Human Wharton's jelly, placenta, umbilical cord blood and adipose tissue samples were obtained from women after they had given birth at Samsung Medical Center (Seoul, South Korea) between September 2016 and September 2024, and were used to isolate MSCs. Each type of tissue was obtained from one individual. All participants provided written informed consent, and the procedures were approved by the Institutional Review Board of Samsung Medical Center (approval no. 2016-07-102-001; Seoul, South Korea). AD-MSCs were isolated from adipose tissues according to a previously reported method (14). Placenta-derived MSCs were isolated from the placenta, following a previously described method (15). Umbilical cord blood-derived MSCs were isolated from umbilical cord blood, as previously reported (16). Wharton's jelly-derived MSCs (WJ-MSCs) were isolated from the separated umbilical cord, as previously described (17). Human bone marrow-derived MSCs (cat. no. PT-2501) were purchased from Lonza Group, Ltd. All MSCs were cultured in α-modified minimum essential medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin (all from Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ incubator. MSCs at passage 7 were used in the experiments.

Cytotoxicity was measured using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.), according to the manufacturer's instructions. AD-MSCs were seeded at a density of 1×10³ cells/well in 96-well culture plates and were incubated overnight. AD-MSCs were treated with different concentrations of NA (0, 40, 80, 120, 160, 200, 240 and 280 μM) or an equal volume of dimethyl sulfoxide (DMSO) as a control, and then incubated for 3 or 7 days at 37°C. For treatment, NA [≥99% (capillary GC); cat. no. 506-37-6; MilliporeSigma] was prepared as a 50 mM stock solution in DMSO (MilliporeSigma). Subsequently, they were incubated with 10 μl CCK-8 at 37°C for 2 h. An equal volume of medium without cells was used as a blank. The absorbance was measured at 450 nm using a microplate reader (Thermo Scientific™ Multiskan SkyHigh Microplate Spectrophotometer; cat. no. A51119600C; Thermo Fisher Scientific, Inc.).

MSCs were seeded in 6-well culture plates at a density of 1×10⁵ cells/well and were incubated until the cells reached 80-90% confluence. For adipogenic differentiation, MSCs were cultured in an adipogenesis differentiation medium (StemPro™ Adipogenesis Differentiation kit; cat. no. A1007001; Gibco; Thermo Fisher Scientific, Inc.) containing different concentrations of NA (0, 40, 80, 120, 160, 200, 240 and 280 μM), according to the manufacturer's instructions. The control group was treated with an equal volume of DMSO in the same differentiation medium. After 7, 14 and 21 days, the differentiated cells were fixed with 4% paraformaldehyde (Biosaesang) for 10 min and stained with Oil Red O solution (MilliporeSigma) for 30 min at 25°C. Subsequently, lipid droplets were observed under a light microscope (CKX41; Olympus Corporation). Oil Red O was quantified by extraction in 100% isopropyl alcohol and the optical density was measured at 540 nm.

Total RNA was obtained from the differentiated AD-MSCs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Next, complementary DNA (cDNA) was synthesized from the total RNA using SuperScript IV Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, and was further purified using a QIAquick PCR Purification kit (Qiagen GmbH). qPCR was performed by adding cDNA to a mixture containing the respective primers and SYBR green master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the QuantiStudio™ 6 Flex Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR conditions were as follows: Pre-denaturation at 50°C for 2 min and 95°C for 10 min; followed by 40 cycles of amplification at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and a melting curve stage at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. The specific primer sequences are listed in Table I. GAPDH was used as the housekeeping gene, and the results were analyzed using the 2^−ΔΔCq method (18).

The following primary antibodies were used for western blotting and immunohistochemistry experiments: PPARγ (cat. no. 2435), CEBPα (cat. no. 2295), adiponectin (cat. no. 2789), phosphorylated (p)-Akt (Ser473; cat. no. 9271), Akt (cat. no. 9272), p-mTOR (Ser2448; cat. no. 5536), mTOR (cat. no. 2983), GSK3β (cat. no. 9315), p-β-catenin (Ser33/37/Thr41; cat. no. 9561), β-catenin (cat. no. 9562), p-Smad1/5 (Ser463/465; cat. no. 9516), Smad1 (cat. no. 9743), p-ERK1/2 (Thr202/Tyr204; cat. no. 9101), ERK1/2 (cat. no. 9102), perilipin-1 (cat. no. 9349) and CD31 (cat. no. 77699), which were purchased from Cell Signaling Technology, Inc.; and lipoprotein lipase (LPL; cat. no. sc-373759) and β-actin (cat. no. sc-47778), which were obtained from Santa Cruz Biotechnology, Inc. HRP-conjugated anti-rabbit (cat. no. abc-5003) or anti-mouse (cat. no. abc-5001) secondary antibodies (AbClon) were used as secondary antibodies in the western blot analysis.

