Abstract

Background

   Mesenchymal stem cells (MSCs) have potential for treating degenerative
   and immune diseases, but their clinical efficacy is limited by
   senescence, characterized by mitochondrial dysfunction, impaired
   mitophagy, and metabolic imbalance. The goal of this study was to
   investigate the effects of dimethyloxalylglycine (DMOG), a
   hypoxia-mimetic agent that stabilizes hypoxia-inducible factor 1 alpha
   (HIF-1α), on rejuvenating senescent MSCs by enhancing mitochondrial
   function, mitophagy, and metabolic reprogramming.

Methods

   Two models of MSC senescence were established: oxidative stress-induced
   senescence using hydrogen peroxide and replicative senescence through
   serial passaging. Umbilical cord derived MSCs were treated with DMOG
   for 48 h under normoxic conditions. Mitochondrial function, mitophagy,
   and metabolism were assessed using assays that measured mitochondrial
   membrane potential, reactive oxygen species levels, ATP production, and
   mitophagy. Western blotting and real-time PCR were employed to analyze
   the expression changes of relevant molecules. RNA sequencing (RNA-seq)
   was performed to identify key genes and pathways regulated by DMOG.
   Additionally, to evaluate the therapeutic potential of rejuvenated
   MSCs, a co-culture system was established, where DMOG-treated senescent
   MSCs were co-cultured with IL-1β-treated chondrocytes.

Results

   DMOG treatment significantly reduced key senescence markers, including
   senescence-associated beta-galactosidase, p53, and p21, in both
   senescence models. DMOG treatment restored mitochondrial morphology and
   function, improving mitochondrial membrane potential, reducing
   mitochondrial reactive oxygen species, and enhancing ATP production.
   DMOG also promoted mitophagy, as evidenced by increased colocalization
   of mitochondria with lysosomes. RNA-seq analysis revealed that DMOG
   activated key pathways, including HIF-1 signaling, calcium signaling,
   and mitophagy-related gene (BNIP3 and BNIP3L). Notably, BNIP3 knockdown
   greatly abolished DMOG-induced mitophagy and its anti-senescence
   effects. Furthermore, DMOG treatment improved metabolic flexibility by
   enhancing both mitochondrial respiration and glycolysis in senescent
   MSCs. Moreover, DMOG-treated senescent MSCs partially restored their
   therapeutic efficacy in an osteoarthritis model by improving
   extracellular matrix regulation in IL-1β-stimulated chondrocytes.

Conclusions

   Short-term DMOG treatment rejuvenates senescent MSCs by enhancing
   mitochondrial function, promoting mitophagy via HIF-1α/BNIP3, and
   improving metabolic reprogramming. DMOG-treated MSCs also showed
   enhanced therapeutic efficacy in co-culture with IL-1β-treated
   chondrocytes, suggesting its potential to improve MSC-based therapies
   in regenerative medicine.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-025-04422-2.

   Keywords: Mesenchymal stem cells (MSCs), Senescence, Hypoxia-Mimetic
   agent, HIF-1α, BNIP3, Mitophagy

Background

   Over the past two decades, mesenchymal stem cells (MSCs) have emerged
   as a promising therapeutic tool for treating immune-related and
   degenerative diseases owing to their regenerative and immunomodulatory
   properties [[44]1, [45]2]. However, the clinical translation of
   MSC-based therapies is limited by their susceptibility to senescence,
   which reduces their proliferative potential, differentiation capacity,
   and therapeutic efficacy [[46]3, [47]4]. Senescence, induced by aging
   or prolonged in vitro expansion, is associated with the development of
   a senescence-associated secretory phenotype (SASP) that compromises the
   regenerative potential of MSCs and hinders their widespread clinical
   application [[48]3, [49]5]. Therefore, understanding and mitigating
   MSCs senescence is essential for maximizing their therapeutic
   potential.

   MSCs senescence is driven by multiple mechanisms, including telomere
   shortening, oxidative stress, and impaired autophagy, with
   mitochondrial dysfunction emerging as a critical hallmark [[50]4,
   [51]6]. Aging MSCs accumulate dysfunctional mitochondria characterized
   by reduced bioenergetic capacity, increased reactive oxygen species
   (ROS) production, and a shift in metabolic activity [[52]7, [53]8].
   These mitochondrial impairments exacerbate oxidative damage and
   contribute to progressive loss of cellular function. Notably,
   mitophagy, a selective form of autophagy that eliminates damaged
   mitochondria, plays a vital role in maintaining mitochondrial quality
   and preventing senescence [[54]7–[55]9]. Impaired mitophagy has been
   implicated in the aging process; however, strategies to enhance
   mitophagy in senescent MSCs remain underexplored.

   Low oxygen levels in the stem cell niche are essential for preserving
   MSC function by limiting oxidative stress and maintaining cellular
   homeostasis [[56]10, [57]11]. Hypoxic conditions activate
   hypoxia-inducible factor-1α (HIF-1α), a master regulator of cellular
   responses to low oxygen levels [[58]12]. HIF-1α drives the expression
   of genes involved in metabolic adaptation, ROS detoxification, and
   autophagy [[59]13]. Numerous studies have demonstrated that hypoxia
   delays the onset of senescence and promotes stem cell function;
   however, culturing cells in hypoxic environments is technically
   challenging and costly [[60]14–[61]19]. Hypoxia-mimetic agents, such as
   dimethyloxalylglycine (DMOG), offer a practical alternative by
   stabilizing HIF-1α under normoxic conditions [[62]20]. Although
   hypoxia-mimetic agents are known to mimic some effects of physical
   hypoxia, their specific role in preventing MSCs senescence and
   modulating mitochondrial function remains unclear.

   In this study, we developed two models of MSCs senescence, oxidative
   stress-induced senescence (via hydrogen peroxide) and replicative
   senescence (via multi-generation expansion), to investigate the effects
   of short-term (48-hour) treatment with DMOG, a hypoxia-mimetic that
   stabilizes HIF-1α. We explored how DMOG modulates mitochondrial
   function, mitophagy, and metabolism to counteract MSCs senescence,
   highlighting its potential to enhance the effectiveness of MSCs based
   therapies.

