Wharton's jelly mesenchymal stem cell-secretome enhances skin rejuvenation via ApoA4 and SERPINH1

Abstract

Introduction

Skin aging arises from intrinsic processes and extrinsic insults (e.g., ultraviolet exposure and oxidative stress). Mesenchymal stromal cell (MSC)-derived secretome offers a cell-free approach to skin regeneration. Wharton's jelly-derived MSCs (WJ-MSCs) may outperform adipose-derived (AD-MSCs) and bone marrow-derived MSCs (BM-MSCs).

Methods

Secretomes from WJ-MSCs, AD-MSCs, and BM-MSCs were compared in vitro for human dermal fibroblast proliferation, scratch-wound closure, extracellular-matrix (ECM) remodeling, and type I procollagen secretion. Anti-inflammatory and antioxidant activities were assessed by IL-6, IL-1β, TNF-α, COX-2 and intracellular reactive oxygen species (ROS). Antibody arrays profiled secreted factors. An exploratory, single-arm human pilot (n = 21; 1-week) evaluated topical WJ-MSC secretome with paired analyses. Safety was monitored, and a separate occlusive patch test (n = 33) was conducted.

Results

The WJ-MSC secretome increased fibroblast proliferation, ECM remodeling, and type I procollagen, and reduced cytokines and ROS, exceeding the effects of AD-MSC and BM-MSC secretomes. Profiling highlighted apolipoprotein A4 (ApoA4) and SERPINH1 as enriched, functionally active mediators; recombinant ApoA4 and SERPINH1 enhanced fibroblast activity, collagen-related readouts, and accelerated in vitro wound closure. In the pilot study, within-subject increases in instrument-derived hydration and elasticity were observed over one week (paired tests). No treatment-related adverse events were noted. Patch testing showed no irritation (ICDRG scores all 0; non-irritant classification).

Conclusions

The WJ-MSC secretome demonstrated consistent in-vitro pro-regenerative, anti-inflammatory, and antioxidant activities, with ApoA4 and SERPINH1 as candidate mediators. Human findings are preliminary/exploratory and suggest potential short-term benefits that require confirmation in adequately powered, controlled trials.

Graphical abstract

Abbreviations

AD-MSC

Adipose tissue-derived mesenchymal stem cells

ApoA4

Apolipoprotein A-IV

BM-MSC

Bone marrow-derived mesenchymal stem cells

CM

Conditioned medium

EV

Extracellular vesicle

ECM

Extracellular matrix

HS68

Human foreskin dermal fibroblast cell line

HaCaT

Human skin keratinocyte cell line

hPL

human platelet lysate

LPS

Lipopolysaccharide

R.A

Retinoic acid

ROS

Reactive oxygen species

SERPINH1

Serpin Family H Member 1

TGF-β

Transforming growth factor-beta

UV

Ultraviolet

VEGF

Vascular endothelial growth factor

WJ-MSC

Wharton's Jelly-derived mesenchymal stem cell

1. Introduction

Skin, the largest organ of the human body, serves as a critical barrier against environmental insults, pathogens, and mechanical injury [1]. Its structural and functional integrity gradually declines due to intrinsic aging and extrinsic factors such as ultraviolet (UV) radiation, oxidative stress, environmental pollutants, and lifestyle-related exposures [2]. These cumulative insults accelerate cellular senescence, extracellular-matrix (ECM) degradation, and inflammation, leading to visible signs of skin aging and impaired tissue regeneration [3,4].

Skin rejuvenation can be viewed as a coordinated therapeutic restoration of aged or damaged skin across complementary dimensions [5]. Clinically, it involves recovery of dermal elasticity, restoration of barrier and moisture-retention function, and normalization of dermal thickness [6]. At the molecular level, it entails activation of collagen synthesis, efficient scavenging of reactive oxygen species (ROS), and down-regulation of pro-inflammatory cytokines [[7], [8], [9]]. From a regenerative perspective, mesenchymal stromal cell (MSC)-derived secretome factors drive paracrine remodeling of the extracellular matrix, stimulating fibroblast proliferation, promoting microvascular network formation, and supporting comprehensive tissue repair [[10], [11], [12]]. This multi-layered framework underpins our evaluation of the skin-rejuvenating potential of the Wharton's jelly (WJ)-MSC secretome in the present study [11]. To capture the multifaceted nature of rejuvenation-structural renewal, barrier recovery, inflammatory modulation, and neovascular support we used human dermal fibroblasts for ECM remodeling (collagen synthesis) and oxidative protection (ROS reduction), keratinocytes for barrier restoration (scratch-wound healing), macrophages for anti-inflammatory signaling, and endothelial cells for angiogenesis [13].

In recent years, MSCs have emerged as a promising strategy for tissue regeneration through secretion of trophic factors, cytokines, and extracellular vesicles (EVs) that modulate inflammation, promote angiogenesis, and enhance tissue repair [10,14]. Among the various MSC sources, Wharton's jelly-derived MSCs (WJ-MSCs) are attractive due to high proliferative capacity, immunomodulatory activity, and low ethical constraints (derivation from umbilical cords) [[15], [16], [17]]. Furthermore, the use of conditioned medium (CM) derived from WJ-MSCs represents a cell-free, scalable therapeutic alternative that preserves the regenerative benefits while circumventing the limitations associated with live-cell therapies [10,18,19].

Prior studies indicate that WJ-MSC secretome can promote dermal fibroblast proliferation, enhance collagen synthesis, and accelerate wound closure [20]. Moreover, its anti-inflammatory and antioxidant properties further support its potential dermatologic applications [19,21].