To determine the levels of adipogenic markers in AD-MSCs, the cells were cultured for 14 days in adipogenesis differentiation medium with either DMSO or NA. The cells were then lysed with RIPA buffer (Biosaesang) + protein inhibitor cocktail (GenDEPOT) + EDTA (GenDEPOT) and centrifuged at 18,000 × g for 30 min at 4°C to collect the lysate. Protein concentration was quantified using the Bradford assay (Bio-Rad Laboratories, Inc.). Protein extracts (10 μg/lane) were then separated by SDS-PAGE on 4-15% polyacrylamide gels (Bio-Rad Laboratories, Inc.). Subsequently, the separated proteins were transferred onto nitrocellulose or polyvinylidene fluoride membranes, which were blocked with 5% skim milk (Difco; BD Biosciences) or 5% bovine serum albumin (BSA) (Thermo Fisher Scientific, Inc.) in TBS-0.2% Tween-20 (TBS-T) and were gently shaken for 60 min at 25°C. The membranes were then incubated with primary antibodies (1:1,000 dilutions in 5% skim milk or 5% BSA) overnight at 4°C with gentle shaking. After three washes in TBS-T (10 min/wash) at 25°C, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5,000 dilutions in 5% skim milk or 5% BSA) at 25°C for 1 h with gentle shaking. Finally, the membranes were washed three times with TBS-T (10 min/wash), were treated with ECL solution (Bio-Rad Laboratories, Inc.) for 1 min, were visualized using an Amersham™ Imager 600 (Cytiva), and were semi-quantified using ImageJ 1.53 software (National Institutes of Health). The primary and secondary antibodies used in western blotting are aforementioned.

Total RNA was isolated for mRNA sequencing from differentiating AD-MSCs at 7, 14 and 21 days using TRIzol^® reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. mRNA sequencing was conducted by Ebiogen Inc. RNA quality was assessed using the TapeStation4000 System (Agilent Technologies, Inc.) and RNA was quantified using an ND-2000 spectrophotometer (Thermo Fisher Scientific, Inc.). NEBNext Ultra II Directional RNA-Seq kit (cat. no. E7760L; New England BioLabs, Inc.) was used to prepare the sequencing libraries from total RNA, and each step was performed using reagents included within the kit. The mRNA was further isolated from total RNA using a Poly(A) RNA Selection kit (cat. no. LEX-157; Lexogen GmbH). Subsequently, cDNA was synthesized from the isolated mRNAs (10 min at 25°C; 15 min at 42°C; 15 min at 70°C for first strand cDNA and 1 h at 16°C for second strand cDNA), and removal of RNA was followed by random-primed synthesis of the complementary strand. The cDNA was sheared according to the manufacturer's instructions of the NEBNext Ultra II Directional RNA-Seq kit. Indexing was performed using Illumina indices 1-12. Library enrichment was performed using PCR. The libraries were checked for quality control using TapeStation HS D1000 Screen Tape (Agilent Technologies, Inc.) to evaluate the mean fragment size. Quantification was performed on the StepOne Real-Time PCR System (Thermo Fisher Scientific, Inc.) using a random primer and NEBNext Ultra II Q5 Master Mix (included in the NEBNext Ultra II Directional RNA-Seq kit). High-throughput sequencing (paired-end 100 bp sequencing) was performed with a loading concentration of 320 pM using NovaSeq 6000 (Illumina, Inc.).

First, FastQC was used for quality control of the raw sequencing data (19). Adapter and low-quality reads (<Q20) were removed using FASTX_Trimmer (20) and BBMap (21). Trimmed reads were mapped to the reference genome using TopHat (22). The read counts were calculated using Cufflinks (23) and the fragments per kb per million reads (FPKM) values were estimated using Cuffdiff. The FPKM values were normalized using Cuffdiff geometric normalization. Data mining and graphic visualization were performed using ExDEGA ver 4.0.3 (ebiogen, Inc.). Data for each sample are available at the Gene Expression Online (GEO) under the accession number GSE269065 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE269065).