Materials and methods

Culture and treatment of MSCs

   Passage 2 (P2) MSCs derived from the umbilical cord were purchased from
   GLABIOLIUS GROUP (Shenyang, China) and cultured in Mesenchymal Stem
   Cell Medium (MSCM) supplemented with 5% fetal bovine serum (FBS)
   (ScienCell, USA). To avoid interference from cytokines, the medium was
   replaced with α-MEM (Invitrogen) for 48 h prior to the experiments.
   Flow cytometry was conducted to confirm the expression of MSC surface
   markers, including CD14, CD34, CD45, CD73, and CD90.

   To examine the anti-aging effects of the hypoxia-mimetic agent
   Dimethyloxallyl Glycine (DMOG), MSCs at passage 5 (P5) and passage 15
   (P15) were treated with 200 µM DMOG (MedChemExpress, China) for 48 h
   under standard culture conditions as previously described in our
   earlier studies [[63]21].

Establishment of MSCs senescence models

   Young MSC controls were defined as passage 5 (P5) cells with minimal
   aging characteristics. Oxidative stress-induced senescence was modeled
   by treating P5 MSCs with 200 µM hydrogen peroxide (H₂O₂) for 24 h.
   Replicative senescence was achieved by serial passaging. Passage 15
   (P15) MSCs were used as the aged group. Both models were used for
   further analysis.

Senescence-Associated β-Galactosidase (SA-β-Gal) staining

   SA-β-Gal staining was performed using an SA-β-Gal Staining Kit
   (Beyotime Biotechnology, China) following the manufacturer’s protocol.
   Cells were seeded into 6-well plates, fixed, and incubated with the
   staining solution overnight at 37 °C. Senescent cells were identified
   based on their blue-green coloration under a light microscope. All
   experiments were biologically replicated at least three times (n = 3).

Western blot analysis

   Total proteins were extracted from MSCs using RIPA lysis buffer
   (Boster, China), separated via SDS-PAGE, and transferred to PVDF
   membranes. The primary antibodies used were p53 (1:1000, BM4309,
   Boster, China), p21 (1:1000, BM3990, Boster, China), HIF-1α (1:1000,
   BM4083, Boster, China), MFN1 (1:1000, BM4882, Boster, China), MFN2
   (1:1000, BM4906, Boster, China), FIS1 (1:1000, A01932-3, Boster,
   China), BNIP3 (1:1000, BA4304-2, Boster, China), and GAPDH (1:5000,
   BM1632, Boster, China) as an internal control. After incubation with
   HRP-conjugated secondary antibodies, protein bands were visualized
   using an enhanced chemiluminescence system.

Real-Time PCR

   Total RNA was extracted using TOROzol Reagent (TOROIVD, China) and
   reverse-transcribed into cDNA using a TOROBlue^® qPCR RT Kit. Real-time
   PCR was performed using TOROGreen^® qPCR Master Mix on an Applied
   Biosystems™ QuantStudio™ 5 System (Thermo Fisher, USA). Gene expression
   was normalized to that of GAPDH and the results were calculated using
   the 2 − ΔΔCt method. Each experiment was conducted in triplicates.

Cell viability assay

   Cell viability was evaluated using the CCK-8 assay and
   Calcein/Propidium Iodide (PI) staining. For CCK-8, MSCs were seeded at
   2,000 cells/well in 96-well plates, treated for 48 h, and incubated
   with the CCK-8 reagent (Boster, China). The absorbance was measured at
   450 nm. For calcein/PI staining, cells were incubated with calcein-AM
   (2 µM) and PI (4 µM) (Beyotime, China) at 37 °C for 30 min Live (green)
   and dead (red) cells were visualized using fluorescence microscopy.

Apoptosis assay

   Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection
   Kit (Beyotime, China). After treatment, the cells were collected,
   stained with Annexin V-FITC and PI, and analyzed on a BD FACSCelesta™
   Flow Cytometer. The percentage of apoptotic cells was quantified using
   the FACSDiva software.

Mitochondrial functional assays

   (1) Membrane Potential: Mitochondrial membrane potential was measured
   using the JC-1 Assay Kit (MCE, USA). MSCs were incubated with JC-1
   solution for 20 min, and fluorescence images were captured using a
   laser confocal microscope.

   (2) ROS Measurement: Mitochondrial ROS levels were assessed using
   MitoSOX Red (Thermo Fisher Scientific, USA). Cells were incubated with
   5 µM MitoSOX for 10 min, washed, counterstained with Hoechst 33,342,
   and analyzed using the CellInsight CX7 Imaging System.

   (3) ATP Production: ATP levels were measured using an ATP Assay Kit
   (Beyotime, China). Luminescence intensity was normalized to the protein
   content and determined using the BCA Assay.

Mitochondrial respiration analysis

   Mitochondrial respiration was analyzed using a Seahorse XFe96 Analyzer
   (Agilent, USA). MSCs were seeded in XF96 assay plates, and sequential
   additions of oligomycin, FCCP, antimycin A, and rotenone was performed
   to assess mitochondrial respiration. Protein normalization was
   performed after the assay.

Mitophagy assay

   Mitophagy was evaluated by the colocalization of mitochondria and
   lysosomes using MitoTracker Green and LysoTracker Red (Beyotime,
   China). MSCs were stained sequentially, counterstained with Hoechst,
   and imaged using the CellInsight CX7 System.

SiRNA transfection

   To knock down BNIP3 expression, MSCs were transfected with siBNIP3 or
   siControl using the jetPRIME^® Transfection Reagent (Polyplus, France)
   in Opti-MEM (Gibco, USA) following the manufacturer’s protocol. After
   6 h, the medium was replaced, and the cells were collected 24 h later
   for analysis. Knockdown efficiency was confirmed using quantitative
   PCR. The siRNA sequences for BNIP3 (siBNIP3) were as follows: sense
   strand, 5’-GGAACACGAGCGUCAUGAA(dT)(dT)-3’ and antisense strand,
   5’-UUCAUGACGCUCGUGUUCC(dT)(dT)-3’.