Despite these findings, the key molecular mediators underlying the regenerative effects of the WJ-MSC secretome remain incompletely defined [22]. Notably, ApoA4 an intestinally secreted apolipoprotein with well-established antioxidant/anti-inflammatory activity and SERPINH1 (HSP47) an ER-resident, collagen-specific chaperone essential for ECM maturation emerged from our secretome profiling [23,24]. Exogenous and EV-associated HSP47 have been reported to act extracellularly and enhance collagen deposition, supporting a plausible ECM-modulatory role in our system [25]. ApoA4 has been associated with regulation of oxidative stress and pro-inflammatory cytokines such as IL-6, COX-2, IL-1β, and TNF-α, while SERPINH1 is essential for collagen processing and may influence hydration-related proteins such as HAS2 and AQP3 [[26], [27], [28]].

To date, few studies have comprehensively investigated the clinical effects of the WJ-MSC secretome in skin rejuvenation. Therefore, this study aims to: (1) elucidate the functional relevance of key bioactive proteins, particularly ApoA4 and SERPINH1, in the WJ-MSC secretome; and (2) assess preliminary clinical signals in an exploratory, single-arm pilot evaluating within-subject changes in skin hydration, elasticity, and brightness following topical application. These findings may advance the development of the WJ-MSC secretome as a next-generation, cell-free therapeutic candidate for dermatologic regeneration.

2. Materials and Methods

2.1. Master cell banking for umbilical cord WJ-MSCs

Umbilical cords were obtained from three healthy donors (<40 years) with informed consent under IRB approval (Samsung Medical Center, IRB No. 2016-07-102-037, approved on August 9, 2022). Donor eligibility was confirmed via immunochromatographic and PCR assays for HBV, HCV, HIV, HTLV-1/2, CMV, EBV, and syphilis, with all results negative. Wharton's jelly was isolated, washed in Dulbecco's phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, USA), minced, and digested with 0.1 % collagenase type I (Sigma-Aldrich, USA) at 37 °C. After 1 h, cells were collected by centrifugation (600×g, 5 min) and cultured in Minimum Essential Medium (MEM; Gibco, Thermo Fisher Scientific) supplemented with 15 % fetal bovine serum (FBS; Gibco). Adherent MSCs were expanded to passage 4, cryopreserved (5.5 × 10⁶ cells/vial), and stored in vapor-phase liquid nitrogen (<−150 °C). Quality control included sterility, endotoxin, viability, Mycoplasma, and adventitious agent testing. Immunophenotyping confirmed ≥90 % expression of CD73, CD90, CD105 and ≤1 % expression of CD34, CD45 (all antibodies from BD Biosciences, USA), consistent with MSC identity.

2.2. Trilineage differentiation of MSCs

To evaluate multipotency, MSCs were subjected to adipogenic, osteogenic, and chondrogenic differentiation.

2.2.1. Adipogenic differentiation

MSCs (1 × 10⁵ cells/well) were cultured for 14 days in adipogenic induction medium containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM hydrocortisone, and 0.1 mM indomethacin (all from Sigma-Aldrich, St. Louis, MO, USA). Lipid droplets were visualized using Oil Red O staining (Sigma-Aldrich).

2.2.2. Osteogenic differentiation

MSCs (2.5 × 10⁵ cells/well) were cultured for 21 days in osteogenic medium (StemPro™ Osteogenesis Differentiation Kit, Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Calcium deposits were detected with Alizarin Red S staining (Sigma-Aldrich).

2.2.3. Chondrogenic differentiation

MSCs (1 × 10⁶ cells/tube) were cultured in 15-mL tubes for 21 days using chondrogenic medium (StemPro™ Chondrogenesis Differentiation Kit, Gibco, Thermo Fisher Scientific). Sulfated glycosaminoglycans were detected by Alcian Blue staining (Sigma-Aldrich).

2.3. Flow cytometric analysis of MSC surface markers

MSCs at passage 6 (5 × 10⁵ cells) were resuspended in PBS with 2 % FBS and stained with fluorochrome-conjugated antibodies against CD73-APC, CD90-FITC, CD105-PE, CD14-PE, CD34-FITC, and CD45-VioGreen (all from BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4 °C in the dark. Isotype controls were included. Fluorescence signals were acquired using a FACSVerse™ flow cytometer (BD Biosciences) and analyzed with CellQuest Pro software.

2.4. MSC culture and secretome preparation

Human mesenchymal stromal cells (MSCs) from three sources-adipose tissue (PromoCell, Heidelberg, Germany; C-12978), bone marrow (Lonza, Walkersville, MD, USA; PT-2501), and Wharton's jelly (Samsung Medical Center GMP Facility, Seoul, Korea)-were maintained in phenol-red-free α-MEM (Gibco, Thermo Fisher Scientific, USA) supplemented with 5 % human platelet lysate (hPL; Helios, Tokyo, Japan) at 37 °C in 5 % CO₂. All MSC lines adhered to plastic, displayed a spindle-shaped morphology, and expressed CD73, CD90, and CD105, but not CD14, CD34, or CD45. For conditioned-medium (CM) production, passage 6 MSCs were plated at 3000 cells/cm² in α-MEM with 5 % hPL (0.3 mL/cm²). After 24 h, the medium was replaced, and cultures were incubated for an additional 48 h. CM was centrifuged (300×g, 10 min) and filtered (0.22 μm; Sartorius). For WJ-MSC secretome, CM from three independent WJ-MSC donor lines (master cell bank) was pooled 1:1:1 prior to processing. By contrast, AD-MSC and BM-MSC secretomes were prepared from single-donor commercial lines (PromoCell and Lonza, respectively). All secretomes were produced at passage 6 and processed identically (48 h conditioning, 300×g clarification, 0.22 μm filtration). The filtered preparations were stored at −80 °C until use.

