-labelled VECTASTAIN ABC (Vector Labs; Burlingame, CA, USA). Bands were visualized and photographed by an imaging system as described above.

Quantitative real-time PCR (qRT-PCR)

Total RNA was prepared using the RNA Pure Tissue & Cell kit (CW Biotech; Beijing, China). The RNA purity was assessed by an A260/A280 ratio of 2.0 and an A260/A230 ratio of 2.1, indicating high-quality, undegraded RNA. Primers were designed using Primer-BLAST software (ncbi.nlm.nih.gov/tools/primer-blast). The first strand of cDNA was synthesized from total RNA using HiScript II Q RT SuperMix (Vazyme). qRT-PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme) [15] and gene expression was quantified using the 2−ΔΔCt method [16]. Primers and shRNAs are listed in the supplemental Table S2.

Cell apoptosis

Cells (2 × 105) were soaked with FITC-coupled membrane-associated protein V (BioLegend; San Diego, CA, USA) for 10 min. After rinsed with PBS, 7-AAD (BioLgend) was added. Early apoptotic cells (annexin V-positive) and late apoptotic cells (annexin V-positive and 7-AAD positive) were quantified by flow cytometry (ACEA Biosciences).

Purification and characterization of sEVs

The sEVs from serum or cells were purified with ultracentrifuge (models Optima x − 100, Beckman Coulter Life Sciences) as described preciously [17]. The sEVs was characterized by transmission electron microscopy (TEM) (Model H-7650; Hitachi; Japan) and Nanoparticle Tracking Analyser (NanoSight LM10, Malvern Instruments; Malvern, UK). Purification and characterization of sEVs were performed by using antibodies of sEVs markers, including antibody CD63 (#A19023, ABclonal, Wuhan, China), TSG101 (#A1692), Alix (#A2215), and negative marker Calnexin (#A15631).

sEVs uptake

Purified sEVs were labeled with ExoTracker probes as described previously [18]. In brief, sEVs were incubated with ExoTracker for 30 min at 37 °C, and unbound ExoTracker were removed using 10-kD centrifugal ultrafiltration filters. Cells were incubated with labeled sEVs for 2 h at 37 °C and analyzed by flow cytometry [17].

Immunoprecipitation (IP) assay

Cells were cultured and lysed as described above. The lysate (1 mg) was incubated with 2 µg of the desired primary antibody at 4 °C for 1 h and added with 20 µl protein A/G Plus-Agarose. The mixture was incubated overnight at 4 °C on a shaker, rinsed with PBS, denatured with loading buffer and analyzed by western blotting.

Proteomic analysis

Protein samples (100 µg) were denatured with 8 M urea, reduced with 5 mM dithiothreitol (DTT) for 1 h at room temperature, then alkylated with 20 mM iodoacetamide (IAM) for 30 min in the dark, diluted with deionized water to a concentration of urea less than 2 M, and digested with lysine endopeptidase (Wako Puro Chemical; Osaka, Japan) at 37 °C for 4 h and digested with trypsin (Promega) at 37 °C overnight. The mixture was acidified to pH < 3 with 10% trifluoroacetic acid (TFA) and purified using a C18 cartridge (Waters Corp.; Taunton, MA, USA). Two-dimensional liquid chromatography/mass spectrometry (LC-MS) analysis and data analysis were performed by LTQ Orbitrap MS (Thermo Fisher Scientific; San Jose, CA, USA), and quantification was performed by the MaxQuant software program V. 1.5.2.8 (www.maxquant.org).

Glycoproteomics analysis

Glycopeptides were prepared according to the protocol described previously [17]. Overall, cells were treated using 8 M urea, 10 mM DTT, and 20 mM IAM (Sigma-Aldrich) with trypsin (Promega; Madison, WI, USA), heated at 37 °C for 4 h. The samples were resuspended with 200 µL of 50% ACN and 1% TFA using an Oasis HLB cartridge (Waters; Milford, MA, USA). The samples were then added to 4 mL of 95% ACN containing 1% TFA, loaded and purified using an Oasis MAX cartridge (Waters) to obtain glycopeptides. The eluate was lyophilized, dissolved in binding buffer (50 mM NH4HCO3, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, pH 7.4), and incubated with 50 µl PHA-E agarose (Vector Labs) overnight at 4 °C. The mixture was rinsed with 1 × PBS, boiled for 10 min to release the peptides, and the glycopeptides with bisecting GlcNAc structures were collected by centrifugation and purified using an Oasis HLB cartridge. Liquid chromatography-tandem mass spectrometry (LC-MS /MS) was performed using LTQ Orbitrap mass spectrometry (Thermo Fisher Scientific; San Jose, CA, USA) with the Byonic software program (Protein Metrics; San Carlos, CA, USA), Glyco-Decipher [19] and GlycoWorkbench 3.0.