Chondrocyte isolation and Co-Culture

   Articular cartilage samples were obtained with ethical approval (PLA
   General Hospital No. 2024KY0132-KS001). The cartilage was minced,
   digested with trypsin (15 min) and 0.2% collagenase II (Gibco, USA)
   overnight, and centrifuged to isolate the chondrocytes. Chondrocytes
   were seeded on sterilized coverslips in 24-well plates. For
   osteoarthritis (OA) modeling, 1 nM IL-1β (Genscript, USA) was added for
   24 h and, then washed with PBS. MSCs were seeded directly into the
   upper compartments of Transwell inserts (Merck, USA) for 48 h of
   co-culture.

Immunofluorescence staining

   Chondrocytes were fixed, permeabilized with 0.1% Triton X-100, blocked
   with 1% BSA, and incubated with primary antibodies against Col2a1,
   MMP13, and Aggrecan (1:200, Boster, China) overnight at 4 °C.
   FITC-conjugated secondary antibodies were applied, and the nuclei were
   stained with DAPI. The fluorescence images were captured using a
   NanoZoomer Scanner under standardized exposure conditions. For
   quantitative analysis, fluorescence intensity was measured using ImageJ
   software (version 1.53, NIH, USA) with the following parameters: (1)
   three independent biological replicates were analyzed per condition;
   (2) five representative fields were captured per replicate; and (3) a
   minimum of 50 cells were quantified per sample to ensure statistical
   robustness. All measurements were normalized to background fluorescence
   and DAPI-stained nuclei counts.

Mitochondrial morphology analysis

   Mitochondrial morphology was analyzed using the Operetta CLS
   High-Content Imaging System (PerkinElmer, USA) with a
   60×water-immersion objective. MitoTracker Red fluorescence was detected
   at 490 nm excitation and 516 nm emission, with z-stack imaging
   performed to ensure optimal focus. For each well, five representative
   regions (top, bottom, left, right, and center) were captured, and
   approximately 100–120 cells per group were analyzed across 15 regions.
   Morphological quantification was performed using Harmony 4.9 software
   (PerkinElmer, USA), assessing key parameters including mitochondrial
   area, perimeter, aspect ratio, and form factor. A fluorescence
   intensity threshold was applied to exclude background noise, and
   automated algorithms quantified fragmentation and elongation. This
   standardized, high-throughput approach ensured objective and
   reproducible analysis of mitochondria.

Transmission Electron Microscopy (TEM) assay

   The adherent UC-MSC in the petri dishes from different experimental
   groups were carefully scraped using a cell scraper and collected by
   centrifugation at 1000 g for 5 min. The cell pellets were fixed
   overnight at 4 °C in 2.5% glutaraldehyde solution (Cat No. P1126,
   Solarbio, China). After fixation, the samples were post-fixed with 1%
   osmium tetroxide in phosphate buffer at room temperature for 1 h to
   enhance structural contrast. The fixed cell samples were then embedded
   in epoxy resin and polymerized at 60 °C for 48 h. Thin
   Sects. (50–100 nm) of the resin blocks were cut and stained with 2%
   uranyl acetate for 10 min, followed by staining with lead citrate for
   5 min to further enhance contrast. Mitochondrial ultrastructure was
   examined using a transmission electron microscope (JEOL JEM-1400, JEOL
   Ltd., Japan) operated at 80 kV. The images were analyzed for
   mitochondrial morphology and structural integrity using ImageJ software
   (NIH, USA).

RNA-seq analysis

   UC-MSCs from the young generation (P5), replicative senescence model
   (P24), and DMOG treatment group were cultured in 10 cm dishes for 48 h,
   and at least 1 × 10^6 cells each group were harvested for RNA
   extraction using TRIzol reagent. Each group has three biological
   replicates. Total RNA was purified via phenol-chloroform extraction,
   with sample purity assessed using a NanoPhotometer^® spectrophotometer
   (IMPLEN, USA). RNA integrity and concentration were determined using an
   Agilent 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, USA).
   Sequencing libraries were prepared using the VAHTS Universal V6 RNA-seq
   Library Prep Kit for Illumina^® (NR604-01/02), incorporating unique
   index tags for multiplexing. High-throughput sequencing was performed
   on an Illumina NovaSeq 6000 platform (Illumina, USA). For
   bioinformatics analysis, clean reads were aligned to the reference
   genome using HISAT2 (v2.1.0) with Bowtie2 (v1.0.1)-generated indexes.
   Differentially expressed genes were identified based on|log2(fold
   change)| and adjusted P-value thresholds. RNA-seq and computational
   analysis were conducted by easyresearch (Beijing, China).

Statistical analysis

   Statistical analyses were performed using GraphPad Prism version 8.0.
   Student’s t-test was used for comparisons between two groups, whereas
   one-way analysis of variance (ANOVA) or two-way ANOVA was applied for
   multiple-group comparisons. Data are presented as mean ± SD, and
   statistical significance was set at p < 0.05.

Results

Short-term DMOG treatment alleviates oxidative stress- and
replication-induced MSCs senescence by activating HIF-1α and reduce apoptosis

   A replicative senescence model was established by serially passaging
   the human umbilical cord derived MSCs. No significant differences in
   the surface markers CD73 and CD90 were observed between P5 and P15 MSCs
   (Fig. [64]S1). In addition, the effect of different concentrations of
   hydrogen peroxide (H₂O₂) on cell viability was evaluated using the
   CCK-8 assay (Fig. [65]S2). Based on these results, we used 200 µM H₂O₂
   to induce oxidative stress-mediated premature senescence in MSCs.
   SA-β-gal staining, a hallmark of cellular senescence, revealed a
   significant increase in the proportion of SA-β-gal-positive cells in
   H₂O₂-treated MSCs, which was markedly reduced after short-term DMOG
   treatment (Fig. [66]1A, B). Quantitative PCR analysis demonstrated that
   DMOG significantly reduced the p53 and p21 mRNA levels (Fig. [67]S3).
   Western blot analysis also demonstrated that H₂O₂ treatment
   significantly upregulated p53 and p21 protein levels, while DMOG
   treatment effectively decreased expression of P53 (Fig. [68]1C).
   Similarly, in the replicative senescence model, DMOG treatment
   significantly decreased the proportion of SA-β-gal-positive cells, with
   a more pronounced reduction observed in the P15 cells (Fig. [69]1D, E).
   Western blot analysis revealed that DMOG treatment effectively
   suppressed p53 and p21 expression, particularly in P15 cells
   (Fig. [70]1F). Furthermore, we assessed the expression levels of
   senescence-associated genes, including IL6, CXCL1, and MMP3, in both
   senescence models. These genes were markedly upregulated in senescent
   MSCs, whereas DMOG treatment significantly downregulated their
   expression (Fig. [71]1G). Because DMOG is a hypoxia mimetic that
   stabilizes HIF-1α, our Western blot analysis showed that short-term
   DMOG treatment significantly increased HIF-1α protein levels in both P5
   and P15 cells, with a more pronounced effect under H₂O₂-induced
   oxidative stress conditions. Protein quantification and RNA analysis
   further confirmed that DMOG increased HIF-1α protein and mRNA levels
   (Fig. [72]1H). These results suggest that short-term DMOG treatment
   alleviates oxidative stress- and replication-induced senescence and
   activates HIF-1α in MSCs.