2.5. Cell cultures

HaCaT keratinocytes (AddexBio, San Diego, CA, USA; T0020001), HS68 human dermal fibroblasts (ATCC, Manassas, VA, USA; CRL-1635), and Raw264.7 murine macrophages (KCLB, Seoul, Korea; 40071) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco) and 1 % penicillin-streptomycin (Gibco) at 37 °C in a humidified 5 % CO₂ incubator. Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD, USA) were maintained in Endothelial Cell Growth Medium (EGM; Lonza) under identical conditions.

2.6. In vitro skin rejuvenation assays

2.6.1. Replication and controls

Unless otherwise stated, n denotes independent experiments conducted on separate days; within each experiment, conditions were run in technical triplicate and averaged to one value. For secretome conditions, freshly thawed aliquots from the same production batch were used for each experiment (WJ-MSC secretome from a three-donor pool; AD-MSC and BM-MSC secretomes from single-donor lines). For secretome versus control comparisons, the negative control was vehicle conditioned medium (Vehicle-CM; α-MEM + 5 % hPL, incubated cell-free for 48 h and processed identically). For recombinant protein gain-of-function dose–response experiments (SERPINH1 and ApoA4), assays were conducted in serum-free assay medium (SFM; DMEM supplemented with 1 % penicillin-streptomycin, without serum or hPL) and SFM served as the baseline control (Vehicle-CM was not used), as specified in each assay/figure legend. Assay-specific n values were: proliferation, n = 5; wound healing, n = 4; type I procollagen ELISA, n = 5; COL1A1/COL3A1 qPCR, n = 5; IL-6 qPCR, n = 3; total antioxidant capacity, n = 5; ROS reduction (DCF-DA), n = 3; tube formation, n = 5.

2.6.2. Statistical Analysis (in vitro)

Data are presented as mean ± SEM unless otherwise indicated. Group comparisons for in-vitro assays were performed using one-way ANOVA with appropriate multiple-comparisons correction (e.g., Tukey for all-pairs or Dunnett versus Vehicle-CM), implemented in GraphPad Prism 8.0 (GraphPad Software). Two-sided α = 0.05 was used for significance. The number of independent experiments (n) for each assay is specified in the “Replication and controls” paragraph.

2.6.3. Cell proliferation assay

Cell proliferation and viability were evaluated using the CCK-8 assay (Dojindo Molecular Technologies, Rockville, MD, USA). HS68 cells (3 × 10³ cells/well) were seeded in 96-well plates (Corning, USA) and incubated overnight. Cells were treated with MSC secretome or recombinant ApoA4/SERPINH1 (R&D Systems, USA) at the indicated concentrations for 24 h. Afterwards, 10 μL of CCK-8 reagent (Dojindo, Japan) was added, and plates were incubated at 37 °C for 2 h. Absorbance at 450 nm was measured on a microplate reader (BioTek, USA) to determine viability.

2.6.4. Wound healing assay

HaCaT cells (2 × 10⁵ cells/well) were plated in 24-well plates (Corning, USA) and grown to 90–100 % confluence. A uniform scratch was made with a sterile 200 μL pipette tip, and debris was removed by washing twice with PBS (Welgene, Korea). Cells were then treated with MSC secretome or recombinant ApoA4/SERPINH1 (R&D Systems, USA) at the indicated concentrations in 1 mL medium and incubated at 37 °C, 5 % CO₂ for 18 h. Wound closure was imaged with an inverted phase-contrast microscope (CKX53; Olympus, Japan) and quantified using ImageJ (v1.53t; NIH, USA).

2.6.5. Collagen synthesis

HS68 fibroblasts (6 × 10⁴ cells/well; ATCC, USA) were seeded in 6-well plates (Corning, USA) overnight and treated with WJ-MSC secretome or recombinant ApoA4/SERPINH1 (R&D Systems, USA) for 24 h. Total RNA was extracted with QIAzol (QIAGEN, Germany) and reverse transcribed using SuperScript III (Invitrogen, USA) COL1A1 and COL3A1 mRNA levels were measured by qPCR (QuantStudio 6 Flex; Thermo Fisher Scientific, USA) with SYBR™ Green Master Mix (Applied Biosystems, USA). Secreted type I procollagen was quantified by ELISA (Takara Bio Inc., Shiga, Japan; MK101). For SERPINH1 effects, cell lysates prepared in PRO-PREP (INTRON Biotechnology, South Korea) were resolved by 10 % SDS-PAGE, transferred to PVDF membranes (Millipore, USA), probed for COL1A1 and fibronectin (Santa Cruz Biotechnology, USA), and visualized using ECL (Thermo Fisher Scientific, USA) on a ChemiDoc Fusion Solo X (Vilber, France).

2.6.6. Antioxidant capacity assay

DCF-DA ROS assay. HS68 human dermal fibroblasts (6 × 10⁴ cells/well) were seeded in 6-well plates (Corning, USA) and allowed to adhere overnight. Cells were treated with WJ-MSC secretome or recombinant ApoA4 (R&D Systems, USA) at the indicated concentrations for 24 h. Intracellular ROS levels were measured by incubation with 10 μM DCF-DA (Thermo Fisher Scientific, USA) for 30 min, followed by fluorescence imaging (Ex/Em 485/535 nm) using an EVOS fluorescence microscope (Thermo Fisher Scientific, USA). Fluorescence intensity was quantified using ImageJ (NIH, USA).