β-galactosidase staining

For β-Galactosidase staining, MSCs were prepared as described previously [20]. In brief, MSCs were plated into 6-well dishes and cultured in complete medium until the cells reached 50% confluence. After being fixed by β-galactosidase fixative for 15 min and rinsed with PBS three times (3 min per time), the cells were stained with freshly prepared β-galactosidase staining working solution as indicated by the kit protocol and incubated overnight at 37 °C. Quantification was carried out by counting the positive stained cells in 10 randomly selected microscopic fields, and then the percentage of β-galactosidase positive cells in each group was obtained.

Enzyme-linked immunosorbent assay (ELISA)

96-well ELISA plates were coated with patient sera and sEVs samples, diluted 1:50 in PBS, incubated at 37 °C with shaking for 2 h, after blocking with 3% BSA at room temperature for 2 h, the plates were washed with TPBS. Primary antibodies against GPNMB (cat # ab188222, Abcam; Cambridge, MA, USA) and biotinylated PHA-E were added separately and incubated at 37 °C with shaking for 2 h, and then washed. Corresponding secondary antibodies and VECTASTAIN ABC (Vector Labs) reagent were added to each well and incubated at room temperature for 30 min, and then washed. TMB substrate kit (Beyotime) was added, followed by an acid stop solution, and the optical density at 450 nm was determined by plate reader.

Data analysis

GraphPad Prism V.8.0 (GraphPad Software; La Jolla, CA, USA) was used for statistical analysis. All experiments were repeated three times. Comparison of data between the two groups was performed using Student’s t-test and presented as mean ± SEM. p < 0.05 was considered a statistically significant difference.

Results

sEVs from sera of the elderly exacerbate the ageing of h-MSCs in vitro

Recent studies have found that treating young cells with sEVs secreted by senescent human fetal lung diploid fibroblasts can induce senescence-like changes in young cells [21]. We hypothesized that serum from aged donors may exacerbate the senescence-related characteristics of young h-MSCs through sEVs. To test our hypothesis, we pooled sEVs isolated from serum of elderly donors (> 60 years old) and young donors (< 45 years old) (termed SEN-sEVs and YOUNG-sEVs, respectively) (Fig. 1A). The serum-sEVs from peripheral blood of young donors demonstrated clear expression of sEVs markers such as CD63, TSG101, and Alix (Fig. 1B), exhibited a narrow size distribution around 70 nm (Fig. 1C), and displayed a sphere-like morphology (Fig. 1D). These sEVs can be efficiently taken up by young h-MSCs (Fig. 1E). SEN-sEVs treated young h-MSCs showed enhanced expression of senescence markers p16, p21, p53, and proinflammatory senescence-associated secretory phenotype (SASP), including MMP9 and interleukin-6 (IL-6) (Fig. 1F-H), presented the increased percentage of senescence-associated SA-β-gal-positive population, and exhibited enlarged and flattened morphologies (Fig. 1I and J). Consistent with previous study on anti-apoptosis in senescent cells [22], the level of apoptosis in young h-MSCs treated with SEN-sEVs was reduced compared to YOUNG-sEVs treated h-MSCs (Fig. 1K).

