Human dental pulp stem cell-derived exosomes inhibit adipogenesis and obesity by activating Wnt/β-catenin signaling

Human dental pulp stem cell-derived exosomes inhibit adipogenesis and obesity by activating Wnt/β-catenin signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Human dental pulp stem cell-derived exosomes inhibit adipogenesis and obesity by activating Wnt/β-catenin signaling Young Kim, Janghyun Kim, Hyekyoung You, Seong Eun Kim, Myeong-Kwan Jih, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8023093/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Obesity is a critical global health challenge linked to cardiovascular disease, type 2 diabetes, metabolic syndrome, and cancers. Current therapies often lack sustained efficacy, prompting interest in molecular strategies targeting adipogenesis. Exosomes, cell-derived extracellular vesicles, exhibit therapeutic potential due to high biocompatibility and ability to mediate intercellular communication. This study explored the anti-adipogenic effects of exosomes derived from human dental pulp stem cells (hDPSC-Exos). hDPSC-Exos were isolated from conditioned media and characterized by TEM, nanoparticle tracking analysis, and Western blotting. Their anti-adipogenic effects were evaluated in vitro using 3T3-L1 and OP9 preadipocytes via Oil Red O staining, RT-qPCR, western blotting and multi-omics analyses (RNA-seq, proteomics). Therapeutic efficacy was further validated in a diet-induced obesity mouse model. Treatment with hDPSC-Exos markedly suppressed adipocyte differentiation in vitro, reducing lipid accumulation and downregulating key adipogenic transcription factors and their downstream targets. Mechanistically, Wnt/β-catenin signaling activation mediated this inhibition. In vivo, hDPSC-Exos administration reduced body weight, fat mass, and adipogenic gene expression in obese mice. These findings reveal a novel regulatory function of hDPSC-Exos in adipogenesis, highlighting their potential as a cell-free therapy for obesity and related metabolic disorders. Biological sciences/Stem cells/Mesenchymal stem cells Health sciences/Medical research/Stem-cell research human dental pulp stem cells exosomes adipogenesis obesity beta-catenin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Over recent decades, obesity has emerged as a major global public health concern, largely attributed to sedentary lifestyles and unhealthy dietary habits. As a chronic condition, it is strongly linked to several metabolic disorders, including type 2 diabetes, cardiovascular diseases, and various malignancies, underscoring the urgency of effective prevention and management 1 . The increasing prevalence of obesity and its associated comorbidities necessitates the development of innovative therapeutic strategies to effectively mitigate its progression 2 . Recent advances in regenerative medicine have focused on natural substances derived from stem cells and regenerative tissues. Among these, human dental pulp stem cells (hDPSCs) offer distinct advantages over other mesenchymal stem cells (MSCs), including easy accessibility from discarded teeth, minimally invasive collection methods, high proliferative capacity, and strong multi-lineage differentiation potential. Moreover, their use is associated with fewer ethical concerns, enhancing their appeal for diverse medical applications 3 – 6 . Notably, hDPSC-derived exosomes (hDPSC-Exos)—small extracellular vesicles enriched with proteins, lipids, and nucleic acids—have shown significant promise in modulating cellular signaling and biological processes 7 , 8 . Although prior studies have demonstrated the therapeutic potential of hDPSC-Exos, their specific role in regulating adipogenesis remains largely unexplored. In this study, the inhibitory effects of hDPSC-Exos on adipogenesis were investigated, with a particular emphasis on their modulation of key adipogenic transcriptional networks. The objective was to elucidate the molecular mechanisms underlying exosome-mediated suppression of adipogenesis and evaluate their potential as a cell-free therapeutic strategy for obesity prevention. Methods Cell culture and adipogenic differentiation 3T3-L1 preadipocytes were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) (LM 001-05, Welgene, Korea) supplemented with 10% bovine calf serum (16170078, Gibco, New Zealand) and 1% penicillin–streptomycin (LS202-02, Welgene, Korea). OP9 mouse stromal cells were maintained in Alpha Modified Eagle's Minimum Essential Medium (12571-071, Gibco) supplemented with 20% fetal bovine serum (FBS) (16000-044, Gibco) and 1% penicillin–streptomycin at 37°C in a humidified incubator with 5% carbon dioxide (CO 2 ). For adipogenic differentiation, cells were seeded in 12-well plates and grown to confluence. Differentiation was induced using a standard adipogenic induction medium comprising DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, I5879, Sigma-Aldrich), 1 µM dexamethasone (D4902, Sigma-Aldrich), and 10 µg/mL insulin (I6634, Sigma-Aldrich, St. Louis, MO, USA). After 2 days, the induction medium was replaced with maintenance medium (DMEM containing 10% FBS and 10 µg/mL insulin), which was refreshed every 2 days until the formation of mature adipocytes with visible lipid droplets. Oil red O staining and dye extraction To evaluate lipid accumulation, differentiated 3T3-L1 and OP9 cells were washed thrice with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 30 min at room temperature. After fixation, cells were washed thrice with PBS (5 min per wash). Oil red O working solution was prepared by diluting the stock solution with distilled water at a 3:2 ratio and filtering it through filter paper. Cells were stained with the working solution for 1 h at room temperature, followed by three PBS washes (5 min per wash). Stained cells were visualized using the Lionheart FX Cell Imager (BioTek, Winooski, VT, USA). Oil red O dye was eluted using 100% isopropanol with gentle shaking for 10 min to quantify lipid content. Optical density was measured at 490 nm using a SYNERGY H1 microplate reader (BioTek, Winooski, VT, USA) with 100% isopropanol as the blank. Isolation, culture, and characterization of hDPSCs Human dental pulp tissues were obtained from supernumerary teeth extracted from seven healthy pediatric donors. hDPSCs were isolated as previously described 9 . Briefly, the extracted teeth were washed with sterile PBS to remove debris. The pulp tissue was then carefully dissected, minced into small fragments, and enzymatically digested with 3 mg/mL collagenase type I (C0130, Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min. The resulting suspension was filtered through a 70 µm cell strainer to obtain a single-cell suspension, washed with PBS, and cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 until reaching approximately 80% confluence. To evaluate osteogenic differentiation potential, hDPSCs were cultured for 7 and 19 days in an osteogenic medium supplemented with 10 mM β-glycerophosphate (G9422, Sigma-Aldrich) and 50 mM L-ascorbic acid (A4544, Sigma-Aldrich, St. Louis, MO, USA). Osteogenic differentiation was assessed by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining using premixed 5-bromo-4-chloro-3-indolyl phosphate ® /nitro blue tetrazolium solution (B6404-100, Sigma-Aldrich, St. Louis, MO, USA) and 2% Alizarin red S solution (0223, ScienCell, San Diego, USA). Stained cells were visualized using a Lionheart FX Cell Imager (BioTek, Winooski, VT, USA). Flow cytometry hDPSCs were harvested and washed thrice using PBS, followed by centrifugation at 300 × g for 10 min at room temperature after each wash. A total of 1 × 10 5 cells per tube were incubated for 20 min at 4°C in the dark with the following antibodies: CD73-FITC, CD105-PE, CD90-FITC, CD45-PE, and CD34-APC (eBioscience, San Diego, CA, USA). After incubation, cells were washed once with PBS to remove unbound antibodies. Flow cytometry was performed using BD AccuriT™ C6 Plus (BD Biosciences, CA, USA). Data analysis and visualization were performed using the flowCore and ggridges packages in R. Exosome extraction To isolate exosomes from hDPSCs, conditioned medium was collected after 48 h of culture. The medium was initially centrifuged at 300 × g for 10 min to remove cellular debris. The supernatant was then processed through the Minimate™ EVO Tangential Flow Filtration system (Cytiva, Wilmington, DE, USA) using a 100 kDa molecular weight cut-off filter. The flow rate was optimized to retain exosomes while allowing smaller proteins and soluble molecules to pass through. The resulting concentrate was further purified by overnight precipitation at 4°C using ExoQuick (EXOTC50A-1, System Biosciences, Palo Alto, CA, USA). The purified exosome pellet was resuspended in PBS and stored at -80°C for subsequent analyses. Nanoparticle tracking analysis The size distribution and concentration of hDPSC-Exos were analyzed using the ZetaView system (Particle Metrix, Germany). Samples were diluted in 0.22 μm-filtered Dulbecco's PBS and analyzed under the following parameters: maximum area, 1000; minimum area, 10; minimum brightness, 30. Measurements were conducted using a 488 nm laser at 40 mW power. Data were processed using ZetaView software (v8.06.01). Transmission electron microscopy (TEM) analysis of exosomes The morphological characteristics of isolated hDPSC-Exos were evaluated using TEM. Exosome samples were fixed in a solution containing 2% glutaraldehyde (v/v) and 2% paraformaldehyde (v/v) in 0.05 M cacodylate buffer (pH 7.2) at room temperature for 4 h. Following fixation, the samples were washed with the same buffer and carefully placed onto a carbon-coated copper grid. TEM imaging was performed using a JEM-ARM200F TEM (JEOL Ltd., Akishima, Japan) operated at an accelerating voltage of 200 kV and a magnification of 100 000×. Exosome labeling using 5-(and-6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) For CFSE staining (V12883, ThermoFisher, USA), a 200 µM working solution was prepared in PBS by diluting a 10 mM dye solution in dimethyl sulfoxide. For exosome labeling, 15 μL of hDPSC-Exos suspended in PBS was mixed with 15 μL of 40 μM CFSE solution and incubated for 10 min at 4°C, unless otherwise indicated. Following staining the exosomes were diluted to 2 mL with DPBS, transferred to Microsep Advance 100k Omega filters (MCP100C41, Pall Corporation, Washington, NY, USA), and centrifuged at 4000 × g for 3 min at 4°C. The exosomes were washed thrice with 2 mL D-PBS and concentrated to a final volume of 50 μL. For cellular uptake studies, CFSE-labeled exosomes were applied to 3T3-L1 cells. After incubation, cells were washed twice with D-PBS, fixed with 4% paraformaldehyde for 15 min, and washed thrice with PBS. Nuclei were counterstained with 2 mg/mL Hoechst 33342 for 5 min. Cells were imaged using a confocal laser scanning microscope (LSM900, Zeiss, Jena, Germany), and the data were analyzed using Zen 3.2 software. Preparation of samples for RNA sequencing For transcriptome analysis, 3T3-L1 preadipocytes were plated and cultured until confluence. Cells were then treated with hDPSC-Exos at a concentration of 20 µg/mL in differentiation media to induce adipogenic differentiation. After 6 days of treatment, total RNA was extracted using RNAiso Plus reagent (9109, Takara, Japan) according to the manufacturer’s protocol. RNA quality was assessed using the Agilent TapeStation4000 system (Agilent Technologies, Amstelveen, Netherlands). Total RNA libraries were constructed using the RiboCop rRNA Depletion Kit (Lexogen Inc., Vienna, Austria) in combination with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on the NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) at e-biogen (Seoul, Republic of Korea). RNA sequencing data processing, differential expression analysis, and data visualization RNA sequencing data were processed following previously established protocols 10 . Briefly, raw FASTQ files were subjected to quality control using Trimmomatic (v0.39) 11 . Cleaned reads were then aligned and quantified against the GENCODE v37 human reference genome 12 using Salmon with default settings (v1.4.0) 13 . Gene-level counts were obtained using the tximport package in R (v1.6.3) 14 , and differential gene expression analysis was conducted using DESeq2 (v1.30.1) 15 . Differentially expressed mRNAs were visualized using