The differentially expressed genes (DEGs) between the DMSO and NA groups that showed a fold change >1.5 and P<0.05 were selected and further analyzed using ExDEGA software and DAVID Bioinformatics Resource 6.8 (NCBI online bioinformatics program) (https://david.ncifcrf.gov/tools.jsp) (24,25) to identify the functions of the selected genes and Gene Ontology. Adjusted P≤0.05 (Fisher's Exact test) was used to select related functional annotations. The principal component analysis (PCA) and the heatmaps of gene expression were generated using Excel 2016 (Microsoft) and ExDEGA software. Additionally, enriched signaling pathways related to the selected DEGs were analyzed using the online Kyoto Encyclopedia of Genes and Genomes (KEGG) mapper tool (https://www.kegg.jp/kegg/mapper.html) (26,27).

Fat transplantation experiments were performed using 5-month-old C57BL/6 mice (ORIENT BIO, Inc.) as both adipose tissue donors and recipients. Both male and female mice were used randomly, which weighed 22-28 g, and had free access to food and water. A total of 33 mice were used for the experiments. Specifically, 15 mice (10 donors, 5 recipients) were used for fat graft measurements and gene expression analysis, and 18 mice (12 donors, 6 recipients) were used for histological analysis. Mice for the experiments were housed in individually ventilated cages with a maximum of five mice per cage. The housing room was air-conditioned with 100% HEPA-filtration. The internal temperature and humidity were maintained at 20-26°C and 30-70%, respectively, and mice were kept under a 12-h light/dark cycle. For fat extraction, mice were anesthetized with 2% isoflurane and allogeneic adipose tissues for transplantation were extracted from the peritoneal fat of mice after laparotomy. The extracted adipose tissues were finely chopped and centrifuged at 2,095 × g for 15 min at 25°C. Subsequently, the upper and lower fractions containing lipids and blood, respectively, were removed to collect pure adipose tissue for engraftment. Thereafter, adipose tissue was transplanted underneath the skin of the back on both sides: Adipose tissue (300 μl) mixed with 160 μM NA was transplanted into the right side of the back, whereas adipose tissue (300 μl) mixed with the same volume of DMSO was transplanted into the left side. For transplantation, mice were anaesthetized with 2% isoflurane. After transplantation, the health and well-being of the mice were assessed three times a week and transplants were harvested and analyzed after 5 weeks. At transplant harvest, the mice were euthanized using CO₂ (35% volume/min) in a chamber for 3-4 min. The death of the mice was confirmed through the cessation of heartbeat and loss of reflex. The graft volume was calculated using the formula reported by Tomayko and Reynolds (28). For histological analysis, the obtained grafts were frozen and sectioned (12 μm) for Oil Red O staining using NovaUltra Oil Red O Stain kit (cat. no. IW-3008; IHC World LLC) according to the manufacturer's instructions. In addition, grafts were embedded in paraffin and sectioned (4 μm) for immunohistochemistry and hematoxylin & eosin (H&E) staining.

The present study was reviewed and approved by the Institutional Animal Care and Use Committee of the Research Institute for Future Medicine (RIFM), belonging to Samsung Medical Center (approval nos. 20230316003 and 20240522001). The RIFM is an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility that abides by the guidelines of the Institute of Laboratory Animal Research (29).

For immunohistochemistry, adipose tissues from mice were embedded in paraffin and sectioned (4 μm) after fixation in 4% paraformaldehyde for 36 h at 25°C. First, the samples were deparaffinized in a gradually decreasing concentration of ethanol and were permeabilized using xylene. Antigen retrieval was then performed using Target Retrieval Solution, Citrate pH 6 (Dako; Agilent Technologies, Inc.), washed and blocked in Dako REAL Peroxidase-Blocking Solution (Dako; Agilent Technologies, Inc.). After washing with PBS three times (5 min/wash), the samples were treated with primary antibodies (1:200) at 4°C. The next day, the primary antibodies were removed, and the samples were washed three times with PBS. They were then treated with Dako Envision + System-HRP Labelled Polymer Anti-Rabbit Antibody (cat. no. K4003, Dako; Agilent Technologies, Inc.) for 1 h and stained with Dako Liquid DAB (Dako; Agilent Technologies, Inc.) for 1 min at 25°C. Finally, the samples were stained with hematoxylin for nuclear staining for 4 sec at 25°C, then dehydrated and cleared before observation. Aperio AT2 (Leica Biosystems) was used for observation, and staining was semi-quantified using ImageJ 1.53 software or QuPath 0.4.4. software (30). The aforementioned primary antibodies were used for immunohistochemistry.

For H&E staining, adipose tissues from mice were embedded in paraffin and sectioned (4 μm) after fixation in 4% paraformaldehyde for 36 h at 25°C. First, 4-μm paraffin-embedded samples were permeabilized using Histoclear (National Diagnostics) for 9 min (3 min × 3), and were rehydrated using 100 and 95% ethanol. The samples were then stained with hematoxylin for 8 min, exposed to HCl for 1 sec and eosin for 40 sec at 25°C, and washed with PBS between each step. Subsequently, the sections were dehydrated with 95 and 100% ethanol, and were treated with Histoclear. Finally, the samples were mounted, and staining was observed using Aperio AT2.