Fig. 1
figure 1

sEVs from sera of the elderly exacerbate the ageing of h-MSCs in vitro (A) Schematic images of young h-MSCs treated with serum sEVs. (B) Western blotting analysis of serum-sEVs marker. (C) Serum-sEVs morphology evaluated by TEM. (D) serum-sEVs analyzed by nanoparticle tracking analysis (NTA). (E) Flow cytometry of uptake of ExoTracker-labeled serum-sEVs in young h-MSCs. (F) Flow cytometry of senescence markers in young h-MSCs treated with YOUNG-sEVs or SEN-sEVs. (G) qRT-PCR of senescence markers in young h-MSCs treated with YOUNG-sEVs or SEN-sEVs. (H) Western blotting analysis and quantification of senescence markers. (I, J) SA-β-gal-positive cell percentage in young h-MSCs treated with YOUNG-sEVs or SEN-sEVs (shown as SEN-sEVs1, SEN-sEVs2, SEN-sEVs3). (K) Apoptosis analysis of young h-MSCs treated with YOUNG-sEVs or SEN-sEVs

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GPNMB exerted a pro-senescence effect on BMMSCs

In D-galactose (D-Gal) induced ageing murine model [23], the characterizations of ageing mice is consistent with previously described (Fig. S1A-F), the mice presented more β-galactosidase-positive population (Fig. 2A, S2A), and up-regulated expression of SASP, including MMP9, IL-6, and IL-1β (Fig. S2B&C). m-MSCs from D-Gal induced ageing mice (termed as D-Gal group) showed the reduced osteogenic differentiation capacity (Fig. S2D&E).

Fig. 2
figure 2

GPNMB exerted a pro-senescence effect on BMMSCs (A) MS analysis of m-MSCs from young and D-Gal groups (schematic). (B) Heatmap of differentially expressed proteins (DEPs) in m-MSCs derived from young and D-Gal groups (fold change > 1.5 or < 0.67; p < 0.05). Red: upregulation. Blue: downregulation. (C) Volcano plot of DEPs. Log10 p-value is plotted against log2 value. (D) PCA analysis. (E) KEGG pathway analysis. (F) LC-MS analysis of GPNMB expression. (G) Western blotting analysis and quantification of GPNMB expression in m-MSCs from young and D-Gal groups. (H) Confocal microscopic analysis of LEPR (red) and GPNMB (green) expression in BM of young group, D-Gal group and ageing group as above. (I) GPNMB expression in MSC-T and MSC-T/OEG cells. (J) qRT-PCR analysis. (K) Western blotting analysis of senescence markers in MSC-T and MSC-T/OEG cells. (L) GPNMB expression in MSC-T, MSC-T/SHG1 and MSC-T/SHG2 cells. (M) qRT-PCR analysis. (N) Western blotting analysis of senescence markers in MSC-T and MSC-T/SHG2 cells. (O) Representative image of SA-β-gal staining and quantification of the percentage of SA-β-gal-positive cells in MSC-T and MSC-T/OEG cells. (P) Representative image of SA-β-gal staining and quantification of the percentage of SA-β-gal-positive cells in MSC-T and MSC-T/SHG2 cells. (Q) Apoptosis of MSC-T, MSC-T/vec, MSC-T/OEG and MSC-T/SHG cells. MSC-T/vec cells were used as the empty vector control group

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Using proteomics analysis, we investigated the differentially expressed proteins of m-MSCs between young and D-Gal groups (Fig. 2A), and identified total 1489 proteins in m-MSCs from young and D-Gal groups (Fig. 2B). Among 371 differentially expressed proteins, a total of 231 were up-regulated and 140 down-regulated (Fig. 2C). The Principal Component Analysis (PCA) analysis showed a significant difference in protein enrichment between young and D-Gal groups (Fig. 2D). And these up-regulated proteins are closely associated with the process of ageing-related neurodegenerative diseases (Fig. 2E).

Consistent with previous study [24], we found that GPNMB expression of m-MSCs in D-Gal group was significantly increased (Fig. 2F), which were confirmed by western blotting (Fig. 2G) and confocal microscopic analysis (Fig. 2H). To investigate the biological function of GPNMB in senescence, we constructed MSC-T with GPNMB overexpression and silencing, designated as MSC-T/OEG and MSC-T/SHG, respectively (Fig. 2I&L). Compared to control group, senescence markers were significantly elevated in MSC-T/OEG cells (Fig. 2J), but reduced in MSC-T/SHG cells (Fig. 2M). GPNMB overexpression in MSC-T increased the expression of senescence markers MMP9, p16 and IL-6 (Fig. 2K), while silencing GPNMB showed the opposite effect (Fig. 2N). GPNMB overexpression also decreased the percentage of SA-β-gal-positive cells and apoptotic cells, whereas silencing GPNMB raised both (Fig. 2O-Q). These results demonstrate that increased GPNMB expression accelerates cellular senescence in BMMSCs.