heatmaps (ComplexHeatmap R package v2.6.2) 16 . Functional and pathway enrichment analysis of differentially expressed genes (DEGs) Gene annotation for DEGs was performed using the clusterProfiler package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (version 6.8; https://david.ncifcrf.gov/home.jsp), applying a threshold of p 2.0. Over-representation analysis (ORA) was conducted using the clusterProfiler package to identify significantly enriched pathways 17 . Proteomic analysis For filter-aided sample preparation digestion of hDPSC-Exos samples, 500 mM Tris (2-carboxyethyl) phosphine was added to a final concentration of 5 mM and incubated at 37°C with shaking at 300 rpm for 30 min. The mixture was transferred to a filtration unit and centrifuged at 14 000 × g for 15 min at 20°C. Protein alkylation was performed by adding 500 mM iodoacetamide to a final concentration of 50 mM, followed by incubation in the dark at 25°C for 1 h, followed by centrifugation under the same conditions. For peptide digestion, trypsin was dissolved in 50 mM ammonium bicarbonate and added at a trypsin-to-protein ratio of 1:50. Samples were incubated at 37°C with shaking at 300 rpm for 12 h. The resulting peptides were desalted using C18 Micro Spin Columns with buffer A (0.1% trifluoroacetic acid) and eluted using a linear gradient of buffer B (80% acetonitrile with 0.1% trifluoroacetic acid) at a flow rate of 300 nL/min. Peptides were then loaded onto a reverse-phase trap column (Acclaim PepMap100 C18, 3 μm, 75 μm × 2 cm, Thermo Scientific) and separated on a C18-reversed phase analytical column (PepMap TM , 75 μm × 50 cm, Thermo Scientific) using buffer A (0.1% formic acid) and a linear gradient of buffer B (80% acetonitrile with 0.1% formic acid) at the same flow rate. Mass spectrometry analysis was conducted using Proteome Discoverer TM (Thermo Scientific) against the UniProt complete human proteome database (version 2018-01-15; 161 584 sequences). RNA isolation and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was extracted using RNAiso Plus reagent (9108, Takara) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using the Maxime RT PreMix Kit (25082, Intronbio). RT-qPCR was performed using TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (RR420A, Takara) on a real-time PCR system to assess mRNA expression of adipogenic marker genes. Relative gene expression levels were calculated using the 2^(- ΔΔ CT) method, with β-actin as the internal control. All primer sequences (Table 1) were designed using Primer3 software. Table 1. All primer sequences used in this study. Gene Primer sequence Pparγg Forward 5'-ATTTGAAAGAAGCGGTGAACC-3' Reverse 5'-GCTGATTCCGAAGTTGGTGG-3' C/ebpαa Forward 5'-TGGACAAGAACAGCAACGAG-3' Reverse 5'-TCACTGGTCAACTCCAGCAC-3' Srebp-1c Forward 5'-TGGACACAGCGGTTTTGAAC-3' Reverse 5'-CGGGAAGTCACTGTCTTGGT-3' Adipoq Forward 5'-CCTGGAGAGAAGGGAGAGAA-3' Reverse 5'-CAGCTCCTGTCATTCCAACA-3' aP2 Forward 5'-AACCTGGAAGCTTGTCTCCA-3' Reverse 5'-ATGATCATGTTGGGCTTGGC-3' CD36 Forward 5'-TGTGTTTGGAGGCATTCTCA-3' Reverse 5'-GGTGCCTGTTTTAACCCAGTT-3' Plin2 Forward 5'-GGCAGTCTTTCCTCCATCCT-3' Reverse 5'-GGCAGTCTTTCCTCCATCCT-3' β-catenin Forward 5'-CTGAAGGTGCTGTCTGTCTG-3' Reverse 5'-TCTGTCAGATGAAGCCCCAG-3' β-actin Forward 5'-TGGCACCACACCTTCTACAA-3' Reverse 5'-TACATGGCTGGGGTGTTGAA-3' Western blot analysis The cells were lysed using RIPA buffer supplemented with cOmplete™ EDTA-free Protease Inhibitor Cocktail (11836153001, Sigma). The whole-cell lysates were cleared by centrifugation at 13 000 × g for 15 min at 4°C. Protein concentrations were determined, and equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by electrophoretic transfer onto nitrocellulose membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies (PPARg, 2435S, Cell signaling, 1:1000; C/EBPa, 8178S, Cell signaling, 1:1000; Adiponectin, 2789S, Cell signaling, 1:1000; Perilipin-1, 9349S, Cell signaling, 1:1000; Non-phospho (Active) β-Catenin (Ser33/37/Thr41), 8814S, Cell signaling, 1:1000; β-Catenin, 9562S, Cell signaling, 1:1000, Anti-β-Actin, K200058M, Solarbio, 1:3000). After washing, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse). Immunoreactive bands were visualized using the LAS-3000 mini imaging system (Fujifilm, Tokyo, Japan) Animal experiments Six-week-old male C57BL/6J mice were housed under a 12-h light/dark cycle with ad libitum access to tap water. Mice were randomly assigned to three groups: standard diet (SD) group, high-fat diet (HFD) group, and HFD supplemented with hDPSC-Exos (HFD+Exos) group. The HFD and HFD+Exos groups were fed an HFD (60% kcal from fat) for 12 weeks. During this period, the HFD+Exos group received intraperitoneal injections of hDPSC-Exos (50 μg/mouse) twice weekly, while the HFD group received equivalent volumes of PBS as a control. Body weight was recorded weekly throughout the study. At the experimental endpoint, mice were anesthetized via intraperitoneal injection of a mixture of alfaxan (45 mg/kg) and xylazine (8 mg/kg), followed by cervical dislocation. Subsequently, white adipose tissues were collected, weighed, and subjected to histological analysis using QuPath 18 . Tissue assessments were conducted by two investigators blinded to group allocation. The work has been reported in line with the ARRIVE guidelines 2.0. Body composition analysis Body composition was assessed using dual-energy X-ray absorptiometry with the InAlyzer system (Medikors Inc., Seongnam, Korea). For the analysis, mice were anesthetized with an intraperitoneal injection of of a mixture of alfaxan (45 mg/kg) and xylazine (8 mg/kg) and placed in a prone position on the scanning platform with limbs extended laterally. Total fat and lean mass were quantified using InAlyzer software following the manufacturer's protocols. Statistical analysis All experiments were performed in triplicate, and data are presented as mean ± standard deviation (SD). All expression statistical analysis was performed using ANOVA with Tukey’s post-hoc analysis in SPSS 21.0 software. Weekly mouse weights data was analyzed by one-way repeated measures ANOVA followed by Tukey's post hoc test for multiple comparisons. A p -value of <0.05 was considered statistically significant. Results hDPSC-Exos inhibits adipogenesis via suppressing Pparγ-C/ebpα expression in 3T3-L1 cells To investigate the effects of hDPSC-Exos on adipogenesis, the quality of the source cells, hDPSCs, was first verified. Flow cytometry analysis confirmed that the isolated hDPSCs expressed mesenchymal stem cell markers CD73, CD90, and CD105, while lacking expression of hematopoietic markers CD34 and CD45 (Fig. 1 A). To further validate their multipotent differentiation potential, the osteogenic capacity was assessed using Alkaline Phosphatase (ALP) activity assay and Alizarin Red S (ARS) staining. hDPSCs exhibited significantly increased alkaline phosphatase activity and enhanced calcium deposition, as indicated by ARS staining, compared to untreated controls (Fig. 1 B). hDPSC-Exos were subsequently isolated from hDPSCs-conditioned media and characterized by TEM, nanoparticle tracking analysis, and Western blotting. Nanoparticle tracking analysis revealed an average particle diameter of 124 nm, while TEM confirmed the presence of vesicles with typical lipid bilayer morphology and appropriate size (Fig. 1 C and 1 D). Western blotting showed enriched expression of established exosomal markers CD9 and CD81 in hDPSC-Exos compared to hDPSC whole-cell lysate (Fig. 1 E). To evaluate cellular uptake, hDPSC-Exos were labeled with CFSE and successfully visualized within 3T3-L1 preadipocytes using confocal fluorescence microscopy (Fig. 1 F). To evaluate the effects of hDPSC-Exos on adipogenesis, 3T3-L1 cells were cultured under adipogenic differentiation conditions with or without hDPSC-Exos for 6 days. Lipid droplet accumulation was assessed using oil red O staining (Fig. 2 A and 2 B). While control differentiated 3T3-L1 cells exhibited substantial lipid accumulation characteristic of mature adipocytes, Exo-treated cells displayed a marked reduction in cytoplasmic lipid droplets (Fig. 2 B). This inhibitory effect was quantitatively confirmed by measuring the absorbance of extracted oil red O dye (Fig. 2 C). To further investigate the underlying mechanisms, the expression levels of key adipogenic markers were examined using RT-qPCR. Treatment with hDPSC-Exos significantly decreased the expression of adipocyte differentiation markers, including Pparγ , C/ebpα , Srebp-1c , Adipoq , aP2, CD36 , and Plin2 , compared to untreated differentiated 3T3-L1 cells (Fig. 2 D). Western blot analysis corroborated these findings at the protein level, showing a similar decrease in adipogenic marker expression (Fig. 2 E). Collectively, the suppression of lipid droplet accumulation and the downregulation of adipogenic markers indicate that hDPSC-Exos exert an inhibitory effect on 3T3-L1 adipocyte differentiation. Although the 3T3-L1 preadipocyte cell line is a well-established model for evaluating adipogenic differentiation, considerable variability in its differentiation potential has been reported 19 . Therefore, the effects of hDPSC-Exos on adipogenesis in an alternative preadipocyte model, OP9 cells. Upon adipogenic induction, control OP9 cells exhibited prominent lipid droplet accumulation, consistent with mature adipocyte morphology. In contrast, hDPSC-Exos-treated OP9 cells showed a marked reduction in lipid accumulation (Fig. S1 A, left). This observation was quantitatively supported by a significant decrease in oil red O absorbance following lipid extraction (Fig. S1 A, right). Furthermore, RT-qPCR and Western blot analyses revealed that hDPSC-Exos treatment significantly suppressed mRNA and protein levels of adipocyte differentiation markers compared to untreated differentiated OP9 cells (Fig. S1 B and 1C). These results, consistent with those observed in 3T3-L1 cells, demonstrate that hDPSC-Exos can inhibit adipocyte differentiation across both cell models. hDPSC-Exos suppresses key adipogenic regulators throughout the differentiation process Previous findings have demonstrated that hDPSC-Exos inhibits lipid accumulation and adipocyte differentiation in 3T3-L1 and OP9 cells. To further elucidate the stage-specific effects of hDPSC-Exos during adipogenesis, 3T3-L1 cells were treated with adipogenic induction medium supplemented with or without hDPSC-Exos at various time points, as illustrated in Fig. 3 A. Consistent with earlier observations, continuous treatment with hDPSC-Exos from day 0 to day 6 significantly decreased the mRNA expression of key adipogenic markers, including Pparγ , C/ebpα , Srebp-1c , Adipoq , aP2, CD36 , and Plin2 . In contrast, when hDPSC-Exos were administered only during early, middle, or late stages of differentiation, the reduction in adipogenic gene expression was less pronounced compared to continuous treatment (Fig. 3 B). These findings suggest that hDPSC-Exos primarily inhibit adipogenesis when present during the continuous and early stages of differentiation. DEGs modulated by hDPSC-Exos during 3T3-L1 adipocyte differentiation To elucidate the molecular mechanisms by which hDPSC-Exos regulate adipocyte differentiation, total RNA was extracted from three 3T3-L1 groups: undifferentiated (Undiff), differentiated (Diff), and differentiated cells treated with hDPSC-Exos (Diff + Exos). Principal Component Analysis (PCA) demonstrated high reproducibility among biological replicates (Fig. 4 A). DEGs between groups were identified using volcano plots (Figs. 4 B and 4 C; Table S1 ), applying thresholds of p 1.0. Notably, hDPSC-Exos reversed differentiation-induced gene expression pattern changes, downregulating 170 genes upregulated during differentiation and upregulating 386 genes that had been downregulated (Fig. 4 D). A heatmap of DEGs based on the same criteria highlighted this reversal (Fig. 4 e). Key adipogenic and lipogenic genes, including Pparγ , C/ebpα , Srebp-1c , Adipoq , aP2, CD36 , and Plin2 , were significantly suppressed by hDPSC-Exos compared to the differentiated group (Fig. 4 E), corroborating RT-qPCR findings. To investigate the functional relevance of these DEGs, GO and KEGG pathway analyses were conducted (Table S2 ). The biological process (BP) terms from the GO analysis revealed that adipogenesis-responsive genes were enriched in lipid metabolic processes and fatty acid metabolic