Research ArticleExperimental Studies
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Proteomic and Metabolomic Analyses of Adipose-derived Mesenchymal Stem Cell Exosomes and Culture Supernatants

TOYOFUMI HIRAKAWA, TAMOTSU KATO, YUMIKO NAKANISHI, DAICHI URUSHIYAMA, KOHEI MIYATA, TSUKASA BABA, HIROSHI OHNO, SHIN’ICHIRO YASUNAGA, FUSANORI YOTSUMOTO and SHINGO MIYAMOTO
Anticancer Research August 2025, 45 (8) 3409-3424; DOI: https://doi.org/10.21873/anticanres.17703

Abstract

Background/Aim: Aberrant differentiation of fertilized eggs during in vitro fertilization is a major contributor to infertility, and adipose-derived mesenchymal stem cells (ASCs) and their exosomes have been reported to facilitate fertilized egg differentiation; however, the underlying mechanisms remain unclear. This study aimed to elucidate how ASC-derived exosomes promote fertilized egg differentiation through proteomic analysis of ASC-derived exosomal proteins and metabolomic profiling of culture supernatants.

Materials and Methods: Extracellular vesicles were isolated from ASC culture supernatants via differential ultracentrifugation, proteins were extracted and digested using a phase-transfer surfactant protocol for LC-MS/MS analysis, and metabolites were analyzed using GC-MS/MS following extraction and derivatization.

Results: Exosomes from early passage ASCs were enriched with various proteins, including alpha and beta proteasome subunits, which exhibit proteasome activity. Gene ontology analysis revealed the presence of proteins associated with transmembrane transport and cytoplasmic translation. Metabolomic profiling of culture supernatants revealed markedly elevated levels of amino acids associated with glycine, serine, and methionine metabolism.

Conclusion: ASC-derived culture supernatants can exert differentiation-promoting effects on fertilized eggs via exosomal proteins and soluble metabolites and may therefore be a novel option for infertility treatment.

Keywords:

Introduction

The incidence of age-associated infertility is increasing owing to societal factors, such as delayed marriage and increasing maternal age at conception. Assisted reproductive technology (ART) encompasses all interventions involving in vitro manipulation of human oocytes, sperm, or embryos for reproductive purposes and constitutes a fundamental aspect of infertility treatment (1). The utilization of ART procedures, including in vitro fertilization (IVF) and embryo transfer, has increased with the increasing incidence of infertility (2). However, the success rate of in vitro embryo development remains suboptimal, necessitating the development of novel strategies to enhance embryonic viability. Hence, various growth factors have been investigated for their potential to promote embryonic development. The supplementation of culture media with epidermal growth factor (EGF) enhances embryonic differentiation rates (3). In addition, insulin-like growth factor-I (IGF-I) increases cell proliferation and improves embryonic differentiation by inhibiting apoptosis (4, 5). Intraovarian administration of platelet-rich plasma (PRP) may enhance chromosomal normality in fertilized eggs (6). However, the efficacy of these factors remains unclear, highlighting the need for further studies.

Mesenchymal stem cells (MSCs) are multipotent stem cells residing in mesoderm-derived connective tissues and are key cellular resources for tissue repair and regeneration. They reside in all tissues of the body and have been successfully isolated from various sources, including the bone marrow, adipose tissue, umbilical cord, cord blood, amniotic fluid, placenta, and dental pulp (7-12). They have garnered significant attention in regenerative medicine and cell therapy because of their capacity to facilitate post-injury tissue repair. They are more suitable for clinical applications than induced pluripotent stem cells (iPSCs) because of their lower tumorigenic potential, ease of production, and relatively low cost. Their therapeutic potential is increasingly recognized, particularly in the treatment of gynecological infertility, including the regeneration of thin endometrium and restoration of aging-associated impaired ovarian function (13, 14). MSC therapy is promising for treating conditions such as endometriosis, thin endometrium, age-related ovarian dysfunction, and impaired fertilized egg differentiation (13-17). However, MSC therapy is associated with potential adverse effects, including microvascular occlusion (18) and the risk of transplanted cells undergoing inappropriate differentiation or malignant transformation (19). Additionally, challenges, such as the long cell processing duration and high costs, further limit its clinical implementation. To overcome these limitations, MSC-derived exosome-based regenerative therapies have emerged as promising alternatives.