Fig. 1.

   [73]Fig. 1
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   Short-term DMOG treatment reduces MSC senescence by activating HIF-1α
   and decreasing apoptosis. (A, B) Representative images of SA-β-gal
   staining showing the proportion of senescent cells in H₂O₂-treated MSCs
   before and after DMOG treatment. Scale bar = 500 μm. (C) Western blot
   analysis of p53 and p21 protein expression during oxidative
   stress-induced senescence. (D, E) SA-β-gal staining in replicative
   senescence (P15) MSCs, demonstrating the effect of DMOG in reducing
   senescence markers. Scale bar = 500 μm. (F) Western blot analysis of
   p53 and p21 protein levels in P5 (young) and P15 (senescent) MSCs. (G)
   Expression levels of senescence-associated genes (IL6, CXCL1, and MMP3)
   in both senescence models as assessed by qRT-PCR. (H) Western blotting
   and qPCR analysis showing increased HIF-1α protein and mRNA levels in
   both senescence models after DMOG treatment. (I) Calcein/PI live-dead
   staining revealed an increase in the proportion of live cells following
   DMOG treatment in both senescence models. (J, K) Flow cytometric
   analysis of apoptosis. Error bars represent the mean ± SD of three
   independent experiments. Statistical significance was set as p < 0.05.
   Full-length blots are presented in Supplementary Materials - WB Raw
   Data

   We further investigated the effects of DMOG on the apoptosis of
   senescent MSCs. Calcein/PI live-dead staining revealed an increase in
   the proportion of dead cells in both senescence models, while
   short-term DMOG treatment markedly increased the proportion of live
   cells (Fig. [75]1I and Fig. [76]S4). Flow cytometry analysis indicated
   that apoptosis levels were significantly elevated in both senescence
   models compared to the controls, whereas DMOG treatment significantly
   reduced the proportion of apoptotic cells (Fig. [77]1J, K). These
   findings demonstrate that short-term DMOG treatment effectively
   counteracted apoptosis in senescent MSCs.

Short-term DMOG treatment significantly improved the mitochondrial morphology
and reduced damage in aged MSCs

   To elucidate the role of mitochondria in DMOG-mediated rejuvenation of
   aged MSCs, we investigated the impact of DMOG treatment on the
   regulation of mitochondrial morphology and function in these cells. In
   this study, mitochondria were labeled with MitoTracker Green and
   analyzed using fluorescence microscopy and transmission electron
   microscopy (TEM) to assess the effects of various treatments on
   mitochondrial structure and function. Fluorescence microscopy revealed
   that mitochondria in both models of senescent MSCs exhibited a
   reduction in number, an increase in size, and morphological changes
   characterized by shortened length and increased diameter (Fig. [78]2A).
   Following DMOG treatment, mitochondrial length, diameter, and
   circularity were significantly improved in senescent MSCs, with a
   notable increase in mitochondrial density and reduction in the area of
   individual mitochondria (Fig. [79]2A-C). TEM analysis of the
   mitochondrial ultrastructure showed that senescent MSCs had larger
   mitochondrial volumes, fewer mitochondria, and reduced inner membrane
   surface area. DMOG treatment effectively reversed these changes,
   leading to a decrease in mitochondrial damage and the restoration of
   mitochondrial structural integrity (Fig. [80]2D, E). Our results
   demonstrated that aging induced alterations in the mitochondrial
   morphology of MSCs, whereas treatment with DMOG effectively reversed
   mitochondrial damage and morphological changes in aged MSCs.

Fig. 2.

   [81]Fig. 2
   [82]Open in a new tab

   Short-term DMOG treatment restores mitochondrial morphology and reduces
   damage in aged MSCs. (A) Representative fluorescence microscopy images
   of mitochondria labeled with Mitotracker Green in senescent MSCs
   (induced by oxidative stress or replicative aging) after DMOG
   treatment. The second row shows the mitochondrial morphology after
   software processing. Scale bar = 20 μm. The locally enlarged portion of
   this is a typical region that reflects the mitochondrial morphology of
   the group. (B, C) Quantitative analysis of mitochondrial morphological
   parameters (length, diameter, circularity, and density) s. (D)
   Transmission electron microscopy (TEM) images of the mitochondrial
   ultrastructure in aged MSCs. (Red box: mitochondrial autophagosomes;
   yellow arrow: dividing mitochondria; blue arrow: damaged and swollen
   mitochondria). (E) Quantitative TEM analysis showing reduced
   mitochondrial damage and restored structural integrity after DMOG
   treatment. Error bars represent the mean ± SD from three independent
   experiments. Statistical significance was set at p < 0.05

Short-term DMOG treatment enhances mitochondrial function and mitophagy in
aged MSCs