Total antioxidant capacity (TAC). After 24 h of treatment, conditioned medium was collected and clarified to remove cell debris. TAC was measured using a Total antioxidant capacity assay kit (Colorimetric) (Abcam, Cambridge, UK; ab65329) according to the manufacturer's instructions. Absorbance was read at 570 nm using a BioTek microplate reader. TAC values were calculated from a Trolox standard curve and expressed as Trolox equivalents (nmol/μL).

2.6.7. Hydration barrier assay

HaCaT keratinocytes (1 × 10⁵ cells/well) were seeded in 6-well plates and grown to 70–80 % confluence. Cells were then incubated in SFM and treated with recombinant SERPINH1 (R&D Systems, USA) at the indicated concentrations for 18 h at 37 °C in a 5 % CO₂ atmosphere. Retinoic acid (RA; 1 μM; vehicle: DMSO; final DMSO concentration ≤0.1 %) was included as a comparator (positive control) where indicated. For molecular markers of barrier hydration, treated HaCaT monolayers were lysed in PRO-PREP (INTRON Biotechnology, South Korea), and protein levels of HAS2 and AQP3 were assessed by Western blot.

2.6.8. Anti-inflammatory assay

RAW 264.7 murine macrophages (5 × 10⁵ cells/well) in 6-well plates (Corning, USA) were stimulated with 2 μg/mL LPS (Sigma-Aldrich, USA) for 2 h, then treated with WJ-MSC secretome or ApoA4 for 24 h. Total RNA was extracted with QIAzol (QIAGEN, Germany) and cDNA synthesized using SuperScript III (Invitrogen, USA). qPCR analysis of IL-6, COX2, IL-1β, and TNF-α was performed using SYBR Green Master Mix (Applied Biosystems, USA) on a QuantStudio 6 Flex system (Thermo Fisher Scientific, USA), with GAPDH as the internal control. The primer sequences used for qPCR are listed in Table S1.

2.6.9. Tube formation assay

Angiogenesis was assessed using a tube formation assay. HUVECs (Lonza) were seeded onto Matrigel-coated six-well plates (Corning, NY, USA) at a density of 6 × 10⁴ cells/well and treated with MSC secretome for 6 h. Tube formation was visualized using an inverted microscope (Olympus) and the number of tubes was quantified using an ImageJ Angiogenesis Analyzer (National Institutes of Health).

2.7. Secretome profiling using antibody arrays

Secretomes were collected from WJ-MSCs (trade name, where applicable: SNE-Secretome®), AD-MSCs, and BM-MSCs after 48 h of culture at 60–70 % confluence. Samples were analyzed using a Human L2000 Antibody Array (RayBiotech, Peachtree Corners, GA, USA) by eBiogen (Seoul, Korea). Data processing included: (1) fold-change calculation (test/control, where control = Vehicle-CM), (2) group-wise averages of normalized data (log₂-transformed), and (3) individual normalized data (log₂ with global normalization). Normalized, log₂-transformed data were visualized as heatmaps in R (v4.4.3; R Foundation for Statistical Computing). To further explore the functional relationships among proteins differentially secreted in MSC-derived Secretome, protein-protein interaction (PPI) network analysis was performed using the STRING database (version 11.5). A high-confidence interaction score cutoff (>0.7) was applied, and only experimentally validated, co-expressed, and curated database-derived interactions were included to enhance reliability. The resulting interaction networks were visualized using Cytoscape software (version 3.9.1).

Functional annotation and enrichment. Differentially abundant proteins were annotated and tested for GO (Biological Process) enrichment using the STRING functional-enrichment module (v11.5). Enrichment p-values were adjusted using Benjamini-Hochberg FDR (significance threshold FDR <0.05). Redundant GO terms were curated to representative processes for clarity.

2.8. ELISA

SERPINH1 (HSP47) and ApoA4 in MSC secretome was quantified using a commercial ELISA kit (RayBiotech, ELH-HSP47-1/ELH-ApoA4-1) according to the manufacturer's protocol. Absorbance was measured at 450 nm using a Synergy™ HTX microplate reader (BioTek Instruments, Winooski, VT, USA).

2.9. siRNA transfection

Small interfering RNAs (siRNAs) targeting SERPINH1 and ApoA4 (Bioneer, Daejeon, Korea) were transfected into cells at a final concentration of 30 nM using standard transfection reagent protocols. After 24 h of incubation, cells were harvested and lysed for subsequent Western blot analysis. A non-targeting control siRNA (siNC) was included as a negative control.

2.10. Clinical study

2.10.1. Primary skin irritation assessment by occlusive patch testing

Primary skin irritation was evaluated by an occlusive patch test in 33 participants using Finn Chambers (8 mm diameter; SmartPractice®, USA). The upper back was cleansed with 70 % ethanol and allowed to dry. Twenty microliters (20 μL) of WJ-MSC secretome was dispensed into each chamber, which was then applied to the designated site and secured under occlusion for 24 h. After patch removal, the application areas were demarcated with a skin-marker pen (DeRoyal, USA). Cutaneous responses were assessed by a board-certified dermatologist at 30 min, 24 h, and 48 h post-removal and graded according to the International Contact Dermatitis Research Group (ICDRG) criteria (Table 1). The irritation potential of the test article was determined based on these scores.

Table 1.

International Contact Dermatitis Research Group (ICDRG) criteria.

Sign	Score	Judging criteria
-	0	Negative
±	0.5	Doubtful or slight reaction with erythema
+	1	Erythema + Induration
++	2	Erythema + Induration + Vesicle
+++	3	Erythema + Induration + Bullae

2.10.2. Study participants and ethical considerations

This clinical study involved 21 healthy adult female participants aged 23–63 years, all of whom provided written informed consent. The protocol was approved by the IRB of the Korea Institute of Dermatological Sciences (IRB No. KIDSIRB-2024-0664, approved on May 13, 2024). All procedures complied with the Declaration of Helsinki.