Effect of bisecting GlcNAc in BMMSCs on cellular senescence

Studies have found that the N-glycosylation changes regularly with ageing and is associated with ageing-related diseases [25, 26]. We found total 141 differentially expressed glycopeptides in m-MSCs from young and D-Gal groups (Fig. S3A). The glycopeptides constituted 26.95% of all structural isoforms, significantly surpassing the proportions of heterozygous (9.21%) and high-mannose (6.38%) N-glycosylations in m-MSCs of young and D-Gal group (Fig. S3B). It was found that bisecting GlcNAc expression, recognized by lectin PHA-E, was significantly higher than branching N-glycan, recognized by lectin PHA-L, in D-Gal induced m-MSCs and natural aging m-MSCs (Fig. 3A). Confocal microscopy confirmed that the bisecting GlcNAc level in D-Gal m-MSCs was significantly higher than in young m-MSCs (Fig. 3B and C). Bisecting GlcNAc is catalyzed by the N-acetylglucosamine aminotransferase (MGAT3) and play a regulatory role in the processing and elongation of N-glycans on proteins [27]. We overexpressed (MSC-T/OEM3) or silenced MGAT3 (MSC-T/SHM3) in MSC-T, respectively (Fig. 3D&E). Upon overexpression of MGAT3 in MSC-T, the senescence factors of MSC-T/OEM3 were up-regulated compared to young MSC-T (Fig. 3F and H). In contrast, silencing MGAT3 in MSC-T showed the opposite results (Fig. 3G and I). We also observed increased percentages of SA-β-gal-positive cells in MSC-T/OEM3, but decreased SA-β-gal-positive cells in MSC-T/SHM3-1 (Fig. 3J&K). Flow cytometry results revealed a substantial reduction in the number of MSC-T/OEM3 apoptotic cells (Fig. 3L). These observations imply that the bisecting GlcNAc modification is involved in ageing process.

Fig. 3
figure 3

Effect of bisecting GlcNAc in mBMMSCs on cellular senescence (A) Lecting blot analysis of glycans in m-MSCs of young group, D-Gal group and ageing group. (B) Confocal microscopic images of bisecting GlcNAc levels in m-MSCs. (C) Confocal microscopic images of branching GlcNAc levels in m-MSCs. (D) MGAT3 expression in MSC-T and MSC-T/OEM3 cells. (E) MGAT3 expression in MSC-T and MSC-T/SHM3-1 and MSC-T/SHM3-2 cells. (F) qRT-PCR results of senescence markers in MSC-T and MSC-T/OEM3 cells. (G) qRT-PCR results of senescence markers in MSC-T and MSC-T/SHM3-1 cells. (H, I) Western blotting analysis of senescence markers. (J, K) SA-β-gal-positive cells in MSC-T, MSC-T/OEM3 and MSC-T/SHM3-1 cells. (L) Apoptosis analysis of MSC-T, MSC-T/vec, MSC-T/OEM3 cells

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Identification and function of bisecting GlcNAc modification sites on GPNMB

Our hypothesis is that GPNMB has bisecting GlcNAc modification. Notably, IP assay showed the elevation of bisecting GlcNAc levels on GPNMB in D-Gal induced m-MSCs, indicating GPNMB can be modified by bisecting GlcNAc (Fig. 4A). Through intact glycoproteomic analysis, utilizing PHA-E enrichment coupled with LC-MS/MS (Fig. 4B), we identified 212 differentially expressed glycoproteins in m-MSCs from young and ageing mice, as depicted in the volcano plot (Fig. 4C), with criteria set at a fold change > 1.5 or < 0.67 and P < 0.05. Among these, 35 were up-regulated and 177 were down-regulated, with GPNMB being among the up-regulated glycoproteins.