processes (Fig. 5 A), whereas hDPSC-Exos-responsive genes were associated with viral response and defense pathways (Fig. 5 B). Similarly, KEGG analysis showed that adipogenesis -responsive genes were enriched in metabolic pathways and non-alcoholic fatty liver disease during differentiation (Fig. S2 A), while hDPSC-Exos-responsive genes showed enrichment in ECM-receptor interaction, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathways (Fig. S2 B). Collectively, RNA sequencing analysis, visualized via heatmaps (Fig. 5 C), demonstrated that hDPSC-Exos significantly downregulate adipogenesis-related genes, supporting their inhibitory role in adipocyte differentiation. Identification of hDPSC-Exos-associated pathways To further elucidate the molecular mechanisms influenced by hDPSC-Exos, ORA was conducted using the Molecular Signatures Database hallmark gene sets, focusing on genes responsive to hDPSC-Exos treatment (Table S2 ). ORA revealed that upregulated genes in response to hDPSC-Exos were significantly enriched in pathways associated with epithelial-mesenchymal transition (EMT), G2M checkpoint, interferon gamma response, mitotic spindle formation, and interferon alpha response (Fig. 5 D, upper). In contrast, downregulated genes were enriched in adipogenesis, fatty acid metabolism, oxidative phosphorylation, and xenobiotic metabolism pathways (Fig. 5 D, lower). These findings further support the upregulation of EMT signaling signatures, with increased expression of pro-EMT genes such as chemokine (c-x-c motif) ligand 5 (Cxcl5), thrombospondin 2 (Tsp2), high-temperature requirement protease A1 (Htra1), microfibril-associated protein 5 (Mfap5), tissue inhibitor of metalloproteinases ( Timp ) 1 , and Timp3 in hDPSC-Exos treated 3T3-L1 cells undergoing differentiation. Conversely, adipogenic signaling was suppressed, as evidenced by the downregulation of key pro-adipogenic genes, including Pparγ , CD36 , Fabp4 , Adipoq , and Plin2 in hDPSC-Exos-treated cells (Fig. 5 E). Proteomic analysis of anti-adipogenic effectors in hDPSC-Exos To elucidate the anti-adipogenic mechanism of hDPSC-Exos, proteomic analysis of their cargo using liquid chromatography-mass spectrometry was performed. A total of 202 proteins were identified as significantly enriched in hDPSC-Exos compared to their parent hDPSCs ( p 1.5; Table S3 ). Of these, 148 overlapped with entries in ExoCarta, a database of exosome-specific proteins (Fig. 6 A). A heatmap comparison of protein abundance between hDPSC-Exos and hDPSCs is presented in Fig. 6 B. GO analysis revealed that the enriched proteins were predominantly involved in extracellular matrix and extracellular structure organization (Fig. 6 C). hDPSC-Exos inhibit adipogenesis through β-catenin signaling pathway activation Among the proteins enriched in hDPSC-Exos, several are established inhibitors of adipogenesis, including AE binding protein 1 (AEBP1) 20 , integrin subunit beta like 1 (ITGBL1), latent transforming growth factor beta binding protein 1 (LTBP1) 21 , latent transforming growth factor beta binding protein 2 (LTBP2) 22 , pleiotrophin (PTN) 23 , transforming growth factor beta 1 (TGFB1) 24 , transforming growth factor beta 2 (TGFB2) 25 , TIMP Metallopeptidase Inhibitor 1 (Timp1) 26 , and TIMP Metallopeptidase Inhibitor 2 (Timp2). Notably, ITGBL1 27 , AEBP1 28 , PTN 23 , and Timp1 26 are known modulators of the Wnt/β-catenin signaling pathway, a key inhibitor of adipocyte differentiation. These findings suggest that hDPSC-Exo might suppress adipogenesis by enhancing β-catenin protein signaling through multiple molecular pathways. To test our hypothesis, β-catenin protein levels were examined in 3T3-L1 cells during adipocyte differentiation, with or without hDPSC-Exos treatment. As expected, β-catenin levels declined under differentiation conditions but were significantly restored following hDPSC-Exos exposure. Notably, hDPSC-Exos treatment increased β-catenin and its active, non-phosphorylated form (serine at position 33/37/threonine at position 41) 29 compared to untreated controls (Fig. 7 A and 7 B). To further validate the role of hDPSC-Exos and their cargo protein Timp1—a known activator of β-catenin—in regulating adipogenesis, key adipogenic markers were assessed using RT-qPCR and Western blot analysis. hDPSC-Exos or Timp1 significantly reduced the mRNA and protein expression of adipocyte differentiation markers compared to untreated differentiated 3T3-L1 cells (Fig. 7Cand 7D). Collectively, these findings indicate that hDPSC-Exos and their cargo protein Timp1 inhibit adipogenesis by upregulating β-catenin expression during 3T3-L1 differentiation. Anti-adipogenic effect of hDPSC-Exos in diet-induced obesity To validate the anti-adipogenic effects of hDPSC-Exos in vivo, a diet-induced obesity mouse model was established. Mice were fed either an SD or an HFD for 12 weeks. A subgroup of HFD-fed mice received intraperitoneal injections of either PBS or HFD + Exos (50 µg/injection, twice weekly) for 12 weeks (Fig. 8 A). As expected, HFD-fed mice exhibited significantly higher body weight compared to SD controls. However, hDPSC-Exos administration significantly attenuated HFD-induced weight gain (Fig. 8 B), primarily by reducing fat mass accumulation (Fig. 8 C). Analysis of gonadal white adipose tissue (gWAT) revealed an approximately five-fold increase in gWAT mass in HFD-fed mice relative to SD-fed controls. Notably, hDPSC-Exos treatment significantly reduced gWAT mass in HFD-fed mice (Fig. 8 D). Histological analysis of gWAT using hematoxylin and eosin staining demonstrated that hDPSC-Exos treatment resulted in a marked decrease in adipocyte size and lipid droplet accumulation in HFD mice (Fig. 8 E). Quantitative analysis confirmed a significant reduction in average adipocyte diameter in the HFD + Exos group compared to HFD-fed mice (Fig. 8 F). At the molecular level, HFD feeding significantly upregulated the expression of genes involved in lipogenesis ( Ppar γ, C/ebpα , and fatty acid synthase (FASN) ), lipid transport ( CD36 and aP2 ), and adipokine signaling (leptin) in gWAT. hDPSC-Exos treatment significantly suppressed the HFD-induced elevation of these genes, except for C/EBPα (Fig. 8 G). Collectively, these findings demonstrated that hDPSC-Exos effectively inhibit white adipose tissue expansion and fatty acid metabolism in vivo. Discussion hDPSCs-Exos have garnered significant attention for their roles in immunomodulation, anti-inflammation, angiogenesis, and apoptosis of tumor cells 30 , 31 . However, the specific role in adipogenesis and obesity remains largely unexplored. In this study, the potential of hDPSC-Exos to regulate adipogenesis and elucidate the underlying mechanisms was investigated. Our findings demonstrated that hDPSC-Exos significantly reduced lipid accumulation during 3T3-L1 adipocyte differentiation. This reduction was accompanied by downregulation of key adipogenic markers, including Pparγ , C/ebpα , Srebp-1c , Adipoq , aP2, CD36 , and Plin2 . Further analysis revealed that continuous and early exposure to hDPSC-Exos during the differentiation period was most effective in suppressing adipogenic marker expression. These findings suggest that hDPSC-Exos exert their anti-adipogenic effects primarily during the early stages of adipocyte differentiation, likely by modulating preadipocyte signaling pathways involved in lineage commitment and differentiation. To investigate the molecular mechanisms by which hDPSC-Exos regulate adipocyte differentiation in 3T3-L1 cells, RNA sequencing was performed on cells treated with or without hDPSC-Exos during adipogenesis. The results showed that hDPSC-Exos treatment significantly reversed the expression of numerous genes associated with adipocyte differentiation. GO analyses revealed that these DEGs were predominantly enriched in lipid metabolic processes. This enrichment underscores the critical role of lipid metabolism in adipocyte function and suggests that hDPSC-Exos inhibit the metabolic adaptations typically involved in adipogenesis. In this study, ORA further revealed that genes associated with EMT, including Cxcl5 , Tsp2 , Htra1 , and Mfap5 , were significantly altered in 3T3-L1 cells following hDPSC-Exos treatment. For instance, CXCL5 inhibits adipocyte differentiation by reducing the transcriptional activity of PPARγ 32 . TSP2 inhibits long-chain fatty acid uptake by binding to CD36 33 . HTRA1 suppresses adipogenesis in human mesenchymal stem cells by promoting the production of matrix metalloproteinases 34 . Similarly, MFAP5 downregulates the expression of downstream molecules associated with Pparγ by inhibiting Staphylococcal nuclease and Tudor domain containing 1 (SND1) expression, a novel coactivator of Pparγ 35 . Notably, the enrichment of these upregulated genes upon hDPSC-Exos treatment suggests a mechanism that not only inhibits adipogenesis but also promotes alternative cellular processes during 3T3-L1 differentiation. Furthermore, proteomic analysis of hDPSC-Exos identified 202 differentially expressed proteins associated with extracellular matrix and structural organization. Several of these proteins, including ITGBL1, AEBP1, PTN, and Timp1, have previously been implicated in modulating adipocyte differentiation. Specifically, ITGBL1 interacts with Wnt receptors and activates c-Jun through a non-canonical pathway, potentially influencing signaling pathways involved in adipogenesis 27 . AEBP1 also binds to Wnt receptors and activates β-catenin, thereby potentially enhancing adipogenic signaling 28 . Additionally, PTN activates β-catenin by binding to the receptor protein tyrosine phosphatase (RPTP) beta/zeta receptor and inhibiting glycogen synthase kinase 3 beta (GSK-3β) 23 , while Timp1 directly activates β-catenin in a matrix metalloproteinase-independent manner 26 . The Wnt signaling pathway, particularly the β-catenin cascade, plays a crucial role in regulating mesenchymal cell fate and differentiation. In preadipocytes, activation of this pathway inhibits adipogenesis by downregulating key adipogenic transcription factors such as Pparγ and C/ebpα 36 . Our findings demonstrate that hDPSC-Exos treatment upregulated total and active β-catenin expression under adipogenic differentiation conditions, indicating that specific cargo proteins within hDPSC-Exos may activate the Wnt/β-catenin pathway. It was further confirmed that Timp1, a protein cargo within hDPSC-Exos, inhibits adipogenesis via β-catenin upregulation. These findings elucidate the mechanism by which hDPSC-Exos modulate adipocyte differentiation through specific signaling pathways and protein interactions. Collectively, this study suggests that hDPSC-Exos might serve as a novel therapeutic strategy for preventing obesity by targeting molecular mechanisms involved in adipogenesis. The diet-induced obesity (DIO) mouse model is a well-established polygenic model that closely mimics many aspects of human obesity. In this study, it was demonstrated that hDPSC-Exos treatment significantly reduced key obesity-related parameters, including body weight, fat mass, and gWAT weight in HFD-fed mice. Furthermore, hDPSC-Exos treatment inhibited adipose tissue maturation and consistently downregulated the expression of lipogenesis- and lipid transport-related genes in the gWAT of the HFD-fed mice. These in vivo results corroborate our in vitro findings and collectively suggest that hDPSC-Exos concurrently suppress de novo lipogenesis and lipid accumulation. This dual mechanism underscores their therapeutic potential for preventing and treating obesity. Conclusion This study provides the first evidence that hDPSC-Exos can effectively inhibit adipogenesis and lipogenesis in vivo and in vitro . While several candidate protein cargos within hDPSC-Exos that exert inhibitory effects during preadipocyte differentiation were identified, further research is warranted to elucidate the complex molecular pathways involved in this anti-obesity mechanism. These findings offer valuable insights into the development of novel cell-free therapeutic strategies for preventing and treating obesity and its associated metabolic disorders. Abbreviations (hDPSCs) : human dental pulp stem cells, MSCs : mesenchymal stem cells, hDPSC-Exos : hDPSC-derived exosomes, ALP : Alkaline Phosphatase, ARS : Alizarin Red S, SD : Standard Diet, HFD : High Fat Diet, gWAT : gonadal White Adipose Tissue, Declarations Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) (no. 2019R1A5A2027521, 2021R1C1C1009601, RS-2023-00247674, RS-2025-00514223, RS-2025-24536036), Chonnam National University Hospital Biomedical Research Institute (BCRI25084) and by the Technology Innovation Program (RS-2024-00406097, Development of customized hydrogel support and manufacturing technology for aggregate production equipped with dental pulp stem cell-derived materials) funded by the Ministry of Trade Industry & Energy (MOTIE, Korea). Author Contributions J.H.K: conceptualization, formal analysis, investigation, writing – original draft. H.K.Y and S.E.K: investigation, visualization. M.G.J.: provided human tissue, data analysis. B.R.L: data analysis. Y.K.: conceptualization, supervision, review and editing. Data availability The RNA-Seq dataset generated during the current study has been deposited in the NCBI Gene Expression Omnibus (GEO) database and is accessible through the accession number GSE298678. All other data generated or analyzed during this study are included in this published article and its supplementary files. Competing interests The authors declare no competing interests. Ethics approval All animal procedures were approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approval number: YB-2024-214; Date of approval: January 22, 2025, Title: Investigation of the anti-obesity effects of human dental pulp stem cell-derived exosomes and their exosomal miRNA in a high fat diet induced animal model). Human dental pulp tissues were obtained from supernumerary teeth extracted from seven healthy pediatric donors. 