2.10.3. Study design and procedure

Participants applied the WJ-MSC secretome formulation evenly over the entire face twice during a 7-day period, with an interval of approximately 3–4 days between applications, following evening facial cleansing. The product was gently massaged until fully absorbed.

2.10.4. Skin elasticity assessment

Skin elasticity was measured using the Cutometer dual MPA 580 (Courage + Khazaka Electronic GmbH, Cologne, Germany). The R2 parameter (overall elasticity, %) was calculated using MPA CTplus software.

2.10.5. Skin hydration assessment

Skin hydration was assessed using the Epsilon E100 (Biox Systems Ltd., UK), a validated instrument designed to measure moisture content of the stratum corneum.

2.10.6. Skin brightness assessment

Skin brightness was evaluated using the VISIA-CR Clinical Research System (Canfield Scientific Inc., USA). Digital image analysis was conducted using ImageJ (NIH) to quantify changes in brightness and pigmentation.

2.10.7. Environmental conditions

All measurements were performed under controlled environmental conditions (temperature: 20–22 °C; relative humidity: 45–55 %) to minimize variability.

2.10.8. Statistical Analysis

For clinical outcomes, normality of within-subject differences was assessed using the Shapiro-Wilk test. Because pre- and post-treatment measurements were obtained from the same individuals, paired statistical tests were used (paired t-test for normally distributed differences; Wilcoxon signed-rank otherwise). We report two-sided p-values, 95 % confidence intervals, and effect sizes (e.g., Cohen's d for paired designs and/or partial η²). Analyses were performed with α = 0.05.

3. Results

3.1. Comparative characterization of MSCs derived from adipose tissue, bone marrow, and Wharton's jelly

Under identical culture conditions, we compared the morphology, tri-lineage differentiation capacity, and immunophenotypic profiles of AD-MSCs, BM-MSCs, and WJ-MSCs (Fig. S1).

All MSCs exhibited plastic adherence and a characteristic spindle-shaped fibroblastic morphology by phase-contrast microscopy (Fig. S1A). While overall morphology was similar, minor differences in cell density and growth patterns were noted across sources.

Adipogenesis was confirmed by Oil Red O staining of cytoplasmic lipid droplets; osteogenesis by Alizarin Red S staining of calcium deposits; and chondrogenesis by Alcian Blue staining of sulfated glycosaminoglycans (Fig. S1B), demonstrating tri-lineage potential consistent with multipotency. Flow cytometry showed uniform expression of canonical MSC markers (CD73, CD90, CD105) and absence of hematopoietic markers (CD14, CD34, CD45) across all sources (Fig. S1C), with no apparent differences among sources, indicating comparable immunophenotypic identity.

3.2. Comparative in-vitro analysis of skin-rejuvenation potential of secretomes from different MSC sources

The skin-rejuvenation potential of secretomes from WJ-MSCs, AD-MSCs, and BM-MSCs was evaluated across multiple functional assays (Fig. 1).

Fig. 1.

Comparative in-vitro analysis of skin-rejuvenation activities of MSC secretomes.

Secretomes from WJ-MSCs, AD-MSCs, and BM-MSCs were compared across multiple functional assays. Unless otherwise stated, the negative control (N.C.) was Vehicle-CM (α-MEM + 5 % human platelet lysate incubated cell-free for 48 h and processed identically). (A) HS68 fibroblast proliferation by CCK-8, normalized to N.C. (=100 %); n = 5. (B) HaCaT keratinocyte scratch-wound closure at 0 h and 18 h; n = 4. (C) Type I procollagen secretion (ELISA) in HS68 fibroblasts; n = 5. (D) ECM-related gene expression (COL1A1, COL3A1) by qPCR (GAPDH-normalized) in HS68 fibroblasts; n = 5. (E) Suppression of pro-inflammatory transcripts (IL-6/COX-2/IL-1β/TNF-α or equivalent cytokine readouts) in LPS-stimulated RAW 264.7 macrophages; n = 3. (F) HUVEC tube-formation assay (6 h); tube number and total length; n = 5. VEGF (100 ng/mL) served as positive control. (G) Intracellular ROS (DCF-DA) after H₂O₂ challenge; secretome treatments versus N.C.; n = 3. Ascorbic acid (Vit. C) served as antioxidant control. (H) Total antioxidant capacity (reported as Trolox equivalents, nmol/μL); n = 5. Vit. C served as positive control. Data are mean ± SEM from independent experiments; statistics by one-way ANOVA with Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). Abbreviations: N.C., negative control (Vehicle-CM); AD, Adipose tissue-derived mesenchymal stem cells secretome; BM, bone marrow-derived mesenchymal stem cells secretome; WJ, Wharton's jelly-derived mesenchymal stem cells secretome; LPS, lipopolysaccharide; VEGF, vascular endothelial growth factor; DCF-DA, 2′,7′-dichlorofluorescein diacetate.

Fibroblast proliferation. In HS68 dermal fibroblasts, the WJ-MSC secretome increased proliferation to 265.74 ± 7.52 % (mean ± SEM, relative to Vehicle-CM = 100 %), exceeding BM-MSC (239.79 ± 8.73 %), AD-MSC (197.67 ± 9.25 %), and control (Vehicle-CM; 100.00 ± 5.78 %). One-way ANOVA with Tukey's multiple-comparisons test showed WJ-MSC > BM-MSC (p = 0.0005) and WJ-MSC > AD-MSC (p < 0.0001) (Fig. 1A).