Graphical illustrations presented in the present study were generated using BioRender (https://www.biorender.com/).

All data are presented as the mean ± SD (n≥3). Excel 2016 (Microsoft Corporation) and GraphPad Prism for Windows ver. 10 (Dotmatics) were used for all analyses. Statistical comparisons between two groups were performed using unpaired Student's t-test, and those between three or more groups were performed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the optimal concentration of NA that induces adipogenesis in AD-MSCs, the formation of oil droplets was examined in AD-MSCs following treatment with various concentrations of NA. First, the cytotoxicity of NA was determined in AD-MSCs using a CCK-8 assay. The optical density of AD-MSCs was measured on days 3 and 7 post-treatment with DMSO or several concentrations of NA (40-280 μM). The viability of AD-MSCs was not significantly altered at day 3 or 7 after NA treatment compared with that in the control group (Fig. 1A). Therefore, NA was confirmed to be non-toxic to AD-MSCs. Notably, AD-MSCs treated with ≥120 μM NA exhibited partial formation of oil droplets at day 3 in culture conditions, indicating that NA is a potential adipogenic inducer (Fig. 1B). Based on these results, AD-MSCs were treated with DMSO or NA during differentiation into the adipogenic lineage for 7 days, and the degree of lipid accumulation was determined using Oil Red O staining. Concentrations of NA up to 160 μM enhanced lipid accumulation in AD-MSCs in a dose-dependent manner (Fig. 1C and D). Thus, 160 μM was considered the optimal concentration of NA for promoting adipogenesis in AD-MSCs and was used in subsequent experiments.

Next, the effect of NA on adipogenic differentiation according to different exposure periods was assessed. Adipogenic conditions were induced for 7, 14 and 21 days, and lipid accumulation was measured using Oil Red O staining (Fig. 1E). AD-MSCs treated with NA exhibited sufficient differentiation from day 14 onward, resulting in a significant increase in lipid accumulation compared with the DMSO control group (Fig. 1F). However, lipid accumulation caused by NA showed the greatest difference on day 7 relative to the control (day 7, 1.94±0.05 fold change; day 14, 1.78±0.03 fold change; day 21, 1.45±0.02 fold change) (Fig. 1G). Because NA promoted the adipogenic differentiation of AD-MSCs, the present study further investigated whether NA could influence the induction of adipogenic genes. RT-qPCR results showed that the master transcription factors PPARG and CEBPA exhibited the highest upregulation on day 14 after NA treatment, whereas the mature adipocyte markers LPL and ADIPOQ were upregulated the most on day 21 after NA treatment (Fig. 1H). These results indicated that NA treatment was effective in accelerating the adipogenic process in AD-MSCs.

The effect of NA on MSCs from different sources was further verified on days 7, 14 and 21. Treatment with NA significantly enhanced lipid accumulation in MSCs isolated from the umbilical cord blood, placenta, Wharton's jelly and bone marrow (Fig. S1), demonstrating that NA had a universal effect on adipogenesis in MSCs, irrespective of their origin.

Because the expression of adipogenic differentiation-related genes was elevated in the NA-treated group, the present study further investigated the expression levels of adipogenesis-related proteins. Consistent with gene expression results, western blotting results indicated that PPARγ, CEBPα, adiponectin and LPL levels were higher in the NA-treated group (2.34±0.69, 1.48±0.17, 2.35±0.55 and 1.53±0.15 fold change, respectively) than in the control group (Fig. 2A). Differentiation of AD-MSCs into mature adipocytes is modulated by the balance of various signaling pathways, such as Smad, MEK/ERK and Akt/mTOR signaling as promoters, and Wnt signaling as an inhibitor (31,32). Since NA promoted the adipogenic differentiation of MSCs, the study aimed to determine the signaling pathway that regulates adipogenesis caused by NA. Western blotting results showed that the phosphorylation of Akt and mTOR, which are positive regulators of adipogenesis, was upregulated after NA treatment (Fig. 2B). Meanwhile, intracellular phosphorylation of β-catenin was significantly upregulated in the NA treatment group (Fig. 2C), indicating that intracellular β-catenin was degraded. Additionally, intracellular GSK3β, which is contained in the destruction complex of β-catenin, was increased (Fig. 2C), which may inhibit Wnt signaling. However, the other positive regulators of adipogenesis, Smad and ERK, were unaffected by NA treatment (Fig. 2D). Collectively, these results indicated that NA may accelerate adipogenesis through activation of the Akt/mTOR pathway and inhibition of Wnt signaling during adipogenesis (Fig. 2E).