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Stem Cells 34 , 1601-1614, doi:10.1002/stem.2297 (2016). Zhang, T. et al. Microfibrillar-associated protein 5 suppresses adipogenesis by inhibiting essential coactivator of PPARgamma. Sci Rep 13 , 5589, doi:10.1038/s41598-023-32868-y (2023). Christodoulides, C., Lagathu, C., Sethi, J. K. & Vidal-Puig, A. Adipogenesis and WNT signalling. Trends Endocrinol Metab 20 , 16-24, doi:10.1016/j.tem.2008.09.002 (2009). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementTable2.xlsx Supplementary Table 2 SupplementaryFig2.tif Supplementary Fig 2 SupplementTable3.xlsx Supplementary Table 3 SupplementTable1.xlsx Supplementary Table 1 SupplementaryFig1.tif Supplementary Fig 1 SupplementaryLegends.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":8937946,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of hDPSCs and hDPSC-Exos. (A) Flow cytometry analysis of positive and negative surface markers of mesenchymal stem cells in hDPSCs. (B) Differentiation potential of hDPSCs assessed by Alkaline Phosphatase (ALP) activity assay and Alizarin Red S (ARS) staining. (C) Nanoparticle Tracking tracking Analysis analysis (NTA) showing the size distribution profile and average diameter of isolated hDPSC-Exos. (D) Representative transmission electron microscopy (TEM) image of isolated hDPSC-Exos, demonstrating their characteristic morphology. Scale bar: 100 nm. (E) Western blot analysis of exosomal marker proteins (CD63, HSP70, CD9, and CD81) in both hDPSCs and hDPSC-Exos. (F) Fluorescence microscopy images showing uptake of fluorescently labeled hDPSC-Exos by 3T3-L1 cells (scale bar: 20 mm). hDPSCs, human dental pulp stem cells; hDPSC-Exos, human dental pulp stem cell-derived exosomes.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/fd63ae84379677b588cf232d.png"},{"id":98171207,"identity":"96db7997-ead5-4e4d-926d-93d06878b1e7","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5641806,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of hDPSC-Exos on adipogenesis in 3T3-L1 cells.\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the experimental timeline for 3T3-L1 cell differentiation with or without hDPSC-Exos. (B) Representative microscopic images of oil red O (ORO) staining showing lipid droplet accumulation in differentiated 3T3-L1 cells treated with or without hDPSC-Exos. (C) Quantification of ORO staining by measuring the absorbance of extracted dye under different conditions. (D) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis showing relative mRNA expression levels of adipogenic markers in response to hDPSC-Exos treatment. (E) Western blot analysis showing protein expression levels of adipogenic markers in response to hDPSC-Exos treatment. Data are presented as mean ± SD. Statistical significance was determined using one-way analysis of variance. ∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/9101dc7099e5f763cd0efe14.png"},{"id":98430540,"identity":"bd2595e1-0e51-4bc4-973c-455c994620cb","added_by":"auto","created_at":"2025-12-17 16:45:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1624736,"visible":true,"origin":"","legend":"\u003cp\u003eTime-dependent effects of hDPSC-Exos on adipogenic differentiation.\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the experimental design showing different treatment periods of hDPSC-Exos during 3T3-L1 adipocyte differentiation. Treatment groups include: continuous (day 0–6), early stage (day 0–2), middle stage (day 2–4), and late stage (day 4–6). (B) The mRNA expression levels of \u003cem\u003ePparg\u003c/em\u003e,\u003cem\u003e C/ebpa\u003c/em\u003e,\u003cem\u003e Srebp-1c, Adipoq\u003c/em\u003e,\u003cem\u003e aP2, CD36\u003c/em\u003e, and\u003cem\u003e Plin2 \u003c/em\u003ein 3T3-L1 cells treated with hDPSC-Exos at different stages of differentiation. Data are presented as mean ± SD. Significance was determined using one-way analysis of variance. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/cb4ee3933fc25b6e224c5bc0.png"},{"id":98432396,"identity":"03c516d8-b80a-4f71-ab7c-a6921da13c1b","added_by":"auto","created_at":"2025-12-17 16:49:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3548733,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed genes (DEGs) regulated by hDPSC-Exos during adipocyte differentiation.\u003c/p\u003e\n\u003cp\u003e(A) PCA plot showing the clustering of RNA sequencing samples, including Undiff, Diff, and Diff+Exos 3T3-L1 cells. (B and C) Volcano plots displaying upregulated and downregulated genes between different experimental conditions. (D) Venn diagram showing DEGs unique to or shared between the Diff and Diff+Exos groups. (E) Heatmap illustrating the expression patterns of DEGs across all three experimental groups. (F) The mRNA expression levels of \u003cem\u003ePparg\u003c/em\u003e,\u003cem\u003e C/ebpa\u003c/em\u003e,\u003cem\u003e Srebp-1c, Adipoq\u003c/em\u003e,\u003cem\u003e aP2, CD36\u003c/em\u003e, and\u003cem\u003e Plin2 \u003c/em\u003efrom based on RNA sequencing data. Data are presented as mean ± SD. Statistical significance was determined using one-way analysis of variance. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eUndiff, undifferentiated; Diff, differentiated, Diff+Exos, differentiated with hDPSC-Exos\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/8e9647b1d9edaa90f7333d44.png"},{"id":98431707,"identity":"7ec2982b-36e0-4815-80b6-778794762587","added_by":"auto","created_at":"2025-12-17 16:48:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1155754,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis of DEGs between differentiated 3T3-L1 adipocytes and hDPSC-Exos-treated differentiated 3T3-L1 adipocytes. (A) GO analysis of adipogenesis-responsive genes. (B) GO analysis of hDPSC-Exos responsive genes. (C) Heatmap showing DEGs associated with adipogenesis, cholesterol metabolism, and fatty acid pathways across the three experimental groups. (D) ORA analysis of MSigDB hallmark gene sets showing positively and negatively enriched pathways. (E) The top significantly enriched pathways identified from ORA results.\u003c/p\u003e\n\u003cp\u003eGO, Gene Ontology; ORA, over-representation analysis; MSigDB, Molecular Signatures Database.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/cdc79d49c3d643e2cc124adc.png"},{"id":98171214,"identity":"70a53c04-067e-4277-9bfb-67f30ea2048e","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":405952,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the protein cargo composition of hDPSC-Exos using liquid chromatography–tandem mass spectrometry. (A) Venn diagram showing the overlap of identified proteins in hDPSC-Exos with the ExoCarta database. (B) Heatmap visualizing comparative protein abundance between hDPSC-Exos and their source cells (hDPSCs), based on proteins with \u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.05 and log2FC \u0026gt;2.0. (C) GO analysis of proteins enriched in hDPSC-Exos.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/88197bfbae4d92cca707d016.png"},{"id":98171215,"identity":"f1ed9322-9654-4dd8-af60-b6970c8b9111","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3052907,"visible":true,"origin":"","legend":"\u003cp\u003ehDPSC-Exos inhibit adipogenesis through β-catenin activation.\u003c/p\u003e\n\u003cp\u003e(A) β-catenin expression levels in 3T3-L1 cells under different treatment conditions. (B) Quantification of protein expression levels normalized to β-actin. (C) The mRNA expression levels of adipocyte markers and \u003cem\u003eβ-catenin\u003c/em\u003eduring 3T3-L1 adipocyte differentiation without or with hDPSC-Exos and tissue inhibitor of metalloproteinases 1 (Timp1). (D) Western blot analysis showing expression of adipocyte markers, active β-catenin, and β-catenin during 3T3-L1 adipocyte differentiation with or without hDPSC-Exos and Timp1. Gene expression levels were normalized to b-actin.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD. Significance was determined using one-way analysis of variance. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/fe56cdf5e251774dba5968d2.png"},{"id":98171218,"identity":"4016b0af-4fc7-4d01-bf57-6455b56bb53e","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":6804752,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of hDPSC-Exos on diet-induced obesity in mice.\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the experimental design for in vivo administration of hDPSC-Exos in a diet-induced obesity model. (B) Body weight changes over 12 weeks in mice fed with SD, HFD, or HFD+Exos (n=16). (C) Body composition analysis across the three experimental groups (n=16). (D) Gonadal white adipose tissue (gWAT) weight after 12 weeks of dietary intervention (n=16). (E and F) Representative hematoxylin and eosin staining and quantification of adipocyte area in gWAT sections from all groups after 12 weeks (n=8). (G) The mRNA expression levels of lipogenesis-related genes in gWAT from the three groups (n=7). Scale bar = 50 μm. Data are presented as mean ± SD. Statistical significance was determined using one-way analysis of variance. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ∗∗\u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003eSD, standard diet; HFD, high-fat diet; HFD+Exos, HFD with Pulp-Exo injection\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/e90a29dc3faef20a97fac8fb.png"},{"id":98775138,"identity":"17f0b639-2914-481a-8ac9-914f2e0b37b4","added_by":"auto","created_at":"2025-12-22 12:18:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30677901,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/bb5daa55-d37b-45fa-8bb8-38e9d4cab67d.pdf"},{"id":98171199,"identity":"13fbe150-2c15-4fe9-b6e9-ad809e8536cb","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":67479,"visible":true,"origin":"","legend":"Supplementary Table 2","description":"","filename":"SupplementTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/d37b7c007f45c8957d8ce22a.xlsx"},{"id":98431018,"identity":"a16069cd-1af3-481c-befb-c2633d418fb2","added_by":"auto","created_at":"2025-12-17 16:46:45","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":958554,"visible":true,"origin":"","legend":"Supplementary Fig 2","description":"","filename":"SupplementaryFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/35d8f4d1f35188a5cd486c61.tif"},{"id":98431261,"identity":"6872f43a-de47-4203-bf5b-aadc42b97d69","added_by":"auto","created_at":"2025-12-17 16:47:23","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21913,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"SupplementTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/c6e1aedf67439eec9cdc94d3.xlsx"},{"id":98171213,"identity":"45b64867-ab3f-470d-bf3c-736e97603349","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":407172,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"SupplementTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/41f9f23d144ea9c1d6b9a30a.xlsx"},{"id":98171221,"identity":"1bfb23fd-8297-443e-b7b3-270e746185f3","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1821498,"visible":true,"origin":"","legend":"Supplementary Fig 1","description":"","filename":"SupplementaryFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/117543653ffde6672d21f444.tif"},{"id":98171211,"identity":"651ca23a-894b-4ec0-946d-dfea5ed8f521","added_by":"auto","created_at":"2025-12-14 16:43:19","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14391,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8023093/v1/673f5e6d6630fef1cdd79229.