Wound healing. In HaCaT scratch assays (18 h), the WJ-MSC secretome accelerated closure to 95.36 ± 4.17 %, surpassing AD-MSC (54.15 ± 5.63 %, p = 0.0013), BM-MSC (46.19 ± 12.58 %, p = 0.0003), and control (38.96 ± 17.69 %, p < 0.0001) (Fig. 1B).

Collagen synthesis. Type I procollagen secretion increased with the WJ-MSC secretome (3.34 ± 0.26 μg/mL) versus BM-MSC (2.68 ± 0.26 μg/mL, p = 0.0142), AD-MSC (2.73 ± 0.31 μg/mL, p = 0.0268), and control (1.49 ± 0.21 μg/mL, p < 0.0001) (Fig. 1C). Consistently, qPCR showed stronger up-regulation of COL1A1 (∼2.4-fold) and COL3A1 (∼2.1-fold) with WJ-MSC secretome than with other groups (all p < 0.01), indicating enhanced ECM-remodeling capacity (Fig. 1D).

Anti-inflammatory effects. In LPS-stimulated RAW 264.7 macrophages, the WJ-MSC secretome produced the greatest reduction in IL-6 expression (−27.34 %) relative to BM-MSC (−15.38 %) and AD-MSC (−18.40 %) secretomes; similar trends were observed for COX-2, IL-1β, and TNF-α (Fig. 1E).

Angiogenesis. In HUVEC tube-formation assays, the WJ-MSC secretome promoted network formation (71 ± 6.3 tubes; total tube length 40,306 ± 3128.8 pixels), comparable to the VEGF (100 ng/mL) positive control and greater than AD-MSC (31.6 ± 22.8 tubes; p < 0.0001) and BM-MSC (19.0 ± 16.0 tubes; p = 0.0429) (Fig. 1F).

Antioxidant effects. DCF-DA assays showed reduced intracellular ROS in WJ-MSC secretome-treated cells (Fig. 1G). Total antioxidant capacity was higher with WJ-MSC (1.20 ± 0.08 nmol/μL) than with BM-MSC (0.78 ± 0.05 nmol/μL) or AD-MSC (0.33 ± 0.003 nmol/μL) secretomes (p < 0.0001) (Fig. 1H).

Collectively, these data indicate that the WJ-MSC secretome exhibits greater pro-regenerative, anti-inflammatory, antioxidant, and pro-angiogenic activity than AD-MSC and BM-MSC secretomes, supporting its potential for skin rejuvenation.

3.3. Integrated results of MSC secretomes

To synthesize the findings across individual functional assays, outcomes were summarized in a comparative analysis (Fig. 2).

Fig. 2.

Integrated summary of in-vitro skin-rejuvenation assays comparing MSC-derived secretomes. Results from the functional assays in Fig. 1 are integrated and summarized. (A) Composite scores were calculated by normalizing each assay to the negative control (Vehicle-CM; α-MEM + 5 % hPL, incubated cell-free 48 h and processed identically) and rescaling to 0–100 (higher = greater activity). Under the conditions tested, the WJ-MSC secretome scored higher than AD-MSC and BM-MSC secretomes in most parameters (fibroblast proliferation, wound closure, type I procollagen, ECM-gene expression, anti-inflammatory activity, antioxidant capacity, angiogenesis). Asterisks indicate pairwise differences from one-way ANOVA with Tukey's multiple-comparisons (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (B) Table reports mean ± SEM and Tukey-adjusted p-values for AD vs WJ and BM vs WJ comparisons. Assay-specific replication (independent experiments): proliferation n = 5; wound healing n = 4; type I procollagen ELISA n = 5; COL1A1/COL3A1 qPCR n = 5; IL-6 qPCR n = 3; ROS reduction n = 3; total antioxidant capacity n = 5; tube formation n = 5. Positive controls, where applicable: TGF-β (ECM/procollagen), VEGF (100 ng/mL) (angiogenesis), ascorbic acid/Trolox (oxidative-stress assays).

Across parameters-fibroblast proliferation, wound closure, type I procollagen secretion, ECM-gene expression, anti-inflammatory activity, antioxidant capacity, and angiogenesis-the WJ-MSC secretome consistently showed higher performance than the AD-MSC and BM-MSC secretomes under the conditions tested. For comparative visualization, each assay was normalized to its corresponding control (Vehicle-CM unless stated otherwise) and expressed as a relative score (higher values indicate greater skin-rejuvenation potential) (Fig. 2A).

Quantitative scoring (Fig. 2A) together with tabulated values (Fig. 2B) highlighted greater pro-regenerative, anti-inflammatory, and antioxidant activity with the WJ-MSC secretome, with statistically significant differences versus AD-MSC and/or BM-MSC secretomes in most assays (p < 0.05).

3.4. Secretome profiling and key bioactive components of WJ-MSC secretome

To identify molecular mediators underlying the regenerative activity of MSC-derived secretomes, we performed antibody-array profiling followed by bioinformatic analyses. Heatmaps (Fig. 3A) showed distinct differences in secreted-protein abundance among AD-MSC, BM-MSC, and WJ-MSC secretomes, with the WJ-MSC secretome enriched for proteins linked to skin-relevant biological processes. STRING-based protein-protein interaction (PPI) analysis (Fig. 3B) grouped upregulated proteins into functional clusters encompassing ECM remodeling, angiogenesis, immune regulation, wound healing, oxidative-stress responses, and skin-barrier integrity. Among proteins with a log₂ fold-change >5, the WJ-MSC secretome was particularly enriched for pro-angiogenic mediators (e.g., PROK1, GREM1, NRCAM, THBS1, TGFBI, GDF2, FN1), consistent with potential support of vascular development and tissue repair. Immune-regulatory proteins (ITK, BTK, LTF, PIK3R1, C8G, PTGDS, ApoA4, ENPP2) were also abundant, suggesting immunomodulatory activity. Wound-healing-related factors (COL5A1, COL3A1, FN1, PLG, NF1) and key ECM components (COL5A1, LAMC1, AHNAK, COL1A1, SERPINH1) associated with elasticity and structural integrity were elevated, as were skin-barrier-related proteins (CLDN4, KLF4, FLG2). Finally, antioxidant-associated molecules (ApoA4, FABP1, APOE, S100A9) were upregulated, indicating enhanced capacity to counter oxidative stress. Collectively, these data indicate that the WJ-MSC secretome is enriched for multiple functional protein clusters aligned with regenerative, anti-inflammatory, antioxidant, and pro-angiogenic properties relevant to skin rejuvenation (Fig. 3C). Among these, SERPINH1 and ApoA4 emerged as representative factors; their functional relevance was examined in subsequent experiments.