   Previous studies have shown that HIF-1α promotes mitochondrial fission
   and regulate functions such as mitophagy [[83]22]. Here, we examined
   the effects of DMOG on mitochondrial function in aged MSCs, including
   mitochondrial membrane potential, ROS levels, mitophagy, ATP
   production, and the expression of fusion and fission-related genes.
   JC-1 fluorescence images revealed that, compared to the P5 group, both
   the H₂O₂-treated group and P15-aged MSCs exhibited significantly
   reduced mitochondrial membrane potential, whereas DMOG treatment
   significantly restored mitochondrial membrane potential in both MSCs
   senescence models (Fig. [84]3A). MitoSox Red staining demonstrated
   increased mitochondrial ROS generation in H₂O₂-treated and P15-aged
   MSCs, which was markedly reduced by DMOG treatment (Fig. [85]3B).
   Quantitative analysis further confirmed that DMOG treatment restored
   the mitochondrial membrane potential and reduced mitochondrial ROS
   production in aged MSCs (Fig. [86]3C, D). Western blot analysis
   revealed the expression levels of the mitochondrial fusion-related
   proteins MFN1 and MFN2, along with the mitochondrial fission-related
   protein Fis1. Notably, DMOG treatment led to significant upregulation
   of Fis1 expression, with the effect being particularly pronounced in
   replicative senescent P15 cells (Fig. [87]3E). Co-staining with
   LysTtracker Red and MitoTracker Green revealed a significant reduction
   in the number of mitochondrial autophagosomes in aged MSCs, whereas
   DMOG treatment notably enhanced the co-localization of mitochondria
   with lysosomes and increased the level of mitophagy in aged MSCs
   (Fig. [88]3F, G). Since the primary function of the mitochondria is ATP
   production, we observed a marked reduction in ATP levels in aging
   cells. However, DMOG treatment significantly restored ATP production in
   both MSC senescence models (Fig. [89]3H). Collectively, these findings
   demonstrate that DMOG restores mitochondrial function in aged MSCs by
   improving the membrane potential, reducing ROS levels, enhancing
   mitophagy, and boosting ATP production.

Fig. 3.

   [90]Fig. 3
   [91]Open in a new tab

   DMOG treatment ameliorates mitochondrial dysfunction and enhances
   mitophagy and ATP production in aged MSCs. (A) Representative JC-1
   fluorescence images showing mitochondrial membrane potential in
   different groups: CCCP (positive control for mitochondrial
   depolarization), P5 MSCs, P5 + H₂O₂-treated MSCs,
   P5 + H₂O₂+DMOG-treated MSCs, P15 MSCs, and P15 + DMOG-treated MSCs.
   JC-1 red fluorescence indicates high mitochondrial membrane potential,
   whereas green fluorescence indicates depolarized mitochondria. Scale
   bar = 100 μm. (B) Representative MitoSox Red fluorescence images
   showing mitochondrial ROS levels in different treatment groups. Scale
   bar = 10 μm. (C, D) Quantitative analysis of the mitochondrial membrane
   potential (C) and mitochondrial ROS levels (D). (E) Western blot
   analysis showing the expression levels of mitophagy-related proteins
   (MFN1, MFN2, and Fis1) in P5 and P15 MSCs with or without H₂O₂ and DMOG
   treatment. GAPDH was used as a loading control. (F) Representative
   confocal images of co-staining with LysoTracker Red (lysosomes) and
   MitoTracker Green (mitochondria) in MSCs under different conditions.
   Scale bar = 10 μm. (G) Quantitative analysis of the number of
   mitophagosomes per cell in the different groups. (H) Quantitative
   analysis of ATP production per cell in the different groups. Data are
   expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01.
   ***p < 0.001. Full-length blots are presented in Supplementary
   Materials - WB Raw Data

Short-term DMOG treatment partially restores mitochondrial respiration and
glycolytic function in senescent MSCs

   We further investigated the impact of DMOG on the metabolic
   reprogramming of senescent MSCs. Mitochondrial respiration and
   glycolytic function in MSCs under various treatment conditions were
   assessed using oxygen consumption rate (OCR) and extracellular
   acidification rate (ECAR). The OCR results demonstrated that MSCs in
   the P5 group exhibited significantly higher baseline mitochondrial
   respiration, ATP production, and maximal respiratory capacity than
   those in the P15 group (Fig. [92]4A, B). Similarly, MSCs in the
   H₂O₂-induced premature senescence group showed markedly lower baseline
   mitochondrial respiration, ATP production, and maximal respiratory
   capacity than those in the P5 group (Fig. [93]4A, B). Notably,
   short-term DMOG treatment significantly enhanced baseline mitochondrial
   respiration and ATP production in the H₂O₂-induced premature senescence
   group, while it had no significant impact on the P15 replicative
   senescence group (Fig. [94]4A, B). In terms of glycolytic function, the
   P5 group showed substantially higher glycolysis, glycolytic capacity,
   and glycolytic reserve than both the P15 group and the H₂O₂-induced
   premature senescence group (Fig. [95]4C, D). This indicates that aged
   MSCs exhibit diminished glycolytic function, including significant
   reductions in basal glycolysis, glycolytic capacity, and glycolytic
   reserves. After DMOG treatment, a notable improvement in glycolysis and
   glycolytic capacity was observed in the H₂O₂-induced premature
   senescence group. The P15 replicative senescence group also experienced
   significant increases in glycolysis and glycolytic capacity, although
   these were still markedly lower than those in P5 cells under all
   treatment conditions (Fig. [96]4C, D). Our results indicated that MSCs
   in the P5 group exhibited superior metabolic activity, whereas aged
   MSCs (P15 and H₂O₂-treated P5 group) displayed significant impairments
   in both mitochondrial oxidative phosphorylation and glycolytic
   functions. Notably, short-term DMOG treatment partially restored the
   OCR and ECAR in senescent MSCs.

Fig. 4.