Fig. 4
figure 4

The bisecting GlcNAc modification on GPNMB. (A) Expression of GPNMB with bisecting GlcNAc in young and D-Gal m-MSCs by IP assay. (B) Intact glycoproteomic analysis by combination of PHA-E enrichment and LC-MS/MS analysis (schematic). (C) Volcano of differentially expressed glycopeptides in young and D-Gal induced m-MSCs. (D) Representative MS/MS spectrum of peptide NDRN#LSDEIFLR of GPNMB with bisecting GlcNAc in m-MSCs. (E, F) Wild-type and mutant GPNMB at Asn 249 were overexpressed in MSC-T, and bisecting GlcNAc on GPNMB in MSC-T/OEG and MSC-T/OEG-MU cells was assayed by IP and western blotting. (G, H) MSC-T/OEM3 and MSC-T/OEG-MU cells were treated with Chlo or MG132 for indicated times, and GPNMB expression was evaluated by western blotting. (I) qRT-PCR results of expression of senescence markers in MSC-T and MSC-T/OEG-MU cells. (J) Western blotting analysis and quantification of senescence markers in MSC-T and MSC-T/OEG-MU cells. (K) Apoptosis of MSC-T, MSC-T/OEG and MSC-T/OEG-MU cells

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Using Glyco-Decipher analysis [19], we identified three N-glycosylation sites on the senescent cell target protein GPNMB (Fig. S3C). Furthermore, a distinctive peptide containing Asn249, NDRN#LSDEIFLR, was characterized as pep + HexNAc3Hex1 (Fig. 4D). We generated wild-type GPNMB-expressing cells (MSC-T/OEG) alongside bisecting GlcNAc-deficient MSCs carrying the N249Q mutation (MSC-T/OEG-MU1, MU2) (Fig. 4E). IP assays indicated that GPNMB expression was significantly decreased, and the protein was rarely modified by bisecting GlcNAc in MSC-T/OEG-MU (Fig. 4F), indicating that Asn 249 is the key site for bisecting GlcNAc modification.

In most cases, extracellular proteins and cell surface proteins enter the cell through endocytosis and are degraded through the lysosomal pathway [28]. GPNMB expression in MSC-T/OEG-MU was enhanced by treatment with lysosomal inhibitor chloroquine, but unaffected by treatment with proteasome inhibitor MG132 (Fig. 4G and H). These findings indicate that bisecting GlcNAc modifi cation affects GPNMB stability, and causes GPNMB degradation via a lysosomal pathway. Upon comparing the senescence profiles, we discovered that MSC-T/OEG-MU showed significantly reduced changes in senescence markers (Fig. 4I&J). Furthermore, apoptosis ability was restored in MSC-T/OEG-MU (Fig. 4K). These findings suggested that bisecting GlcNAc influences GPNMB stability.

Bisecting GlcNAc modified GPNMB enriched in senescent sEVs

We isolated sEVs from the supernatants of young and D-Gal induced m-MSCs (termed young-sEVs and D-Gal-sEVs) (Fig. 5A and B). A quantitative proteomics of sEVs from young and D-Gal m-MSCs showed a total of 1778 differentially expressed proteins in the two groups of sEVs. We found that GPNMB was included in the up-regulated proteins of these proteins (Fig. 5C). GPNMB expression was significantly up-regulated in sEVs from D-Gal m-MSCs (Fig. 5D&E). Western blotting revealed that GPNMB and bisecting GlcNAc levels were both elevated in D-Gal-sEVs compared to young-sEVs (Fig. 5F). IP assay revealed that GPNMB presented higher levels of bisecting GlcNAc in D-Gal-sEVs (Fig. 5G). Furthermore, the clinical samples confirmed the higher expression of GPNMB and biecting GlcNAc in serum and serum-sEVs of older adults (Fig. 5H and K).

Fig. 5
figure 5

Bisecting GlcNAc modified GPNMB enriched in senescent sEVs. (A) sEVs morphology evaluated by TEM. (B) Particle size of sEVs analyzed by NTA. (C) Venn diagram showing the intersection of differential proteins of sEVs between young and D-Gal m-MSCs. (D) Heatmap of 30 differential proteins co-enriched by the two sets of sEVs. Red: upregulation. Blue: downregulation. (E) LC-MS analysis of GPNMB expression sEVs in young and D-Gal m-MSCs. (F) GPNMB and bisecting GlcNAc levels in sEVs of young and D-Gal m-MSCs by western blotting. (G) Expression and bisecting GlcNAcylation of GPNMB from Young-sEVs and D-Gal-sEVs evaluated by immunoprecipitation (IP) assay and western blotting. (H, I) ELISA for the determination of GPNMB in young and senescent serum and serum-sEVs of clinical samples. (J, K) ELISA for the determination of bisecting GlcNAc in young and senescent serum and serum-sEVs of clinical samples