MSC-derived exosomes exhibit therapeutic effects comparable with those of MSCs (20). Exosomes, approximately 100 nm in diameter, are lipid bilayer-enclosed vesicles, secreted by cells (21). They mediate intercellular communication and facilitate the transfer of proteins and genetic materials such as microRNAs, mRNAs, and DNA between cells, thereby modulating recipient cell characteristics and regulating biological processes (22). Furthermore, exosome-based treatments do not pose the risk of microvascular complications or aberrant cell differentiation as MSC-based therapies do. Moreover, MSC-derived exosomes can be preserved for long term through cryopreservation or lyophilization, making them more accessible and cost effective (23). Consequently, MSC-exosome therapy is a potentially safer, more economical, and more efficacious alternative to conventional MSC-based cell therapies. Following uptake by recipient cells, exosomes release miRNAs and mRNAs that can be translated into novel proteins or regulate specific gene expression. Certain proteins and lipids within exosomes may also activate intracellular signaling pathways, thereby influencing cellular behavior, metabolism, and differentiation. Consequently, exosomes modulate the function of recipient cells through multiple mechanisms. The component microRNAs are key mediators of the physiological effects of exosomes (24-28). However, miRNAs within exosomes may not reach biologically relevant concentrations, and their effects may, therefore, remain uncertain. Conversely, proteins within MSC-derived exosomes play pivotal roles in a variety of critical biological processes, including cell communication, structural integrity, inflammation, exosome biogenesis, development, tissue repair, regeneration, and metabolism (29). Similar to the miRNAs, the exosomal proteins can regulate numerous biological processes involved in disease pathogenesis and tissue repair and regeneration.

We reported the embryogenesis-enhancing effects of adipose tissue-derived mesenchymal stem cells (ASCs), a subclass of MSCs, and observed that the co-culture of early passage ASCs with fertilized mouse oocytes using 400 nm pore-size transwells significantly enhanced the differentiation rate of fertilized eggs (15). The exosomes from early passage ASCs were enriched with miR-127, which is implicated in early embryonic and placental development, and miR-181 and miR-200, which are associated with antioxidant effects (15). These findings suggested that the microRNA content of ASC-derived exosomes may influence their role in promoting fertilized egg differentiation. Although both microRNAs and exosomal proteins contribute to this biological activity, research has primarily focused on the role of microRNAs, while the functions of other exosomal components, such as proteins, lipids, other nucleic acids (e.g., mRNAs or DNA fragments), and the water-soluble metabolites in ASC culture supernatants, remain largely unexplored.

Hence, this study aimed to identify factors other than exosome-derived miRNAs that facilitate the differentiation of fertilized mouse eggs by conducting a proteomic analysis of ASC-derived exosomes and a comprehensive metabolomic assessment of water-soluble metabolites in adipose stem cell culture supernatants.

Materials and Methods

Collection of cell culture supernatants for exosome isolation. The cell culture supernatants were collected according to a previously established protocol (15). Briefly, to obtain supernatants from early passage ASCs (ASC-P3: third passage) and late-passage ASCs (ASC-P20: twentieth passage), ASCs were cultured in 10 ml of MEMα/GlutaMax medium (Gibco, Grand Island, NY, USA). supplemented with 10% Exo-FBS (System Biosciences, Palo Alto, CA, USA), and 1% antibiotic-antimycotic (Gibco) on 100 mm culture plates under controlled conditions of 37°C and 5% CO2. After 48 h of incubation, the supernatants were carefully collected and filtered through a 0.22 μm membrane to remove detached cells and particulate debris (N=3).

Isolation of extracellular vesicles (EVs). EVs were isolated from cell culture supernatants using differential ultracentrifugation and a sucrose cushion (30). Briefly, the cell culture supernatant was centrifuged at 300 × g for 10 min to remove large debris. Thereafter, the supernatants were passed through a 0.22 μm spin filter (Agilent Technologies, Santa Clara, CA, USA) and centrifuged on a 30% sucrose/D2O cushion at 100,000 × g for 90 min. The collected cushion was subsequently ultracentrifuged twice at 100,000 × g for 70 min, and the supernatants were removed.