   [97]Fig. 4
   [98]Open in a new tab

   DMOG treatment improves mitochondrial respiration and glycolytic
   function in senescent MSCs. (A) Oxygen consumption rate (OCR) profiles
   representing mitochondrial respiration in MSCs under various
   conditions. (B) Quantitative analysis of mitochondrial respiration
   parameters, including baseline respiration, ATP production, and maximal
   respiratory capacity of MSCs. (C) Extracellular acidification rate
   (ECAR) profiles representing the glycolytic function in MSCs under
   various conditions. (D) Quantitative analysis of glycolytic parameters,
   including glycolysis, glycolytic capacity, and glycolytic reserves.
   Data are expressed as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01,
   ***p < 0.001

Transcriptomic analysis reveals key genes and signaling pathways involved in
the rejuvenation of aged MSCs by short-term DMOG treatment

   To gain deeper insights into the molecular mechanisms underlying the
   rejuvenation of aged MSCs by short-term DMOG treatment, we cultured P5
   MSCs through successive passages until P24, followed by DMOG treatment.
   RNA sequencing and subsequent bioinformatic analyses were conducted to
   investigate the transcriptional changes induced by DMOG. Volcano plots
   (Fig. [99]5A, C) show the gene expression profiles of P5 and P24 MSCs
   following DMOG treatment. Notably, the number of differentially
   expressed genes regulated by DMOG in P24-aged MSCs was significantly
   lower than that in P5 MSCs. Furthermore, key mitophagy-related genes,
   such as BNIP3 and BNIP3L, were consistently upregulated in P5 and P24
   MSCs after DMOG treatment. KEGG pathway analysis (Fig. [100]5B, D)
   revealed that in P5 young MSCs, DMOG primarily activated the calcium,
   HIF-1, MAPK glycolysis/gluconeogenesis, and mitophagy-related pathways.
   In contrast, in P24-aged MSCs, DMOG predominantly activated the HIF-1
   signaling, glycolysis/gluconeogenesis, fructose and mannose metabolism,
   and mitophagy-related pathways. Heatmaps and KEGG analysis of the four
   groups (Fig. [101]5E, F) further demonstrated that differentially
   expressed genes were enriched in the calcium, HIF-1, MAPK, and
   mitophagy-related pathways. The heatmap (Fig. [102]5G) highlights the
   expression levels of key mitophagy signaling genes, including HIF-1α,
   ATG9A, BNIP3, and BNIP3L, following DMOG treatment. A Venn diagram
   (Fig. [103]5H) highlights both shared and group-specific DEGs,
   revealing differences in gene expression between young and aged MSCs in
   response to DMOG treatment. GO analysis of the 79 genes co-regulated by
   DMOG in both P5 and P24 MSCs showed significant enrichment in processes
   such as cellular response to hypoxia, metabolic reprogramming, and
   mitophagy-related pathways (Fig. [104]5I-K). Pathway analysis confirmed
   their involvement in HIF-1 signaling, glycolysis/gluconeogenesis, and
   arginine and proline metabolism. GO analysis further emphasized terms,
   such as cellular response to decreased oxygen levels, peptidyl-proline
   hydroxylation, and metabolic processes. These findings suggest that
   DMOG rejuvenates aged MSCs by activating hypoxia- and mitophagy-related
   genes such as HIF-1α, BNIP3, and BNIP3L, while enhancing metabolic
   reprogramming and adaptive stress responses.

Fig. 5.

   [105]Fig. 5
   [106]Open in a new tab

   DMOG treatment activates hypoxia- and mitophagy-related pathways in
   aged MSCs. (A, C) Volcano plots of differentially expressed genes
   (DEGs) in P5 (A) and P24 (C) MSCs after DMOG treatment. Key
   mitophagy-related genes, BNIP3 and BNIP3L, were consistently
   upregulated in both groups. (B, D) KEGG pathway enrichment analysis in
   P5 (B) and P24 (D) MSCs showing that DMOG activated HIF-1 signaling,
   glycolysis/gluconeogenesis, and mitophagy-related pathways in both
   young and aged MSCs. (E) Heatmap of DEGs in the P5, P5 + DMOG, P24, and
   P24 + DMOG groups. Each group has three biological replicates. (F) KEGG
   pathway analysis of all groups highlights the key pathways activated by
   DMOG, including HIF-1 signaling and glycolysis. (G) Heatmap showing the
   expression levels of key mitophagy-related genes (e.g., BNIP3, BNIP3L,
   HIF-1 A) upregulated by DMOG treatment. (H) Venn diagram illustrating
   the shared and unique DEGs across comparisons. A total of 79 genes were
   co-regulated by DMOG in both P5 and P24 MSCs. (I-K) GO and pathway
   analyses of co-regulated genes revealed enrichment in hypoxia response,
   glycolysis, and other metabolic processes

BNIP3-mediated mitophagy is essential for the rejuvenation effects of DMOG on
senescent MSCs

   RNA-seq analysis revealed that DMOG significantly upregulated the
   expression of mitophagy-related genes, BNIP3 and BNIP3L, which is
   essential for the rejuvenating effects of DMOG on senescent MSCs.
   Western blotting results confirmed that the expression of BNIP3 and
   BNIP3L was markedly downregulated in both H₂O₂-induced premature
   senescence and replicative senescence models, whereas DMOG treatment
   effectively restored their expression in senescent MSCs (Fig. [107]6A,
   B). Previous studies have identified BNIP3 as a direct target of
   HIF-1α. Given that BNIP3 is a known direct target of HIF-1α, we
   investigated whether BNIP3-mediated mitophagy was critical for the
   rejuvenating effects of DMOG on senescent MSCs. To this end, BNIP3 was
   knocked down in a P15 replicative senescence model using BNIP3-specific
   siRNA. Western blot analysis demonstrated that DMOG significantly
   increased BNIP3 expression in senescent MSCs; however, this
   upregulation was notably suppressed upon BNIP3 knockdown (Fig. [108]6C,
   D). Consistently, real-time PCR results corroborated that BNIP3
   knockdown abolished DMOG-induced upregulation of BNIP3 (Fig. [109]6E).
   Functional assessments using SA-β-gal staining revealed that BNIP3
   knockdown increased the proportion of senescent cells in P15 BMSCs,
   whereas DMOG treatment significantly reduced the percentage of
   SA-β-gal-positive cells. The anti-senescence effect of DMOG was
   markedly attenuated by BNIP3 knockdown (Fig. [110]6F, G). Furthermore,
   colocalization analyses using LysoTracker Red and MitoTracker Green
   staining indicated that DMOG significantly enhanced the interaction
   between mitochondria and lysosomes, reflecting increased mitophagy, an
   effect that was substantially suppressed by BNIP3 knockdown
   (Fig. [111]6H, I). Collectively, these findings highlight the pivotal
   role of BNIP3-mediated mitophagy in the rejuvenating effects of DMOG on
   senescent MSCs, providing novel insights into the underlying
   mechanisms.