Fig. 3.

Secretome profiling and key bioactive components of the WJ-MSC secretome.

(A) Antibody-array heatmap of secreted proteins from AD-, BM-, and WJ-MSC secretomes; values are log₂-transformed, normalized intensities expressed as fold-change vs Vehicle-CM (color scale: blue = lower, red = higher). (B) STRING (v11.5) protein-protein interaction networks of proteins enriched in the WJ-MSC secretome; nodes are colored by log₂ fold-change (WJ vs AD/BM) and grouped into GO Biological Process clusters relevant to skin rejuvenation (angiogenesis, immune regulation, wound healing, collagen/ECM organization & elasticity, skin-barrier function, oxidative-stress response). Only high-confidence edges (score >0.7) are shown. (C) GO-based functional classification of significantly upregulated proteins (log₂ fold-change >5), highlighting ApoA4 and SERPINH1 as candidates taken forward for functional validation.

3.5. Quantitative analysis of ApoA4 and SERPINH1 expression

To probe the molecular basis for the greater regenerative activity observed with WJ-MSCs under our conditions, we quantified ApoA4 and SERPINH1 at the mRNA level in MSCs and at the protein level in their secretomes. ApoA4 mRNA was highest in WJ-MSCs (149.83 ± 22.33, relative units), compared with AD-MSCs (100.00 ± 17.92, p = 0.0055 vs WJ-MSCs) and BM-MSCs (36.59 ± 2.94, p < 0.0001) (Fig. 4A). SERPINH1 mRNA showed a similar pattern, with WJ-MSCs (218.07 ± 6.29) exceeding AD-MSCs (100.00 ± 0.84, p < 0.0001) and BM-MSCs (97.75 ± 2.34, p < 0.0001) (Fig. 4A). One-way ANOVA with Tukey's multiple-comparisons test indicated significant group differences, particularly WJ-MSCs > AD-/BM-MSCs. By ELISA, ApoA4 concentration was higher in the WJ-MSC secretome (3.59 ± 0.18 ng/mL) than in BM-MSC (2.85 ± 0.06 ng/mL, p = 0.0006) and AD-MSC (1.85 ± 0.07 ng/mL, p < 0.0001) secretomes (Fig. 4B). SERPINH1 protein levels were also greatest in the WJ-MSC secretome (11.99 ± 0.41 pg/mL) versus BM-MSC (9.05 ± 0.27 pg/mL, p < 0.0001) and AD-MSC (8.28 ± 0.21 pg/mL, p < 0.0001) secretomes; the AD-vs BM-MSC difference was not significant (p = 0.0503) (Fig. 4B).

Fig. 4.

Gene and protein expression of ApoA4 and SERPINH1 across MSC sources.

(A) Relative mRNA expression of APOA4 and SERPINH1 in AD-MSCs, BM-MSCs, and WJ-MSCs by qPCR (GAPDH-normalized; calibration as in Methods). (B) Secreted protein levels of ApoA4 and SERPINH1 in the corresponding secretomes by ELISA. Under the conditions tested, WJ (WJ-MSC secretome) showed higher APOA4/SERPINH1 mRNA and higher ApoA4/SERPINH1 protein than AD (AD-MSC secretome) or BM (BM-MSC secretome). Data are mean ± SEM; one-way ANOVA with Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

These data indicate that WJ-MSCs exhibit higher ApoA4 and SERPINH1 mRNA expression, and that their secretome contains higher levels of the corresponding proteins, suggesting a source-associated enrichment of these factors under the conditions tested.

3.6. SERPINH1 in skin rejuvenation

To evaluate the functional role of SERPINH1 in skin rejuvenation, we performed in-vitro assays quantifying cell proliferation and migration, extracellular-matrix (ECM) remodeling, hydration-associated protein expression, and signaling.

Cell proliferation. In HS68 fibroblasts, recombinant SERPINH1 increased proliferation in a concentration-dependent manner. Relative to untreated controls (101.28 ± 2.26 %), viability rose to 124.36 ± 3.44 % at 0.2 μg/mL (p = 0.0003) and 142.67 ± 5.78 % at 0.5 μg/mL (p < 0.0001) (Fig. 5A). Immunoblotting confirmed a corresponding rise in cell-associated SERPINH1 (2.10 ± 0.59-fold and 2.63 ± 0.94-fold at 0.2 and 0.5 μg/mL, respectively; p = 0.0343 vs control) (Fig. 5B).

Fig. 5.

SERPINH1 in vitro functions relevant to skin rejuvenation.