Sample preparation for liquid chromatography with tandem mass spectrometry (LC-MS/MS). Protein extraction and proteolytic digestion were performed using a phase-transfer surfactant (PTS) protocol (31). Each sample was dissolved in PTS buffer, transferred into a 1.5 mL tube, and heated at 95°C for 5 min. The samples were then treated with 10 mM TCEP for reduction, followed by alkylation with 20 mM iodoacetamide. Protein purification was carried out using the SP3 protocol (32). The purified protein was enzymatically digested with trypsin and Lys-C at a ratio of 1:50 (enzyme to protein) for 16 h at 37°C. The resulting peptides were desalted and purified using C18 StageTips (33), dried in a SpeedVac, and reconstituted in 0.1% formic acid with 2% acetonitrile.

LC-MS/MS analyses. LC-MS/MS analysis was conducted by integrating an UltiMate 3000 Nano LC system (Thermo Scientific, Bremen, Germany) with an HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland). Sample injection was facilitated using a trap column (0.075×20 mm, Acclaim PepMap RSLC Nano-Trap Column; Thermo Fisher Scientific), and peptide ionization was achieved via nano-electrospray ionization (ESI) in positive ion mode using a nano LC-MS interface (AMR, Tokyo, Japan). The ionized peptides were subsequently analyzed on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific). Separation was performed using an analytical column (75 μm × 30 cm) packed in-house with ReproSil-Pur C18-AQ resin (1.9 μm, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 280 nl/min. A 105-min gradient was applied, increasing solvent B from 5% to 30% (solvent A: 0.1% FA and 2% acetonitrile; solvent B: 0.1% FA and 90% acetonitrile). The Q Exactive instrument was operated in data-independent acquisition (DIA) mode. Full mass spectra were recorded across a mass range of 398-802 m/z with the following settings: resolution of 140,000, auto gain control (AGC) target of 3×106, maximum injection time of 200 ms, and normalized collision energy of 27. MS2 spectra were obtained using an 8 m/z isolation window at a resolution of 70,000, AGC target of 3×106 ions, maximum injection time set to “auto” (overlapping window patterns), and normalized collision energy of 27.

Data processing and visualization of LC-MS/MS analyses. Raw MS data were analyzed using DIA-NN software (version 1.7.12). The database search encompassed all entries from the Homo sapiens UniProt database, along with known contaminants (34). Additionally, a decoy database was generated by appending reversed sequences of all proteins. The search parameters were set as follows: allowance for up to two missed cleavage sites, peptide length restricted to 7-30 amino acids, carbamidomethylation of cysteine residues (+57.021 Da) designated as a fixed modification, protein grouping based on FASTA-derived names, robust LC (high precision) selected as the quantification strategy, and cross-run normalization performed in an RT-dependent manner. Precursor ion filtering was applied using a 1% false discovery rate (FDR).

Collection of cell culture supernatants for analysis of metabolites. The cell culture supernatants were collected as described previously (15). In summary, Transwell filters (Greiner Bio-One, 24-well, 0.4 μm pore size) were used to separate the upper and lower chambers for nanoparticle transport studies. ASCs were plated in the upper chamber at a density of 1×105 cells per well in 100 μl of MEMα/GlutaMax medium supplemented with 10% Exo-FBS and 1% antibiotic-antimycotic solution. Simultaneously, frozen two-cell embryos were cultured in the lower chamber containing 600 μl of potassium simplex optimized medium (KSOM) (ARK Resource) under controlled conditions of 37°C and 5% CO2. The medium in both chambers was replaced 48 h after the initiation of fertilized egg culture. Supernatants from the lower chamber were collected 48 and 72 h after incubation for subsequent metabolite analysis. Frozen two-cell embryos cultured in KSOM without a Transwell filter served as the KSOM control (KSOM-C), whereas the wells without cells in the upper chamber served as the control group. ASCs at early (third) and late (twentieth) passages seeded in the upper chamber were categorized into ASC-P3 and ASC-P20 groups, respectively. Supernatants collected at 48 and 72 h were labeled with the suffix “-48h” and “-72h,” respectively. The KSOM without cell culture was designated as the KSOM group. All the experimental groups were set at n=5.