Fig. 6.

   [112]Fig. 6
   [113]Open in a new tab

   DMOG rejuvenates senescent MSCs through BNIP3-dependent mitophagy. (A,
   B) Western blot analysis (A) and quantification (B) showing the
   expression of mitophagy-related proteins BNIP3 and BNIP3L in P5, P15,
   and H₂O₂-treated P5 MSCs with or without DMOG treatment. (C, D) Western
   blot analysis (C) and quantification (D) of BNIP3 expression in P15
   MSCs after BNIP3 knockdown with siRNA. (E) Real-time PCR analysis of
   BNIP3 expression in P15 MSCs with or without DMOG treatment and BNIP3
   knockdown. (F, G) SA-β-gal staining (F) and quantification (G) showing
   the percentage of senescent cells in P15 MSCs. Scale bar = 500 μm. (H,
   I) Confocal images (H) and quantification (I) of colocalization between
   mitochondria (Mitotracker Green) and lysosomes (Lysotracker Red) in P15
   MSCs. Scale bar = 10 μm. Data are expressed as mean ± SEM (n = 3).
   **p < 0.01, ***p < 0.001. Full-length blots are presented in
   Supplementary Materials - WB Raw Data

DMOG restores the therapeutic potential of senescent MSCs to alleviate
metabolic dysfunction in OA chondrocytes

   To evaluate whether DMOG treatment could restore the therapeutic
   potential of senescent MSCs for osteoarthritis (OA), we established an
   in vitro co-culture system to investigate the effects of DMOG on MSCs
   in modulating the expression of cartilage extracellular matrix
   (ECM)-related genes and proteins under IL-1β stimulation
   (Fig. [114]7A). OA-like conditions were induced in isolated human
   articular chondrocytes through IL-1β treatment, and these cells were
   then co-cultured with MSCs from different experimental groups using a
   Transwell system. Immunofluorescence staining and quantitative analysis
   revealed a substantial reduction in the expression of key ECM
   molecules, including Collagen II (Col2a1) and aggrecan (AGC), in
   IL-1β-stimulated OA chondrocytes, along with significant upregulation
   of MMP13 expression (Fig. [115]7B, C). Young MSCs at passage 5 (P5)
   effectively ameliorated the anabolic and catabolic imbalance induced by
   IL-1β in OA chondrocytes. However, both H₂O₂-induced prematurely
   senescent MSCs and replicatively senescent MSCs failed to mitigate
   metabolic dysregulation in OA chondrocytes (Fig. [116]7B, C). Notably,
   short-term DMOG treatment partially restored the therapeutic efficacy
   of both H₂O₂-induced prematurely senescent and replicatively senescent
   MSCs. Although the level of improvement did not fully match that of P5
   young MSCs, the observed effects were statistically significant
   compared with the untreated senescent MSC groups (Fig. [117]7B, C).
   Quantitative real-time PCR (qRT-PCR) analysis further confirmed that
   DMOG-treated senescent MSCs significantly increased the mRNA expression
   of Col2a1 and AGC in OA chondrocytes, while concurrently suppressing
   MMP13 mRNA expression (Fig. [118]7D). In addition to restoring the
   paracrine regulatory function of senescent MSCs in OA chondrocytes,
   DMOG treatment also ameliorated their compromised multipotent
   differentiation capacity—effectively suppressing aberrant adipogenic
   differentiation while partially rescuing osteogenic and chondrogenic
   potential in hydrogen peroxide-induced aged MSCs (Fig. [119]S5). These
   findings suggest that DMOG may enhance the therapeutic potential of
   senescent MSCs, possibly by partially restoring their multipotent
   differentiation capacity and modulating their ability to regulate
   ECM-related gene and protein expression, which could contribute to
   mitigating the metabolic dysfunction in OA chondrocytes.

Fig. 7.

   [120]Fig. 7
   [121]Open in a new tab

   DMOG enhances the therapeutic potential of senescent MSCs to mitigate
   metabolic dysfunction in OA chondrocytes. (A) Schematic diagram of the
   co-culture system. Human articular chondrocytes were treated with IL-1β
   to induce OA-like conditions and co-cultured with MSCs from various
   experimental groups using a Transwell system. (B) Representative
   immunofluorescence staining of ECM-related proteins, including Collagen
   II (Col2a1), Aggrecan (AGC), and MMP13, in OA chondrocytes co-cultured
   with MSCs from different experimental groups. Scale bar = 50 μm. (C)
   Quantitative analysis of immunofluorescence intensity for Col2a1, AGC,
   and MMP13. (D) Quantitative real-time PCR (qRT-PCR) analysis of mRNA
   levels for Col2a1, AGC, and MMP13 in OA chondrocytes co-cultured with
   MSCs. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01,
   ***p < 0.001

Discussion

   This study demonstrates that short-term DMOG treatment effectively
   rejuvenates senescent MSCs by targeting mitochondrial dysfunction and
   enhancing mitophagy possibly through the HIF-1α/BNIP3 signaling
   pathway. Hypoxia plays a critical role in maintaining the stem cell
   niche by reducing oxidative stress and preserving the properties
   [[122]10, [123]23, [124]24]. Studies have shown that hypoxic
   preconditioning can delay MSC senescence and improve their
   proliferation and differentiation capacities [[125]11, [126]13,
   [127]14, [128]19, [129]25, [130]26]. However, the effects of hypoxia on
   MSC senescence remain inconsistent. While most studies have suggested
   that hypoxia has anti-aging benefits, some have reported potential
   adverse effects [[131]27, [132]28]. These inconsistencies in previous
   findings may be attributed to differences in experimental conditions,
   such as hypoxic environment settings, oxygen concentrations, and MSC
   sources. As a hypoxia-mimetic agent, DMOG provides a reproducible and
   practical alternative by stabilizing HIF-1α under normoxic conditions,
   thereby eliminating the need for complex hypoxic culture systems
   [[133]16, [134]29]. This study is for the first to demonstrate that
   short-term DMOG treatment reverses aging-associated phenotypes in
   umbilical cord-derived MSCs in both oxidative stress-induced and
   replicative senescence models. It significantly reduces aging markers
   such as SA-β-gal, p53, and p21, while decreasing apoptosis and
   enhancing cell viability. These findings underscore the translational
   potential of DMOG as a robust strategy for delaying MSC senescence and
   restoring their functionality.