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Human dental pulp stem cell-derived exosomes inhibit adipogenesis and obesity by activating Wnt/β-catenin signaling","fulltext":[{"header":"Background","content":"\u003cp\u003eOver recent decades, obesity has emerged as a major global public health concern, largely attributed to sedentary lifestyles and unhealthy dietary habits. As a chronic condition, it is strongly linked to several metabolic disorders, including type 2 diabetes, cardiovascular diseases, and various malignancies, underscoring the urgency of effective prevention and management\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The increasing prevalence of obesity and its associated comorbidities necessitates the development of innovative therapeutic strategies to effectively mitigate its progression\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent advances in regenerative medicine have focused on natural substances derived from stem cells and regenerative tissues. Among these, human dental pulp stem cells (hDPSCs) offer distinct advantages over other mesenchymal stem cells (MSCs), including easy accessibility from discarded teeth, minimally invasive collection methods, high proliferative capacity, and strong multi-lineage differentiation potential. Moreover, their use is associated with fewer ethical concerns, enhancing their appeal for diverse medical applications\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Notably, hDPSC-derived exosomes (hDPSC-Exos)\u0026mdash;small extracellular vesicles enriched with proteins, lipids, and nucleic acids\u0026mdash;have shown significant promise in modulating cellular signaling and biological processes\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although prior studies have demonstrated the therapeutic potential of hDPSC-Exos, their specific role in regulating adipogenesis remains largely unexplored.\u003c/p\u003e\u003cp\u003eIn this study, the inhibitory effects of hDPSC-Exos on adipogenesis were investigated, with a particular emphasis on their modulation of key adipogenic transcriptional networks. The objective was to elucidate the molecular mechanisms underlying exosome-mediated suppression of adipogenesis and evaluate their potential as a cell-free therapeutic strategy for obesity prevention.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and adipogenic differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3T3-L1 preadipocytes were cultured in high-glucose Dulbecco\u0026apos;s Modified Eagle Medium (DMEM) (LM 001-05, Welgene, Korea) supplemented with 10% bovine calf serum (16170078, Gibco, New Zealand) and 1% penicillin\u0026ndash;streptomycin (LS202-02, Welgene, Korea). OP9 mouse stromal cells were maintained in Alpha Modified Eagle\u0026apos;s Minimum Essential Medium (12571-071, Gibco) supplemented with 20% fetal bovine serum (FBS) (16000-044, Gibco) and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified incubator with 5% carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e). For adipogenic differentiation, cells were seeded in 12-well plates and grown to confluence. Differentiation was induced using a standard adipogenic induction medium comprising DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, I5879, Sigma-Aldrich), 1 \u0026micro;M dexamethasone (D4902, Sigma-Aldrich), and 10 \u0026micro;g/mL insulin (I6634, Sigma-Aldrich, St. Louis, MO, USA). After 2 days, the induction medium was replaced with maintenance medium (DMEM containing 10% FBS and 10 \u0026micro;g/mL insulin), which was refreshed every 2 days until the formation of mature adipocytes with visible lipid droplets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOil red O staining and dye extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate lipid accumulation, differentiated 3T3-L1 and OP9 cells were washed thrice with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 30 min at room temperature. After fixation, cells were washed thrice with PBS (5 min per wash). Oil red O working solution was prepared by diluting the stock solution with distilled water at a 3:2 ratio and filtering it through filter paper. Cells were stained with the working solution for 1 h at room temperature, followed by three PBS washes (5 min per wash). Stained cells were visualized using the Lionheart FX Cell Imager (BioTek, Winooski, VT, USA). Oil red O dye was eluted using 100% isopropanol with gentle shaking for 10 min to quantify lipid content. Optical density was measured at 490 nm using a SYNERGY H1 microplate reader (BioTek, Winooski, VT, USA) with 100% isopropanol as the blank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, culture, and characterization of hDPSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman dental pulp tissues were obtained from supernumerary teeth extracted from seven healthy pediatric donors. hDPSCs were isolated as previously described\u003csup\u003e9\u003c/sup\u003e. Briefly, the extracted teeth were washed with sterile PBS to remove debris. The pulp tissue was then carefully dissected, minced into small fragments, and enzymatically digested with 3 mg/mL collagenase type I (C0130, Sigma-Aldrich, St. Louis, MO, USA) at 37\u0026deg;C for 30 min. The resulting suspension was filtered through a 70 \u0026micro;m cell strainer to obtain a single-cell suspension, washed with PBS, and cultured in DMEM supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003euntil reaching approximately 80% confluence. To evaluate osteogenic differentiation potential, hDPSCs were cultured for 7 and 19 days in an osteogenic medium supplemented with 10 mM \u0026beta;-glycerophosphate (G9422, Sigma-Aldrich) and 50\u0026nbsp;mM L-ascorbic acid (A4544, Sigma-Aldrich, St. Louis, MO, USA). Osteogenic differentiation was assessed by alkaline phosphatase\u0026nbsp;(ALP)\u0026nbsp;and Alizarin Red S (ARS) staining using premixed 5-bromo-4-chloro-3-indolyl phosphate\u003csup\u003e\u0026reg;\u003c/sup\u003e/nitro blue tetrazolium solution (B6404-100, Sigma-Aldrich, St. Louis, MO, USA) and 2% Alizarin red S solution (0223, ScienCell, San Diego, USA). Stained cells were visualized using a Lionheart FX Cell Imager (BioTek, Winooski, VT, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehDPSCs were harvested and washed thrice using PBS, followed by centrifugation at 300 \u0026times; g for 10 min at room temperature after each wash. A total of 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e cells per tube were incubated for 20 min at 4\u0026deg;C in the dark with the following antibodies: CD73-FITC, CD105-PE, CD90-FITC, CD45-PE, and CD34-APC (eBioscience, San Diego, CA, USA). After incubation, cells were washed once with PBS to remove unbound antibodies. Flow cytometry was performed using BD AccuriT\u0026trade; C6 Plus (BD Biosciences, CA, USA). Data analysis and visualization were performed using the flowCore and ggridges packages in R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo isolate exosomes from hDPSCs, conditioned medium was collected after 48 h of culture. The medium was initially centrifuged at 300 \u0026times; g for 10 min to remove cellular debris. The supernatant was then processed through the Minimate\u0026trade; EVO Tangential Flow Filtration system (Cytiva, Wilmington, DE, USA) using a 100 kDa molecular weight cut-off filter. The flow rate was optimized to retain exosomes while allowing smaller proteins and soluble molecules to pass through. The resulting concentrate was further purified by overnight precipitation at 4\u0026deg;C using ExoQuick (EXOTC50A-1, System Biosciences, Palo Alto, CA, USA). The purified exosome pellet was resuspended in PBS and stored at -80\u0026deg;C for subsequent analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNanoparticle tracking analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe size distribution and concentration of hDPSC-Exos were analyzed using the ZetaView system (Particle Metrix, Germany). Samples were diluted in 0.22 \u0026mu;m-filtered Dulbecco\u0026apos;s PBS and analyzed under the following parameters: maximum area, 1000; minimum area, 10; minimum brightness, 30. Measurements were conducted using a 488 nm laser at 40 mW power. Data were processed using ZetaView software (v8.06.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM) analysis of exosomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphological characteristics of isolated hDPSC-Exos were evaluated using TEM. Exosome samples were fixed in a solution containing 2% glutaraldehyde (v/v) and 2% paraformaldehyde (v/v) in 0.05 M cacodylate buffer (pH 7.2) at room temperature for 4 h. Following fixation, the samples were washed with the same buffer and carefully placed onto a carbon-coated copper grid. TEM imaging was performed using a JEM-ARM200F TEM (JEOL Ltd., Akishima, Japan) operated at an accelerating voltage of 200 kV and a magnification of 100 000\u0026times;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome labeling using 5-(and-6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor CFSE staining (V12883, ThermoFisher, USA), a 200\u0026thinsp;\u0026micro;M working solution was prepared in PBS by diluting a 10\u0026thinsp;mM dye solution in dimethyl sulfoxide. For exosome labeling, 15\u0026thinsp;\u0026mu;L of hDPSC-Exos suspended in PBS was mixed with 15\u0026thinsp;\u0026mu;L of 40\u0026thinsp;\u0026mu;M CFSE solution and incubated for 10 min at 4\u0026deg;C, unless otherwise indicated. Following staining the exosomes were diluted to 2 mL with DPBS, transferred to Microsep Advance 100k Omega filters (MCP100C41, Pall Corporation, Washington, NY, USA), and centrifuged at 4000 \u0026times; g for 3 min at 4\u0026deg;C. The exosomes were washed thrice with 2 mL D-PBS and concentrated to a final volume of 50 \u0026mu;L. For cellular uptake studies, CFSE-labeled exosomes were applied to 3T3-L1 cells. After incubation, cells were washed twice with D-PBS, fixed with 4% paraformaldehyde for 15 min, and washed thrice with PBS. Nuclei were counterstained with 2\u0026thinsp;mg/mL Hoechst 33342 for 5\u0026thinsp;min. Cells were imaged using a confocal laser scanning microscope (LSM900, Zeiss, Jena, Germany), and the data were analyzed using Zen 3.2 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of samples for RNA sequencing\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transcriptome analysis, 3T3-L1 preadipocytes were plated and cultured until confluence. Cells were then treated with hDPSC-Exos at a concentration of 20 \u0026micro;g/mL in differentiation media to induce adipogenic differentiation. After 6 days of treatment, total RNA was extracted using RNAiso Plus reagent (9109, Takara, Japan) according to the manufacturer\u0026rsquo;s protocol. RNA quality was assessed using the Agilent TapeStation4000 system (Agilent Technologies, Amstelveen, Netherlands). Total RNA libraries were constructed using the RiboCop rRNA Depletion Kit (Lexogen Inc., Vienna, Austria) in combination with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on the NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) at e-biogen (Seoul, Republic of Korea).