Metabolomic analysis. For the extraction and measurement of hydrophilic metabolites and SCFAs in fecal and medium samples, we followed the previously described hydrophilic metabolite extraction and measurement methods (35), with some modifications. Briefly, 50 μl aliquot of medium was extracted using 5:5:2 methanol:water:chloroform. The extracted solution was centrifugally filtered through a 5 kDa cutoff filter (UltrafreeMC-PLHCC 250/pk, Human Metabolome Technologies) to remove proteins. The filtrate was evaporated to dryness using a vacuum evaporator and lyophilized using a freeze dryer. The dried extract was derivatized prior to gas chromatography–tandem mass spectrometry (GC-MS/MS) analysis. SCFA extraction and measurement were then performed as previously described (35), with some modifications. A 90 μl aliquot of medium was added to 10 μl Milli-Q water containing internal standards (2 mM [1,2-13C2] acetate, 2 mM [2H7] butyrate, and 2 mM crotonate). The solutions were extracted and derivatized before GC-S/MS analysis. The GC-MS/MS analysis program was set as described previously (35). The analysis was performed using a GC-MSplatform on a Shimadzu GCMS-TQ8040 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a capillary column (BPX5) (SGE Analytical Science). The GC-MS/MS data were processed, and concentrations were calculated using LabSolutions Insight (Shimadzu Corporation, Kyoto, Japan). Enrichment analysis of the metabolite data was performed using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca).

Data availability. The datasets produced and/or examined in this study can be obtained from the corresponding author upon reasonable request.

Results

To provide a comprehensive overview of the experimental design, a schematic workflow of the study is presented in Figure 1. Proteomic and metabolomic analyses were performed on different experimental systems. Proteomic analysis was conducted on extracellular vesicles (EVs) isolated from the conditioned medium of ASC monocultures (P3 and P20). Meanwhile, metabolomic profiling was performed using the supernatant from transwell co-cultures of ASCs and fertilized oocytes, to better reflect the embryo–ASC interaction environment.

Figure 1.
Figure 1.

Experimental workflow. Adipose-derived mesenchymal stem cells (ASCs) at early (P3) and late (P20) passages were cultured in medium supplemented with 10% Exo-FBS. For proteomic analysis, conditioned medium from ASC monocultures was collected and subjected to ultracentrifugation to isolate extracellular vesicles (EVs), which were analyzed by LC-MS/MS. In parallel, for metabolomic analysis, ASCs were co-cultured with fertilized embryos in a transwell system, and the supernatant from the co-culture was analyzed using GC-MS.

Proteomic analysis of ASC exosomes. The average number of identified proteins per sample (mean±SD, N=3) was 1,613±53 in ASC-P3 and 1,402±5 in ASC-P20 exosome samples. The ASC-P3 and ASC-P20 exosomes differed in protein composition, and the E-ASC exosomes were enriched in 131 different proteins (Figure 2A), including proteins with proteasomal activity, such as alpha and beta proteasome subunits. Gene ontology analysis showed that the proteins significantly contained in the exosomes of ASC-P3 were associated with the following terms: ‘Ion transmembrane transport’, ‘Transmembrane transport’, ‘Anion transmembrane transport’, and ‘Cytoplasmic translation’ for biological processes; ‘Structural constituents of the ribosome’, ‘Transmembrane transporter activity’, and ‘Organic anion transmembrane transporter activity’ for molecular functions; and ‘Intrinsic component of plasma membrane’, ‘Integral component of plasma membrane’, ‘Cytosolic ribosome’, and ‘Ribosomal subunit’ for cellular components (Figure 2B).

Figure 2.
Figure 2.
Figure 2.

Characteristics of proteins highly expressed in the exosomes of early passage of adipose-derived mesenchymal stem cells (ASCs). (A) Volcano plot of expression levels of proteins in early passage ASC-derived exosomes and late-passage ASC-derived exosomes. Red dots are those expressed highly in early passage ASC-derived exosomes, and blue dots represent those highly expressed in late-passage ASC-derived exosomes. (B) Characterization of proteins upregulated in the early passage ASC-derived exosomes compared with their expression in the late passage ASCs.