   A key mechanism underlying DMOG-induced rejuvenation is its ability to
   restore mitochondrial function, which is a hallmark of cellular aging
   and a primary driver of MSC senescence. Senescent MSCs exhibit
   increased ROS production, loss of mitochondrial membrane potential, and
   impaired ATP generation [[135]8, [136]30]. This study demonstrated that
   DMOG can reverse structural and functional mitochondrial damage.
   Transmission electron microscopy and functional analyses showed that
   DMOG improved the mitochondrial membrane potential, reduced ROS levels,
   enhanced ATP production, increased mitochondrial respiratory
   efficiency, and promoted mitophagy. These findings highlight the
   importance of mitochondrial quality control in combating senescence,
   consistent with previous studies linking mitochondrial homeostasis to
   stem cell rejuvenation [[137]7, [138]8]. Moreover, the role of
   mitochondrial dysfunction in aging is further underscored by previous
   studies showing that Ndufs6 deficiency in the mitochondria accelerates
   stem cell aging through excessive ROS accumulation and activation of
   the p53/p21 pathway [[139]31]. Previous studies have suggested that
   senescent MSCs exhibit significant declines in both oxidative
   phosphorylation and glycolytic capacity [[140]32]. This study
   demonstrated that DMOG partially restores oxidative phosphorylation and
   glycolytic capacity, improves the metabolic flexibility of senescent
   MSCs and supports the energy requirements of rejuvenated cells.

   Another key mechanism by which DMOG induces cellular rejuvenation is
   the activation of the HIF-1α/BNIP3 pathway, thereby regulating
   mitophagy. Mitophagy, a selective process for the removal of damaged
   mitochondria to maintain mitochondrial quality, protects bone
   marrow-derived mesenchymal stem cells (MSCs) against oxidative damage
   [[141]7, [142]33]. Previous studies have demonstrated that DMOG
   regulates the HIF-1α signaling pathway and enhances the biological
   functions of MSCs [[143]29]. Hypoxia protects nucleus pulposus-derived
   stem cells (NPSCs) under compression by increasing
   macroautophagy/autophagy levels, whereas HIF-1α alleviates
   compression-induced apoptosis in NPSCs through autophagy upregulation
   [[144]34]. BNIP3, a hypoxia-responsive gene regulated by HIF-1α, is
   critical for maintaining metabolic homeostasis and mitochondrial
   function in nucleus pulposus cells of intervertebral discs [[145]35].
   In vivo animal experiments further confirmed that DMOG delays
   osteoarthritis (OA) progression in mice by stabilizing HIF-1α and
   enhancing mitophagy [[146]36]. This study demonstrated that DMOG
   significantly upregulates BNIP3 expression by stabilizing HIF-1α,
   thereby promoting mitophagy, improving mitochondrial quality, and
   reducing ROS accumulation. Transcriptomic and functional analyses
   further revealed that DMOG activates the HIF-1α/BNIP3 signaling
   pathway, which enhances mitophagy and improves the structural and
   functional integrity of the mitochondria. Notably, BNIP3 knockdown
   markedly attenuated the anti-aging effects and pro-mitophagy activities
   of DMOG, confirming the indispensable role of BNIP3 in the anti-aging
   mechanisms of DMOG. This finding aligns with previous studies, further
   establishing the critical regulatory role of the HIF-1α/BNIP3 axis in
   mitochondrial function and stress responses in senescent cells.
   Moreover, this highlights the potential of mitophagy as a therapeutic
   target for anti-aging interventions.

   The translational relevance of effects of DMOG on MSCs was further
   validated in an osteoarthritis (OA) co-culture model. Untreated
   senescent MSCs failed to restore the catabolic-anabolic balance in OA
   chondrocytes, whereas DMOG-treated MSCs showed significantly enhanced
   therapeutic efficacy. DMOG-treated MSCs promoted the expression of
   anabolic markers, including collagen II and aggrecan, while suppressing
   the catabolic marker MMP13, demonstrating their capacity to restore the
   therapeutic potential of senescent MSCs in degenerative disease
   contexts. Consistent with previous reports, hypoxia and hypoxia mimetic
   agents are promising priming approaches to enhance the potential of
   mesenchymal stem cells [[147]16]. Meanwhile, HIF-1α was stabilized and
   its nuclear localization enhanced by releasing DMOG from mesenchymal
   stem cell (MSC)-loaded alginate hydrogels, which was associated with
   both enhanced chondrogenesis and reduced expression of chondrocyte
   hypertrophy-related markers [[148]37]. However, the long-term effects
   of DMOG treatment and its potential for off-target effects require
   further investigation. Additionally, the observed differences in
   responses between replicative and oxidative stress-induced senescence
   emphasize the complexity of cellular aging and the need for tailored
   therapeutic strategies.

Conclusions

   This study demonstrated for the first time that DMOG rejuvenates the
   senescence-associated phenotype of MSCs via HIF-1α/BNIP3-mediated
   mitophagy. By enhancing mitochondrial function, promoting mitophagy,
   and reprogramming cellular metabolism, DMOG has emerged as a practical
   and effective hypoxia-mimetic strategy to delay MSC senescence and
   preserve their functional capacity. This mechanistic study lays a
   theoretical foundation for developing innovative stem cell rejuvenation
   strategies and improving the therapeutic efficacy of MSCs in
   regenerative medicine.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [149]Supplementary Material 1^ (3.2MB, tif)
   [150]Supplementary Material 2^ (1.1MB, tif)
   [151]Supplementary Material 3^ (1.1MB, tif)
   [152]Supplementary Material 4^ (6.8MB, tif)
   [153]Supplementary Material 5^ (8.8MB, tif)
   [154]Supplementary Material 6^ (13.5MB, tif)
   [155]Supplementary Material 7^ (4MB, xlsx)
   [156]Supplementary Material 8^ (4.7MB, xlsx)
   [157]Supplementary Material 9^ (1.6MB, docx)

Acknowledgements