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing data processing, differential expression analysis, and data visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing data were processed following previously established protocols\u003csup\u003e10\u003c/sup\u003e. Briefly, raw FASTQ files were subjected to quality control using Trimmomatic (v0.39)\u003csup\u003e11\u003c/sup\u003e. Cleaned reads were then aligned and quantified against the GENCODE v37 human reference genome\u003csup\u003e12\u003c/sup\u003e using Salmon with default settings (v1.4.0)\u003csup\u003e13\u003c/sup\u003e. Gene-level counts were obtained using the tximport package in R (v1.6.3)\u003csup\u003e14\u003c/sup\u003e, and differential gene expression analysis was conducted using DESeq2 (v1.30.1)\u003csup\u003e15\u003c/sup\u003e. Differentially expressed mRNAs were visualized using heatmaps (ComplexHeatmap R package v2.6.2)\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional and pathway enrichment analysis of differentially expressed genes (DEGs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene annotation for DEGs was performed using the clusterProfiler package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (version 6.8; https://david.ncifcrf.gov/home.jsp), applying a threshold of \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 and |log2FC| \u0026gt;2.0. Over-representation analysis (ORA) was conducted using the clusterProfiler package to identify significantly enriched pathways\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor filter-aided sample preparation digestion of hDPSC-Exos samples, 500 mM Tris (2-carboxyethyl) phosphine was added to a final concentration of 5 mM and incubated at 37\u0026deg;C with shaking at 300 rpm for 30 min. The mixture was transferred to a filtration unit and centrifuged at 14 000 \u0026times; g for 15 min at 20\u0026deg;C. Protein alkylation was performed by adding 500 mM iodoacetamide to a final concentration of 50 mM, followed by incubation in the dark at 25\u0026deg;C for 1 h, followed by centrifugation under the same conditions. For peptide digestion, trypsin was dissolved in 50 mM ammonium bicarbonate and added at a trypsin-to-protein ratio of 1:50. Samples were incubated at 37\u0026deg;C with shaking at 300 rpm for 12 h. The resulting peptides were desalted using C18 Micro Spin Columns with buffer A (0.1% trifluoroacetic acid) and eluted using a linear gradient of buffer B (80% acetonitrile with 0.1% trifluoroacetic acid) at a flow rate of 300 nL/min. Peptides were then loaded onto a reverse-phase trap column (Acclaim PepMap100 C18, 3 \u0026mu;m, 75 \u0026mu;m \u0026times; 2 cm, Thermo Scientific) and separated on a C18-reversed phase analytical column (PepMap\u003csup\u003eTM\u003c/sup\u003e, 75 \u0026mu;m \u0026times; 50 cm, Thermo Scientific) using buffer A (0.1% formic acid) and a linear gradient of buffer B (80% acetonitrile with 0.1% formic acid) at the same flow rate. Mass spectrometry analysis was conducted using Proteome Discoverer\u003csup\u003eTM\u003c/sup\u003e (Thermo Scientific) against the UniProt complete human proteome database (version 2018-01-15; 161 584 sequences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis\u003c/strong\u003e\u003cbr\u003eTotal RNA was extracted using RNAiso Plus reagent (9108, Takara) according to the manufacturer\u0026apos;s instructions. Complementary DNA (cDNA) was synthesized using the Maxime RT PreMix Kit (25082, Intronbio). RT-qPCR was performed using TB Green\u0026reg; Premix Ex Taq\u0026trade; (Tli RNaseH Plus) (RR420A, Takara) on a real-time PCR system to assess mRNA expression of adipogenic marker genes. Relative gene expression levels were calculated using the 2^(-\u003csup\u003e\u0026Delta;\u0026Delta;\u003c/sup\u003eCT) method, with \u0026beta;-actin as the internal control. All primer sequences (Table 1) were designed using Primer3 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eAll primer sequences used in this study.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"519\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 416px;\"\u003e\n \u003cp\u003ePrimer sequence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003ePpar\u0026gamma;g\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-ATTTGAAAGAAGCGGTGAACC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-GCTGATTCCGAAGTTGGTGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003eC/ebp\u0026alpha;a\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TGGACAAGAACAGCAACGAG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TCACTGGTCAACTCCAGCAC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003eSrebp-1c\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TGGACACAGCGGTTTTGAAC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-CGGGAAGTCACTGTCTTGGT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003eAdipoq\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-CCTGGAGAGAAGGGAGAGAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-CAGCTCCTGTCATTCCAACA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003eaP2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-AACCTGGAAGCTTGTCTCCA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-ATGATCATGTTGGGCTTGGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003eCD36\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TGTGTTTGGAGGCATTCTCA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-GGTGCCTGTTTTAACCCAGTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003ePlin2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-GGCAGTCTTTCCTCCATCCT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-GGCAGTCTTTCCTCCATCCT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026beta;-catenin\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-CTGAAGGTGCTGTCTGTCTG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TCTGTCAGATGAAGCCCCAG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026beta;-actin\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TGGCACCACACCTTCTACAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 282px;\"\u003e\n \u003cp\u003e5\u0026apos;-TACATGGCTGGGGTGTTGAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were lysed using RIPA buffer supplemented with cOmplete\u0026trade; EDTA-free Protease Inhibitor Cocktail (11836153001, Sigma). The whole-cell lysates were cleared by centrifugation at 13 000 \u0026times; g for 15 min at 4\u0026deg;C. Protein concentrations were determined, and equal amounts of protein were subjected to sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis, followed by electrophoretic transfer onto nitrocellulose membranes. Membranes were blocked and incubated overnight at 4\u0026deg;C with primary antibodies (PPARg, 2435S, Cell signaling, 1:1000; C/EBPa, 8178S, Cell signaling, 1:1000; Adiponectin, 2789S, Cell signaling, 1:1000; Perilipin-1, 9349S, Cell signaling, 1:1000; Non-phospho (Active) \u0026beta;-Catenin (Ser33/37/Thr41), 8814S, Cell signaling, 1:1000; \u0026beta;-Catenin, 9562S, Cell signaling, 1:1000, Anti-\u0026beta;-Actin, K200058M, Solarbio, 1:3000). After washing, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse). Immunoreactive bands were visualized using the LAS-3000 mini imaging system (Fujifilm, Tokyo, Japan)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal experiments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old male C57BL/6J mice were housed under a 12-h light/dark cycle with ad libitum access to tap water. Mice were randomly assigned to three groups: standard diet (SD) group, high-fat diet (HFD) group, and HFD supplemented with hDPSC-Exos (HFD+Exos) group. The HFD and HFD+Exos groups were fed an HFD (60% kcal from fat) for 12 weeks. During this period, the HFD+Exos group received intraperitoneal injections of hDPSC-Exos (50 \u0026mu;g/mouse) twice weekly, while the HFD group received equivalent volumes of PBS as a control. Body weight was recorded weekly throughout the study. At the experimental endpoint, mice were anesthetized via intraperitoneal injection of a mixture of alfaxan (45 mg/kg) and xylazine (8 mg/kg), followed by cervical dislocation. Subsequently, white adipose tissues were collected, weighed, and subjected to histological analysis using QuPath\u003csup\u003e18\u003c/sup\u003e. Tissue assessments were conducted by two investigators blinded to group allocation. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBody composition analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody composition was assessed using dual-energy X-ray absorptiometry with the InAlyzer system (Medikors Inc., Seongnam, Korea). For the analysis, mice were anesthetized with an intraperitoneal injection of of a mixture of alfaxan (45 mg/kg) and xylazine (8 mg/kg) and placed in a prone position on the scanning platform with limbs extended laterally. Total fat and lean mass were quantified using InAlyzer software following the manufacturer\u0026apos;s protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in triplicate, and data are presented as mean \u0026plusmn; standard deviation (SD). All expression statistical analysis was performed using ANOVA with Tukey\u0026rsquo;s post-hoc analysis in SPSS 21.0 software. Weekly mouse weights data was analyzed by one-way repeated measures ANOVA followed by Tukey\u0026apos;s post hoc test for multiple comparisons. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ehDPSC-Exos inhibits adipogenesis via suppressing\u003c/b\u003e \u003cb\u003ePparγ-C/ebpα\u003c/b\u003e \u003cb\u003eexpression in 3T3-L1 cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effects of hDPSC-Exos on adipogenesis, the quality of the source cells, hDPSCs, was first verified. Flow cytometry analysis confirmed that the isolated hDPSCs expressed mesenchymal stem cell markers CD73, CD90, and CD105, while lacking expression of hematopoietic markers CD34 and CD45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further validate their multipotent differentiation potential, the osteogenic capacity was assessed using Alkaline Phosphatase (ALP) activity assay and Alizarin Red S (ARS) staining. hDPSCs exhibited significantly increased alkaline phosphatase activity and enhanced calcium deposition, as indicated by ARS staining, compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). hDPSC-Exos were subsequently isolated from hDPSCs-conditioned media and characterized by TEM, nanoparticle tracking analysis, and Western blotting. Nanoparticle tracking analysis revealed an average particle diameter of 124 nm, while TEM confirmed the presence of vesicles with typical lipid bilayer morphology and appropriate size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Western blotting showed enriched expression of established exosomal markers CD9 and CD81 in hDPSC-Exos compared to hDPSC whole-cell lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To evaluate cellular uptake, hDPSC-Exos were labeled with CFSE and successfully visualized within 3T3-L1 preadipocytes using confocal fluorescence microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effects of hDPSC-Exos on adipogenesis, 3T3-L1 cells were cultured under adipogenic differentiation conditions with or without hDPSC-Exos for 6 days. Lipid droplet accumulation was assessed using oil red O staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). While control differentiated 3T3-L1 cells exhibited substantial lipid accumulation characteristic of mature adipocytes, Exo-treated cells displayed a marked reduction in cytoplasmic lipid droplets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This inhibitory effect was quantitatively confirmed by measuring the absorbance of extracted oil red O dye (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To further investigate the underlying mechanisms, the expression levels of key adipogenic markers were examined using RT-qPCR. Treatment with hDPSC-Exos significantly decreased the expression of adipocyte differentiation markers, including \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003eC/ebpα\u003c/em\u003e, \u003cem\u003eSrebp-1c\u003c/em\u003e, \u003cem\u003eAdipoq\u003c/em\u003e, \u003cem\u003eaP2, CD36\u003c/em\u003e, and \u003cem\u003ePlin2\u003c/em\u003e, compared to untreated differentiated 3T3-L1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Western blot analysis corroborated these findings at the protein level, showing a similar decrease in adipogenic marker expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, the suppression of lipid droplet accumulation and the downregulation of adipogenic markers indicate that hDPSC-Exos exert an inhibitory effect on 3T3-L1 adipocyte differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the 3T3-L1 preadipocyte cell line is a well-established model for evaluating adipogenic differentiation, considerable variability in its differentiation potential has been reported\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Therefore, the effects of hDPSC-Exos on adipogenesis in an alternative preadipocyte model, OP9 cells. Upon adipogenic induction, control OP9 cells exhibited prominent lipid droplet accumulation, consistent with mature adipocyte morphology. In contrast, hDPSC-Exos-treated OP9 cells showed a marked reduction in lipid accumulation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, left). This observation was quantitatively supported by a significant decrease in oil red O absorbance following lipid extraction (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, right). Furthermore, RT-qPCR and Western blot analyses revealed that hDPSC-Exos treatment significantly suppressed mRNA and protein levels of adipocyte differentiation markers compared to untreated differentiated OP9 cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB and 1C). These results, consistent with those observed in 3T3-L1 cells, demonstrate that hDPSC-Exos can inhibit adipocyte differentiation across both cell models.