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sEVs carrying bisecting GlcNAcylated GPNMB promote senescence in young m-MSCs

Our data suggested GPNMB expression was increased in young m-MSCs after treated with sen-sEVs, indicating GPNMB can be transferred by sEVs (Fig. S3F&G). To investigate the impact of sEVs containing bisecting GlcNAcylated GPNMB on cellular senescence, young m-MSCs were treated with sEVs from young MSC-T, MSC-T/OEG, and MSC-T/OEG-MU, termed young sEVs, MSC-T/OEG-sEVs, and MSC-T/OEG-MU-sEVs, respectively (Fig. 6A). Bisecting GlcNAc levels were higher in sEVs from MSC-T/OEG than from young MSC-T (Fig. 6B). Efficient internalization of sEVs by recipient cells was confirmed (Fig. 6C). MSC-T/OEG-sEVs treatment induced a senescence phenotype in young m-MSCs, marked by increased SA-β-gal-positive cells and a morphological shift towards senescent cells (Fig. 6D&E). Flow cytometry analysis revealed that MSC-T/OEG-sEVs significantly increased the expression of senescence markers MMP9, p16, p21, as well as IL-6 in recipient m-MSCs (Fig. 6F). In contrast, young m-MSCs treated with MSC-T/OEG-MU-sEVs maintained minimal changes in senescence-related traits (Fig. 6G). MSC-T/OEG-sEVs treatment reduced m-MSCs apoptosis, whereas no significant change was observed with MSC-T/OEG-MU-sEVs treatment (Fig. 6H). These findings collectively suggest that sEVs from MSC-T/OEG can induce cellular senescence in young m-MSCs and confirm that bisecting GlcNAc modification stabilizes vesicular GPNMB.

Fig. 6
figure 6

sEVs carrying bisecting GlcNAc-modified GPNMB promote senescence in young mMSCs (A) Treatment of young m-MSCs using different sEVs (schematic). (B) Bisecting GlcNAc levels of young-sEVs and MSC-T/OEG-sEVs. (C) FACS analysis of exotracker-labelled sEVs taken up by recipient cells. (D, E) SA-β-gal-positive cell percentage in MSC-T/OEG-sEVs and MSC-T/OEG-MU-sEVs treated young m-MSCs. (F, G) FACS analysis of senescence markers and quantification in young m-MSCs treated with MSC-T/OEG-sEVs and MSC-T/OEG-MU-sEVs. (H) Apoptosis of young m-MSCs treated with MSC-T/OEG-sEVs and MSC-T/OEG-MU-sEVs

Full size image

Discussion

The concept of restoring or ameliorating ageing has become a research hotspot. sEVs can mediate intercellular communication and allow exchange of their cargoes between source cells and target cells. Recent study suggests that sEVs play a role in regulating systemic senescence and the onset of age-related diseases [29]. It has been found that sEVs produced by MSCs isolated from neonatal umbilical cords (UCMSC) are rich in anti-ageing signals and can rejuvenate senescent adult bone marrow-derived MSCs. sEVs from UCMSC also promote regenerative capacity in terms of bone formation, wound healing and angiogenesis [30]. In aged mice for intravenous injection of sEVs of UCMSC also exerted anti-ageing effects, including reduction of skeletal and renal degeneration [8]. Liu et al. found that iPSC-sEVs had anti-ageing properties which can slow down the senescence properties of MSCs by translocating peroxiredoxin antioxidant enzymes [31].