mRNA sequencing was performed to confirm the transcriptomic changes induced by NA during adipogenesis. PCA implied that genetic changes induced by NA occurred most significantly on day 7 post-adipogenesis induction, and the difference decreased with increasing differentiation period (Fig. 3A). On day 7, 688 genes were upregulated and 387 genes were downregulated in the NA treatment group (fold change >1.5). Gene Ontology analysis revealed that genes upregulated by NA were mainly involved in signaling pathways such as 'GO:0045444~Fat cell differentiation' and 'GO:0060612~Adipose tissue development', which are related to adipogenesis, and 'GO:0008286~Insulin receptor signaling pathway' and 'GO:0031929~TOR signaling', which were partially involved in fat differentiation (Fig. 3B). In addition, the 'hsa04151:PI3K-Akt signaling pathway' and 'hsa04150:mTOR signaling pathway' were included in the signaling cascades affected by NA, which was consistent with the results of protein expression (Figs. S2 and 2B). Furthermore, NA-treated MSCs exhibited upregulated expression patterns for adipose tissue development, mTOR signaling, angiogenesis and VEGF production (Fig. 3C and D). To summarize, NA treatment may enhance Akt/mTOR signaling in MSCs and thereby increase the expression of genes involved in angiogenesis and fat differentiation.

As aforementioned, it is crucial to create a stable microenvironment within the graft for successful transplantation outcomes (4,5). Since NA demonstrated adipogenesis-promoting effects in vitro, the present study further assessed whether NA treatment could improve outcomes after allogeneic fat engraftment in mice. Allogeneic C57BL/6 mouse adipose tissues treated with DMSO or NA were engrafted subcutaneously in mice and histologically analyzed after 5 weeks (Fig. 4A and B). Fig. 4C shows the fat grafts after 5 weeks of fat transplantation (n=5/group). The fat grafts treated with NA showed a significant increase in long and short axes lengths, volume and weight compared with those treated with DMSO (Fig. 4D-F). Lipid accumulation in the fat graft was confirmed by Oil Red O staining. Notably, while the fat grafts treated with DMSO had a number of unstained empty spaces after 5 weeks, the fat grafts treated with NA exhibited much higher staining with completely lipid-filled spaces, indicating that supplementation with NA helped mature adipocytes maintain greater stability after engraftment (Fig. 4G and H). In addition, the mRNA expression levels of Pparg, Lep and Lpl were elevated in the NA-treated fat grafts (Fig. 4I). Taken together, these results suggested that NA may effectively reduce the resorption of transplanted fat and maintain stability after tissue engraftment.

Vacuoles (cysts) in fat grafts are formed by the necrosis of adipocytes after fat transplantation, and the proportion of vacuoles in the graft is considered an important indicator for determining the outcome of fat transplantation (33). Therefore, the present study examined vacuole formation in the transplanted fat treated with or without NA. Histological analysis showed that oil cysts or vacuoles with diameters >120 μm (34) were largely formed in the grafts treated with DMSO (Fig. 5A). To confirm the effect of NA on the survival of adipocytes in fat grafts, viable adipocytes were identified using immunohistochemistry for perilipin-1 (Fig. 5B). The fat grafts treated with NA had more perilipin-1⁺ adipocytes with a lower proportion of vacuoles (Fig. 5D and E), verifying that NA may preserve more viable adipocytes. Notably, relatively large portions of small, perilipin-1⁺ adipocytes, which are distinguishable from mature adipocytes, can be seen in the NA-treated grafts (Fig. S3). Subsequently, the degree of graft vascularization was assessed. CD31⁺ vessel structures were more frequently observed in the NA-treated group, with a notable increase in the proportion of the CD31⁺ area (Fig. 5C and F). Moreover, blood vessels in the NA-treated group were relatively evenly distributed between viable adipocytes, improving their survival.

RT-qPCR analysis showed that pro-inflammatory Tnf was downregulated, whereas the anti-inflammatory marker Il10 was elevated (Fig. 5G), and angiogenic-promoting genes Angpt1, Tek and Vegfa were upregulated in grafts supplemented with NA (Fig. 5H). Given that inflammation and neovascularization are critical elements for the successful engraftment of external tissue, these findings indicated that NA may improve the outcomes of adipose tissue engraftment by enhancing angiogenesis and suppressing inflammation, resulting in a relatively well-reorganized microenvironment in the grafts.