\u003c/p\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ehDPSC-Exos suppresses key adipogenic regulators throughout the differentiation process\u003c/h2\u003e\u003cp\u003ePrevious findings have demonstrated that hDPSC-Exos inhibits lipid accumulation and adipocyte differentiation in 3T3-L1 and OP9 cells. To further elucidate the stage-specific effects of hDPSC-Exos during adipogenesis, 3T3-L1 cells were treated with adipogenic induction medium supplemented with or without hDPSC-Exos at various time points, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Consistent with earlier observations, continuous treatment with hDPSC-Exos from day 0 to day 6 significantly decreased the mRNA expression of key adipogenic markers, including \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003eC/ebpα\u003c/em\u003e, \u003cem\u003eSrebp-1c\u003c/em\u003e, \u003cem\u003eAdipoq\u003c/em\u003e, \u003cem\u003eaP2, CD36\u003c/em\u003e, and \u003cem\u003ePlin2\u003c/em\u003e. In contrast, when hDPSC-Exos were administered only during early, middle, or late stages of differentiation, the reduction in adipogenic gene expression was less pronounced compared to continuous treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings suggest that hDPSC-Exos primarily inhibit adipogenesis when present during the continuous and early stages of differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eDEGs modulated by hDPSC-Exos during 3T3-L1 adipocyte differentiation\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular mechanisms by which hDPSC-Exos regulate adipocyte differentiation, total RNA was extracted from three 3T3-L1 groups: undifferentiated (Undiff), differentiated (Diff), and differentiated cells treated with hDPSC-Exos (Diff\u0026thinsp;+\u0026thinsp;Exos). Principal Component Analysis (PCA) demonstrated high reproducibility among biological replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). DEGs between groups were identified using volcano plots (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), applying thresholds of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |Log2FC| \u0026gt;1.0. Notably, hDPSC-Exos reversed differentiation-induced gene expression pattern changes, downregulating 170 genes upregulated during differentiation and upregulating 386 genes that had been downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). A heatmap of DEGs based on the same criteria highlighted this reversal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Key adipogenic and lipogenic genes, including \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003eC/ebpα\u003c/em\u003e, \u003cem\u003eSrebp-1c\u003c/em\u003e, \u003cem\u003eAdipoq\u003c/em\u003e, \u003cem\u003eaP2, CD36\u003c/em\u003e, and \u003cem\u003ePlin2\u003c/em\u003e, were significantly suppressed by hDPSC-Exos compared to the differentiated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), corroborating RT-qPCR findings. To investigate the functional relevance of these DEGs, GO and KEGG pathway analyses were conducted (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The biological process (BP) terms from the GO analysis revealed that adipogenesis-responsive genes were enriched in lipid metabolic processes and fatty acid metabolic processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), whereas hDPSC-Exos-responsive genes were associated with viral response and defense pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similarly, KEGG analysis showed that adipogenesis -responsive genes were enriched in metabolic pathways and non-alcoholic fatty liver disease during differentiation (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), while hDPSC-Exos-responsive genes showed enrichment in ECM-receptor interaction, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathways (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Collectively, RNA sequencing analysis, visualized via heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), demonstrated that hDPSC-Exos significantly downregulate adipogenesis-related genes, supporting their inhibitory role in adipocyte differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eIdentification of hDPSC-Exos-associated pathways\u003c/h2\u003e\u003cp\u003eTo further elucidate the molecular mechanisms influenced by hDPSC-Exos, ORA was conducted using the Molecular Signatures Database hallmark gene sets, focusing on genes responsive to hDPSC-Exos treatment (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). ORA revealed that upregulated genes in response to hDPSC-Exos were significantly enriched in pathways associated with epithelial-mesenchymal transition (EMT), G2M checkpoint, interferon gamma response, mitotic spindle formation, and interferon alpha response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, upper). In contrast, downregulated genes were enriched in adipogenesis, fatty acid metabolism, oxidative phosphorylation, and xenobiotic metabolism pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, lower). These findings further support the upregulation of EMT signaling signatures, with increased expression of pro-EMT genes such as chemokine (c-x-c motif) ligand 5 (Cxcl5), thrombospondin 2 (Tsp2), high-temperature requirement protease A1 (Htra1), microfibril-associated protein 5 (Mfap5), tissue inhibitor of metalloproteinases (\u003cem\u003eTimp\u003c/em\u003e) \u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eTimp3\u003c/em\u003e in hDPSC-Exos treated 3T3-L1 cells undergoing differentiation. Conversely, adipogenic signaling was suppressed, as evidenced by the downregulation of key pro-adipogenic genes, including \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003eCD36\u003c/em\u003e, \u003cem\u003eFabp4\u003c/em\u003e, \u003cem\u003eAdipoq\u003c/em\u003e, and \u003cem\u003ePlin2\u003c/em\u003e in hDPSC-Exos-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eProteomic analysis of anti-adipogenic effectors in hDPSC-Exos\u003c/h2\u003e\u003cp\u003eTo elucidate the anti-adipogenic mechanism of hDPSC-Exos, proteomic analysis of their cargo using liquid chromatography-mass spectrometry was performed. A total of 202 proteins were identified as significantly enriched in hDPSC-Exos compared to their parent hDPSCs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5; Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Of these, 148 overlapped with entries in ExoCarta, a database of exosome-specific proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). A heatmap comparison of protein abundance between hDPSC-Exos and hDPSCs is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. GO analysis revealed that the enriched proteins were predominantly involved in extracellular matrix and extracellular structure organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003ehDPSC-Exos inhibit adipogenesis through β-catenin signaling pathway activation\u003c/h2\u003e\u003cp\u003eAmong the proteins enriched in hDPSC-Exos, several are established inhibitors of adipogenesis, including AE binding protein 1 (AEBP1)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, integrin subunit beta like 1 (ITGBL1), latent transforming growth factor beta binding protein 1 (LTBP1)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, latent transforming growth factor beta binding protein 2 (LTBP2)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, pleiotrophin (PTN)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, transforming growth factor beta 1 (TGFB1)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, transforming growth factor beta 2 (TGFB2)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, TIMP Metallopeptidase Inhibitor 1 (Timp1)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and TIMP Metallopeptidase Inhibitor 2 (Timp2). Notably, ITGBL1\u003csup\u003e27\u003c/sup\u003e, AEBP1\u003csup\u003e28\u003c/sup\u003e, PTN\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and Timp1\u003csup\u003e26\u003c/sup\u003e are known modulators of the Wnt/β-catenin signaling pathway, a key inhibitor of adipocyte differentiation. These findings suggest that hDPSC-Exo might suppress adipogenesis by enhancing β-catenin protein signaling through multiple molecular pathways.\u003c/p\u003e\u003cp\u003eTo test our hypothesis, β-catenin protein levels were examined in 3T3-L1 cells during adipocyte differentiation, with or without hDPSC-Exos treatment. As expected, β-catenin levels declined under differentiation conditions but were significantly restored following hDPSC-Exos exposure. Notably, hDPSC-Exos treatment increased β-catenin and its active, non-phosphorylated form (serine at position 33/37/threonine at position 41)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e compared to untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). To further validate the role of hDPSC-Exos and their cargo protein Timp1\u0026mdash;a known activator of β-catenin\u0026mdash;in regulating adipogenesis, key adipogenic markers were assessed using RT-qPCR and Western blot analysis. hDPSC-Exos or Timp1 significantly reduced the mRNA and protein expression of adipocyte differentiation markers compared to untreated differentiated 3T3-L1 cells (Fig.\u0026nbsp;7Cand 7D). Collectively, these findings indicate that hDPSC-Exos and their cargo protein Timp1 inhibit adipogenesis by upregulating β-catenin expression during 3T3-L1 differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eAnti-adipogenic effect of hDPSC-Exos in diet-induced obesity\u003c/h2\u003e\u003cp\u003eTo validate the anti-adipogenic effects of hDPSC-Exos in vivo, a diet-induced obesity mouse model was established. Mice were fed either an SD or an HFD for 12 weeks. A subgroup of HFD-fed mice received intraperitoneal injections of either PBS or HFD\u0026thinsp;+\u0026thinsp;Exos (50 \u0026micro;g/injection, twice weekly) for 12 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). As expected, HFD-fed mice exhibited significantly higher body weight compared to SD controls. However, hDPSC-Exos administration significantly attenuated HFD-induced weight gain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), primarily by reducing fat mass accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Analysis of gonadal white adipose tissue (gWAT) revealed an approximately five-fold increase in gWAT mass in HFD-fed mice relative to SD-fed controls. Notably, hDPSC-Exos treatment significantly reduced gWAT mass in HFD-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Histological analysis of gWAT using hematoxylin and eosin staining demonstrated that hDPSC-Exos treatment resulted in a marked decrease in adipocyte size and lipid droplet accumulation in HFD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Quantitative analysis confirmed a significant reduction in average adipocyte diameter in the HFD\u0026thinsp;+\u0026thinsp;Exos group compared to HFD-fed mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). At the molecular level, HFD feeding significantly upregulated the expression of genes involved in lipogenesis (\u003cem\u003ePpar\u003c/em\u003eγ, \u003cem\u003eC/ebpα\u003c/em\u003e, and \u003cem\u003efatty acid synthase (FASN)\u003c/em\u003e), lipid transport (\u003cem\u003eCD36\u003c/em\u003e and \u003cem\u003eaP2\u003c/em\u003e), and adipokine signaling (leptin) in gWAT. hDPSC-Exos treatment significantly suppressed the HFD-induced elevation of these genes, except for \u003cem\u003eC/EBPα\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Collectively, these findings demonstrated that hDPSC-Exos effectively inhibit white adipose tissue expansion and fatty acid metabolism in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ehDPSCs-Exos have garnered significant attention for their roles in immunomodulation, anti-inflammation, angiogenesis, and apoptosis of tumor cells\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, the specific role in adipogenesis and obesity remains largely unexplored. In this study, the potential of hDPSC-Exos to regulate adipogenesis and elucidate the underlying mechanisms was investigated. Our findings demonstrated that hDPSC-Exos significantly reduced lipid accumulation during 3T3-L1 adipocyte differentiation. This reduction was accompanied by downregulation of key adipogenic markers, including \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003eC/ebpα\u003c/em\u003e, \u003cem\u003eSrebp-1c\u003c/em\u003e, \u003cem\u003eAdipoq\u003c/em\u003e, \u003cem\u003eaP2, CD36\u003c/em\u003e, and \u003cem\u003ePlin2\u003c/em\u003e. Further analysis revealed that continuous and early exposure to hDPSC-Exos during the differentiation period was most effective in suppressing adipogenic marker expression. These findings suggest that hDPSC-Exos exert their anti-adipogenic effects primarily during the early stages of adipocyte differentiation, likely by modulating preadipocyte signaling pathways involved in lineage commitment and differentiation.