Fig. 7
figure 7

MSC derived sEVs promotes senescence depend on bisecting GlcNAcylated GPNMB

Full size image

GPNMB is a specific senescent cell surface protein, and knockout of GPNMB in obese mice significantly reduces the proportion of senescent cells and slows down metabolic disorders [9]. It was found to be upregulated in a mouse model of senescent AD [9], and it has been described as a new AD-related marker expressed in a subpopulation of activated microglia [32]. In addition, GPNMB is thought to play a role in neuroinflammation [33], and a recent immunohistochemical study confirmed its localization in microglia in the brains of patients with AD disease [11]. It was found that GPNMB can be packaged into sEVs derived from hepatocellular carcinoma cells, and GPNMB on the sEVs inhibits the activation of CD8 + T cells by binding to the surface receptor syndecan-4 (SDC4) of CD8 + T cells, leading to cancer immune evasion [12]. The post-translational modification of GPNMB is crucial to its function. It has been found that GPNMB can be phosphorylated, and this modification significantly reduces stemness, tumor growth, and cell migration in breast cancer [34]. Han et al. found that the loss of Asn 134-glycosylation on GPNMB can significantly inhibit the binding of GPNMB to mutant epidermal growth factor, block its downstream signaling, and ultimately inhibit cancer metastasis in non-small cell lung cancer [35]. We identified GPNMB, the transmembrane glycoprotein, as a bisecting GlcNAcylated target protein in MSC and its derived sEVs. We found GPNMB expression was strongly enhanced when the level of bisecting GlcNAc was high, reflecting the fact that bisecting GlcNAc modification is crucial to its expression. Our data showed bisecting GlcNAc can regulate the translocation of GPNMB and loss of bisecting GlcNAc induced its degradation via the lysosomal pathway. Glycopeptide mass spectrometry analysis identified Asn 249 on GPNMB as a key bisecting GlcNAc site. Mutation of N-glycosylation at Asn 249 on GPNMB rarely boosted cellular senescence in BMMSCs, suggesting site Asn 249 determined the localization of GPNMB to the cell membrane.

In conclusion, senescent MSCs promote the ageing of young MSCs through sEVs secretion, including an increase in SASP, upregulation of senescence factors, an increase in the number of SA-β-positive cells, and a decrease in the number of apoptotic cells (Fig. 7). Our study shows that GPNMB with bisecting GlcNAc modification plays a critical role in sEVs-mediated ageing of MSCs. These findings will facilitate targeted treatments of glycosylated GPNMB, such as the development of drugs or biologics targeting GPNMB to modulate the ageing process of MSCs.

Data availability

The data supporting the conclusions of this article have been given in this article and its additional files. The mass spec Data have been deposited in Jianguoyun. (https://www.jianguoyun.com/p/DXOu3_cQzc2CDRin7egFIAA). All data contained within this article are available from the corresponding authors upon reasonable request.

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Funding

This study was supported by the National Science Foundation of China (No. 92478123, 32071274, 82370147, 82172828,3 2471333), Shaanxi Innovation Team Project (2023-CX-TD-58)and Shaanxi Fundamental Science Research Project for Chemistry & Biology (22JHQ077). The First Affiliated Hospital of Northwest University (Xi’an NO.1 hospital) Research Funding (2025001). Authors declare that they have not use AI-generated work in this manuscript.

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Authors and Affiliations

  1. Key Laboratory of Resource Biology and Biotechnology in Western China, Provincial Key Laboratory of Biotechnology, College of Life Sciences, Ministry of Education, Northwest University, Xi’an, China

    Yihan Ma, Chongfu Zhao, Jingjing Feng, Junjie Gou, Feng Guan & Xiang Li

  2. Xi’an Gaoxin No.1 High School International Division, Xi’an, Shaanxi, China

    Enci Kang

  3. The First Affiliated Hospital of Northwest University, Xi’an No.1 Hospital, Xi’an, China

    Qiong Wu

  4. Provincial Key Laboratory of Biotechnology of Shaanxi, Key Laboratory of Resource Biology and Modern Biotechnology in Western China, Faculty of Life Science, Northwest University, Xi’an, China

    Qiong Wu

  5. Institute of Hematology, Provincial Key Laboratory of Biotechnology, School of Medicine, Northwest University, Xi’an, China

    Xiang Li

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Contributions

XL, FG and QW conceived the study. YM, JF, CZ, JG, EK performed the experiments and data analysis. YM, FG and XL supervised research and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Qiong Wu or Xiang Li.

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The animal study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of the Northwest University Research Ethics Committee. The project titled “MSC-sEVs exacerbate senescence by transferring bisecting GlcNAcylated GPNMB” was approved under resolution number NWU-AWC-20240104 M, on January 15, 2024.

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Informed written consents were obtained from all participating subjects and all procedures were performed as per the guidelines and regulations approved by the ethics committee.

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Ma, Y., Zhao, C., Feng, J. et al. MSC-sEVs exacerbate senescence by transferring bisecting GlcNAcylated GPNMB. Stem Cell Res Ther 16, 23 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04140-9

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Stem Cell Research & Therapy

ISSN: 1757-6512