Metabolomic analysis of ASC supernatant. Principal component analysis showed that the composition of water-soluble metabolites differed between groups (Figure 3A). The average number of identified metabolites per sample (mean±SD, n=5) was 133.4±1.8 in ASC_P3_48h, 133.0±1.9 in ASC_P3_72h, 134.8±1.1 in ASC_P20_48h, 132.2±1.3 in ASC_P20_72h, 127.8±2.6 in Control_48h, 125.6±2.4 in Control_72h, 82.6±1.9 in KSOM_0h, 94.0±6.3 in KSOM_C_48h, and 86.4±2.2 in KSOM_C_72h. A heat map analysis was performed on 50 representative water-soluble metabolites that differed in content between the groups (Figure 3B). Putrescine, 4-aminobultynic acid, xylulose, glucuronic acid, ribonolactone, hypotaurine, malic acid, ribose, and xylitol were more abundant in the ASC-P3 group than in the other groups. In addition, 4-hydroxyphenyllactic acid, 2-ketoglutanic acid, 2-aminopimelic acid, citric acid, isovalerate, propionate, 3-hydroxyisobutyric acid, galactose, 2-ketoisovaleric acid, 2-ketoisocaproic acid, and isobutyrate were more abundant in ASC-P20 than in the other groups (Figure 3B).

Figure 3.
Figure 3.

Characteristics of metabolites in cell culture supernatants of ASCs. (A) Principal component analysis plot showing overall clustering of metabolomic profiles. Each color represents a specific experimental group, as indicated in the plot legend. (B) Heat map of the top 50 proteins with significant differences. ASC-P3: 3rd (Early) passage of ASCs; ASC-P20: 20th (Late) passage of ASCs.

Next, the metabolites significantly enriched in the ASC-P3 group were examined, and 20 metabolites, including putrescine, 2-ketoisocaproic acid, and 2-hydroxyisocaproic acid, were found more abundant in ASC-P3-48h than in the control group. Enrichment analysis showed that ASC-P3-48h was enriched with glycine and serine metabolism, methionine metabolism, phenylalanine and tyrosine metabolism, homocysteine degradation, and cysteine metabolism (Figure 4A). Eighteen metabolites, including cytosine, 3-hydroxyisobutyric acid, 4-aminobutyric acid, propionate, xylitol, and putrescine, were more abundant in the ASC-P3-72h group than in the control group. Enrichment analysis showed that the metabolites were involved in aspartate metabolism, urea cycle, glycine and serine metabolism, ammonia recycling, and arginine and proline metabolism (Figure 4B).

Figure 4.
Figure 4.
Figure 4.
Figure 4.
Figure 4.

Metabolites elevated in supernatants co-cultured with adipose stem cells (ASCs) and their characteristics. (A) ASC-P3-48h vs. control. (B) ASC-P3-72h vs. control. (C) ASC-P20-48h vs. control. (D) ASC-P20-72h vs. control. In all volcano plots, red dots indicate significantly up-regulated metabolites in ASC-derived samples compared to control, whereas blue dots indicate significantly down-regulated metabolites. ASC-P3: 3rd (Early) passage of ASCs; ASC-P20: 20th (Late) passage of ASCs.

In addition, the metabolites significantly enriched in the ASC-P20 group were examined. A total of 22 metabolites, including putrescine, isovalerate, 3-hydroxyisobutyric acid, 4-aminobutyric acid, isobutyrate, and citric acid, were more abundant in the ASC-P20-48h group than in the control group. Enrichment analysis showed that ASC-P20-48h was enriched with glycine and serine metabolism, aspartate metabolism, urea cycle, ammonia recycling, and methionine metabolism (Figure 4C). Propionate, 2-ketoisocaproic acid, citric acid, and 4-aminobutyric acid were more abundant in ASC-P20-72 h than in the control group. Enrichment analysis showed that the following pathways were also present: glycine and serine metabolism, urea cycle, aspartate metabolism, ammonia recycling, and methionine metabolism (Figure 4D).