\u003c/p\u003e\u003cp\u003eTo investigate the molecular mechanisms by which hDPSC-Exos regulate adipocyte differentiation in 3T3-L1 cells, RNA sequencing was performed on cells treated with or without hDPSC-Exos during adipogenesis. The results showed that hDPSC-Exos treatment significantly reversed the expression of numerous genes associated with adipocyte differentiation. GO analyses revealed that these DEGs were predominantly enriched in lipid metabolic processes. This enrichment underscores the critical role of lipid metabolism in adipocyte function and suggests that hDPSC-Exos inhibit the metabolic adaptations typically involved in adipogenesis.\u003c/p\u003e\u003cp\u003eIn this study, ORA further revealed that genes associated with EMT, including \u003cem\u003eCxcl5\u003c/em\u003e, \u003cem\u003eTsp2\u003c/em\u003e, \u003cem\u003eHtra1\u003c/em\u003e, and \u003cem\u003eMfap5\u003c/em\u003e, were significantly altered in 3T3-L1 cells following hDPSC-Exos treatment. For instance, CXCL5 inhibits adipocyte differentiation by reducing the transcriptional activity of PPARγ\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. TSP2 inhibits long-chain fatty acid uptake by binding to CD36\u003csup\u003e33\u003c/sup\u003e. HTRA1 suppresses adipogenesis in human mesenchymal stem cells by promoting the production of matrix metalloproteinases\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Similarly, MFAP5 downregulates the expression of downstream molecules associated with Pparγ by inhibiting Staphylococcal nuclease and Tudor domain containing 1 (SND1) expression, a novel coactivator of Pparγ\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Notably, the enrichment of these upregulated genes upon hDPSC-Exos treatment suggests a mechanism that not only inhibits adipogenesis but also promotes alternative cellular processes during 3T3-L1 differentiation.\u003c/p\u003e\u003cp\u003eFurthermore, proteomic analysis of hDPSC-Exos identified 202 differentially expressed proteins associated with extracellular matrix and structural organization. Several of these proteins, including ITGBL1, AEBP1, PTN, and Timp1, have previously been implicated in modulating adipocyte differentiation. Specifically, ITGBL1 interacts with Wnt receptors and activates c-Jun through a non-canonical pathway, potentially influencing signaling pathways involved in adipogenesis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. AEBP1 also binds to Wnt receptors and activates β-catenin, thereby potentially enhancing adipogenic signaling\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Additionally, PTN activates β-catenin by binding to the receptor protein tyrosine phosphatase (RPTP) beta/zeta receptor and inhibiting glycogen synthase kinase 3 beta (GSK-3β)\u003csup\u003e23\u003c/sup\u003e, while Timp1 directly activates β-catenin in a matrix metalloproteinase-independent manner\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe Wnt signaling pathway, particularly the β-catenin cascade, plays a crucial role in regulating mesenchymal cell fate and differentiation. In preadipocytes, activation of this pathway inhibits adipogenesis by downregulating key adipogenic transcription factors such as \u003cem\u003ePparγ\u003c/em\u003e and \u003cem\u003eC/ebpα\u003c/em\u003e\u003csup\u003e36\u003c/sup\u003e. Our findings demonstrate that hDPSC-Exos treatment upregulated total and active β-catenin expression under adipogenic differentiation conditions, indicating that specific cargo proteins within hDPSC-Exos may activate the Wnt/β-catenin pathway. It was further confirmed that Timp1, a protein cargo within hDPSC-Exos, inhibits adipogenesis via β-catenin upregulation. These findings elucidate the mechanism by which hDPSC-Exos modulate adipocyte differentiation through specific signaling pathways and protein interactions. Collectively, this study suggests that hDPSC-Exos might serve as a novel therapeutic strategy for preventing obesity by targeting molecular mechanisms involved in adipogenesis.\u003c/p\u003e\u003cp\u003eThe diet-induced obesity (DIO) mouse model is a well-established polygenic model that closely mimics many aspects of human obesity. In this study, it was demonstrated that hDPSC-Exos treatment significantly reduced key obesity-related parameters, including body weight, fat mass, and gWAT weight in HFD-fed mice. Furthermore, hDPSC-Exos treatment inhibited adipose tissue maturation and consistently downregulated the expression of lipogenesis- and lipid transport-related genes in the gWAT of the HFD-fed mice. These in vivo results corroborate our in vitro findings and collectively suggest that hDPSC-Exos concurrently suppress \u003cem\u003ede novo\u003c/em\u003e lipogenesis and lipid accumulation. This dual mechanism underscores their therapeutic potential for preventing and treating obesity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first evidence that hDPSC-Exos can effectively inhibit adipogenesis and lipogenesis \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. While several candidate protein cargos within hDPSC-Exos that exert inhibitory effects during preadipocyte differentiation were identified, further research is warranted to elucidate the complex molecular pathways involved in this anti-obesity mechanism. These findings offer valuable insights into the development of novel cell-free therapeutic strategies for preventing and treating obesity and its associated metabolic disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e(hDPSCs) : human dental pulp stem cells, MSCs : mesenchymal stem cells, hDPSC-Exos : hDPSC-derived exosomes, ALP : Alkaline Phosphatase, ARS : Alizarin Red S, SD : Standard Diet, HFD : High Fat Diet, gWAT : gonadal White Adipose Tissue,\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) (no. 2019R1A5A2027521, 2021R1C1C1009601, RS-2023-00247674, RS-2025-00514223, RS-2025-24536036), Chonnam National University Hospital Biomedical Research Institute (BCRI25084) and by the Technology Innovation Program (RS-2024-00406097, Development of customized hydrogel support and manufacturing technology for aggregate production equipped with dental pulp stem cell-derived materials) funded by the Ministry of Trade Industry \u0026amp; Energy (MOTIE, Korea).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.H.K: conceptualization, formal analysis, investigation, writing \u0026ndash; original draft. H.K.Y and S.E.K: investigation, visualization. M.G.J.: provided human tissue, data analysis. B.R.L: data analysis. Y.K.: conceptualization, supervision, review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq dataset generated during the current study has been deposited in the NCBI Gene Expression Omnibus (GEO) database and is accessible through the accession number GSE298678. All other data generated or analyzed during this study are included in this published article and its supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee of Chonnam National University (Approval number: YB-2024-214; Date of approval: January 22,\u0026nbsp;2025, Title:\u0026nbsp;Investigation of the\u0026nbsp;anti-obesity effects of human dental pulp stem cell-derived exosomes and their exosomal miRNA in a high fat diet induced animal model).\u003c/p\u003e\n\u003cp\u003eHuman dental pulp tissues were obtained from supernumerary teeth extracted from seven healthy pediatric donors. This study was approved by the Ethics Committee of Chonnam National University Hospital (Approval number: CNUH-2025-092; Date of approval: April 07, 2025, Title: The inhibitory effects of dental pulp cell-derived exosomes in adipocyte differentiation), and written informed consent was obtained from each patient\u0026apos;s legal guardian.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBluher, M. Obesity: global epidemiology and pathogenesis. \u003cem\u003eNat Rev Endocrinol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 288-298, doi:10.1038/s41574-019-0176-8 (2019).\u003c/li\u003e\n\u003cli\u003eKumar, S. \u0026amp; Kelly, A. S. 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Adipogenesis and WNT signalling. \u003cem\u003eTrends Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 16-24, doi:10.1016/j.tem.2008.09.002 (2009).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"human dental pulp stem cells, exosomes, adipogenesis, obesity, beta-catenin","lastPublishedDoi":"10.21203/rs.3.rs-8023093/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8023093/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity is a critical global health challenge linked to cardiovascular disease, type 2 diabetes, metabolic syndrome, and cancers. Current therapies often lack sustained efficacy, prompting interest in molecular strategies targeting adipogenesis. Exosomes, cell-derived extracellular vesicles, exhibit therapeutic potential due to high biocompatibility and ability to mediate intercellular communication. This study explored the anti-adipogenic effects of exosomes derived from human dental pulp stem cells (hDPSC-Exos). hDPSC-Exos were isolated from conditioned media and characterized by TEM, nanoparticle tracking analysis, and Western blotting. Their anti-adipogenic effects were evaluated in vitro using 3T3-L1 and OP9 preadipocytes via Oil Red O staining, RT-qPCR, western blotting and multi-omics analyses (RNA-seq, proteomics). Therapeutic efficacy was further validated in a diet-induced obesity mouse model. Treatment with hDPSC-Exos markedly suppressed adipocyte differentiation in vitro, reducing lipid accumulation and downregulating key adipogenic transcription factors and their downstream targets. Mechanistically, Wnt/β-catenin signaling activation mediated this inhibition. In vivo, hDPSC-Exos administration reduced body weight, fat mass, and adipogenic gene expression in obese mice. These findings reveal a novel regulatory function of hDPSC-Exos in adipogenesis, highlighting their potential as a cell-free therapy for obesity and related metabolic disorders.\u003c/p\u003e","manuscriptTitle":"Human dental pulp stem cell-derived exosomes inhibit adipogenesis and obesity by activating Wnt/β-catenin signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-14 16:43:14","doi":"10.21203/rs.3.rs-8023093/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b5f92d55-9d3f-4c85-986a-9b1614d9dad0","owner":[],"postedDate":"December 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":59320398,"name":"Biological sciences/Stem cells/Mesenchymal stem cells"},{"id":59320399,"name":"Health sciences/Medical research/Stem-cell research"}],"tags":[],"updatedAt":"2026-01-22T18:45:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-14 16:43:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8023093","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8023093","identity":"rs-8023093","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}