Discussion

In this study, we investigated the potential mechanisms through which adipose-derived stem cell culture supernatants promote fertilized egg differentiation by analyzing the proteomic profile of exosomal proteins and the metabolomic composition of water-soluble metabolites in the supernatants of ASCs culture medium. These findings suggest that the differentiation-promoting effects of adipose stem cell culture supernatants may result from the synergistic interplay of multiple bioactive components within exosomes, including miRNAs, proteins, and water-soluble metabolites. Ei et al. similarly demonstrated the therapeutic potential of stem cell-derived exosomes in reproductive systems, reinforcing the relevance of exosome-mediated paracrine effects in embryonic development (36).

MSCs have garnered significant attention for their potential applications in regenerative medicine (37). MSC-based cell therapies have hence been extensively studied for a range of diseases, including graft-versus-host disease, and have demonstrated efficacy in tissue functional restoration (38-40). Potential applications of MSC therapy are expanding in the fields of obstetrics and gynecology. Female infertility can arise from factors related to the ovaries, fallopian tubes, uterus, or immune system. In the present study, we found that MSC exosomes were enriched in proteasome-active proteins, which likely contribute to the regulation of cellular functions, such as proteostasis maintenance and signal transduction, through the selective degradation of both aberrant and redundant functional proteins. The activation of transmembrane proteins may also facilitate signaling responses and induce structural and functional alterations within and outside the cell.

Exosomes have been implicated in a wide array of biological processes, including cancer development and progression. For instance, exosomes contribute to the metastasis and progression of pancreatic and breast cancers (41, 42). Moreover, exosomes released by stem cells under oxidative stress conditions promote breast cancer cell proliferation by inducing the expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition-related markers (43). Exosomes may also be involved in resistance to anticancer therapies. Biliary tract cancer cell lines acquire resistance to gemcitabine via exosome-mediated transfer of miR-141-3p (44). Conversely, exosomes have potential therapeutic applications in cancer. For example, exosomes derived from adipose stem cells inhibit skin cancer progression via the miR-199a-5p/SOX4 signaling pathway (45). In addition, exosomes derived from cancer-associated fibroblasts, particularly those positive for CD9, inhibit malignant melanoma cell growth (46). Furthermore, melatonin suppresses gastric cancer cell proliferation by downregulating miR-27b-3p in exosomes (47). Thus, existing research underscores the dual role of exosomes in cancer progression and therapeutic intervention.

Amino acids are organic compounds characterized by the presence of one or more amino and carboxyl groups. They serve not only as integral components of proteins but also as precursors for the synthesis of various nitrogen-containing compounds and as substrates for energy production, contributing to the formation of sugars and lipids. Our analysis revealed a diverse array of amino acids was identified in the ASC culture supernatant. Notably, gene ontology analysis revealed significantly elevated levels of amino acids involved in glycine, serine, and methionine metabolism. Although our study focused on embryo-supportive metabolic changes in vitro, similar regenerative effects of ASC-derived components have been demonstrated in vivo. For example, Mori et al. reported that human adipose-derived stem cells improved defecation function and sphincter tissue regeneration in a mouse model of anal sphincter injury, suggesting that ASC-secreted factors may exert tissue-supportive effects through diverse mechanisms (48). These findings support the broader relevance of ASC-based interventions in both reproductive and non-reproductive regenerative contexts. These amino acids are involved in proliferation, metabolism, and differentiation of pluripotent stem cells. Serine and glycine exhibit reversible interconversion and play crucial roles in the epigenetic regulation of stem cells through the carbon metabolic pathway. In undifferentiated cells, LIN28 enhances serine synthesis from glucose and its conversion to S-adenosylmethionine, thereby contributing to the maintenance of the undifferentiated state (49). Methionine in the culture medium of mouse small intestinal organoid cells reduces undifferentiated marker expression and promotes cellular differentiation (50).

The findings of this study indicate that cell culture supernatants derived from ASCs, enriched with exosomes, hold promise for application in assisted reproductive technologies, particularly in vitro fertilization. However, several critical challenges must be addressed to facilitate clinical translation. One major obstacle in therapies based on stem cell-derived exosomes is the insufficient assessment of their safety, biological properties, and optimal dosage. For instance, impurities or unanticipated components in exosome preparations may provoke severe immune responses, including allergic reactions or graft rejection. Moreover, the characteristics of exosomes vary depending on their cellular origin and extraction process, posing significant challenges in ensuring batch-to-batch consistency and quality control. The biological functions of MSC-derived exosomes differ based on their source, despite shared fundamental properties. Notably, exosomes from adipose tissue-derived MSCs exhibit superior angiogenic potential compared to those derived from bone marrow (51). Conversely, exosomes from bone marrow-derived MSCs demonstrate stronger immunomodulatory and anti-inflammatory effects (52). Consequently, efforts have been made to isolate homogeneous exosome populations using immortalized cell lines; however, a standardized methodology has yet to be firmly established (53). Therefore, the optimal source of MSC-derived exosomes for therapeutic applications may vary depending on the specific target disease, necessitating meticulous evaluation and strategic selection. Furthermore, comprehensive data on the long-term effects of exosome-based therapies remain limited, and concerns regarding their safety and potential risks persist. To ensure the safe and effective clinical implementation of exosomes, rigorous control over the manufacturing process, establishment of stringent quality assessment criteria, and extensive longitudinal clinical studies are imperative.

Thus, miRNAs, proteins, and water-soluble metabolites present in ASC-derived exosomes may contribute to the differentiation of fertilized eggs. By elucidating the underlying mechanisms, this effect may be replicated by adding specific active compounds to the cell culture supernatants, thus obviating the need for co-culture. Furthermore, addressing future challenges related to the therapeutic use of cell culture supernatants necessitates a thorough investigation of safety concerns and optimal dosage parameters.

Conclusion

ASCs may promote fertilized egg differentiation by the action of proteins and soluble metabolites contained within exosomes. Although the precise underlying mechanisms remain to be elucidated, various factors, including miRNAs, proteins, and water-soluble metabolites within exosomes collaboratively contribute to fertilized egg differentiation in a complex and multifaceted manner.

Acknowledgements

We would like to thank Editage (www.editage.com) for the English language editing.

Footnotes

  • Authors’ Contributions

    Conceptualization: Toyofumi Hirakawa and Shingo Miyamoto; Resources and Data curation: Toyofumi Hirakawa, Daichi Urushiyama, Kohei Miyata, and Fusanori Yotsumoto; Formal analysis: Toyofumi Hirakawa, Tamotsu Kato, and Yumiko Nakanishi; Writing - original draft: Toyofumi Hirakawa and Shingo Miyamoto; Critical revision of the article for important intellectual content: Toyofumi Hirakawa, Tsukasa Baba, Hiroshi Ohno, Shinichiro Yasunaga, Fusanori Yotsumoto, and Shingo Miyamoto; Final approval of the version to be published: Toyofumi Hirakawa and Shingo Miyamoto. All Authors read and approved the final version of the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Funding

    This work was supported in part by grants-in-aid from the Kakihara Science and Technology Foundation, Fukuoka, Japan (grant number 230340); Seiichi Imai Memorial Foundation, Fukuoka, Japan (grant number 220469); Medical Care Education Research Foundation, Fukuoka, Japan (grant number 220530); Grant of the Clinical Research Promotion Foundation, Fukuoka, Japan (grant number 220362); Kaibara Morikazu Medical Science Promotion Foundation, Fukuoka, Japan (grant number 220553); and from the Scholarship Fund for Young Researchers, Tokyo, Japan (grant number G23001) and a funding from Fukuoka University (grant number 226004), all of which were awarded to T. Hirakawa.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received May 23, 2025.
  • Revision received June 13, 2025.
  • Accepted June 18, 2025.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Proteomic and Metabolomic Analyses of Adipose-derived Mesenchymal Stem Cell Exosomes and Culture Supernatants
TOYOFUMI HIRAKAWA, TAMOTSU KATO, YUMIKO NAKANISHI, DAICHI URUSHIYAMA, KOHEI MIYATA, TSUKASA BABA, HIROSHI OHNO, SHIN’ICHIRO YASUNAGA, FUSANORI YOTSUMOTO, SHINGO MIYAMOTO
Anticancer Research Aug 2025, 45 (8) 3409-3424; DOI: 10.21873/anticanres.17703
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