Baseline control: serum-free assay medium (SFM); Vehicle-CM not used. (A) HS68 fibroblast proliferation (CCK-8) after recombinant SERPINH1 treatment. (B) Immunoblot confirming increased cell-associated SERPINH1 after treatment. (C) HaCaT scratch-wound closure at 18 h; EGF (20 ng/mL) as positive control. (D) Immunoblot of ECM proteins (COL1A1, fibronectin). (E) Immunoblot of hydration-related proteins (HAS2, AQP3); retinoic acid as comparator (positive control). (F) siRNA-mediated SERPINH1 knockdown impairs HaCaT migration. (G) Knockdown reduces TGF-β, TGFβR1, p-Smad2/3, COL1A1, COL3A1, and fibronectin, consistent with dampened TGF-β/Smad signaling and ECM remodeling. Data are mean ± SEM; one-way ANOVA with Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Wound healing. Scratch-wound closure accelerated in a concentration-dependent manner after SERPINH1 treatment; at 18 h, closure reached 96.74 ± 4.85 % at 0.5 μg/mL (p < 0.0001), comparable to EGF (20 ng/mL; 89.33 ± 1.52 %) and greater than untreated controls (32.40 ± 5.84 %) (Fig. 5C).

ECM remodeling. SERPINH1 increased ECM proteins, with COL1A1 (2.18 ± 0.47-fold; p = 0.0112) and fibronectin (2.27 ± 0.51-fold; p = 0.0273) upregulated at 0.5 μg/mL, comparable to TGF-β (Fig. 5D).

Hydration-associated proteins. SERPINH1 upregulated HAS2 (4.63 ± 0.10-fold; p = 0.0032) and AQP3 (2.01 ± 0.30-fold; p = 0.0185) at 0.5 μg/mL, comparable to or exceeding retinoic acid (RA) (Fig. 5E).

siRNA knockdown. siRNA-mediated SERPINH1 silencing reduced keratinocyte migration in scratch-wound assays (Fig. 5F) and decreased TGF-β, TGFβR1/2, phosphorylated Smad2/3, COL1A1, COL3A1, and fibronectin compared with siNC (Fig. 5G).

Collectively, SERPINH1 promotes fibroblast proliferation, accelerates wound closure, enhances ECM protein accumulation, and increases hydration-related proteins in a concentration-dependent manner, whereas its silencing impairs migration and attenuates TGF-β/Smad signaling. These findings implicate SERPINH1 as a functional mediator of skin-regenerative responses under the conditions tested.

3.7. ApoA4 in skin rejuvenation

To assess the role of ApoA4 in skin regeneration, we performed in-vitro assays of keratinocyte migration, inflammatory-gene expression, oxidative stress, total antioxidant capacity, and loss-of-function validation.

Wound healing. In HaCaT scratch-wound assays (18 h), ApoA4 increased closure in a concentration-dependent manner. Compared with the control (52.63 ± 12.22 %), closure reached 57.42 ± 10.66 % at 0.1 μg/mL (p = 0.9379), 77.04 ± 1.17 % at 0.5 μg/mL (p = 0.0212), and 89.34 ± 5.96 % at 1.0 μg/mL (p = 0.0013), with the strongest effects at 0.5 and 1.0 μg/mL (Fig. 6A).

Fig. 6.

ApoA4 in vitro functions relevant to skin rejuvenation.

Baseline control: SFM (no serum/hPL); Vehicle-CM not used. (A) HaCaT scratch-wound assay showing concentration-dependent increases in migration/closure with ApoA4. (B) In LPS-stimulated RAW 264.7 macrophages, ApoA4 reduces mRNA levels of IL-6, COX-2, IL-1β, and TNF-α. (C) DCF-DA staining shows decreased intracellular ROS with ApoA4; representative images confirm fewer ROS-positive cells, comparable to ascorbic acid (10 μM). (D) Immunoblot verifying siApoA4 knockdown. (E) Total antioxidant capacity increased with ApoA4; siApoA4 reduced capacity toward control levels. Total antioxidant capacity was calculated from a Trolox standard curve and is presented as Trolox equivalents (nmol/μL). Data are mean ± SEM; one-way ANOVA with Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Anti-inflammatory activity. In LPS-stimulated RAW 264.7 macrophages, ApoA4 (0.5 μg/mL) reduced mRNA levels of IL-6 (−48.73 %, p < 0.0001), COX-2 (−53.50 %, p < 0.0001), IL-1β (−54.64 %, p = 0.0003), and TNF-α (−53.18 %, p = 0.0003) relative to the LPS-only group (Fig. 6B).

Oxidative stress. DCF-DA staining showed that H₂O₂ increased intracellular ROS, whereas ApoA4 co-treatment reduced ROS in a concentration-dependent manner (Fig. 6C). Immunofluorescence confirmed fewer ROS-positive cells in ApoA4-treated groups, comparable to the antioxidant control (ascorbic acid, 10 μM).

Total antioxidant capacity. ApoA4 increased total antioxidant capacity from 2.43 ± 0.05 nmol/μL (control) to 2.80 ± 0.06, 2.82 ± 0.07, and 2.86 ± 0.01 nmol/μL at 0.1, 0.5, and 1.0 μg/mL, respectively (all p < 0.0001) (Fig. 6E).

Loss-of-function validation. siRNA-mediated ApoA4 knockdown (siApoA4) reduced ApoA4 protein by immunoblot (Fig. 6D). Consistently, siApoA4-transfected fibroblasts displayed antioxidant levels similar to untreated controls, indicating loss of ApoA4-mediated ROS-scavenging activity (Fig. 6E). Moreover, siApoA4 in HaCaT keratinocytes significantly impaired scratch-wound closure compared with siNC (Fig. 6F).

Collectively, ApoA4 enhances keratinocyte migration, suppresses inflammatory cytokines, reduces oxidative stress, and elevates antioxidant capacity, whereas knockdown attenuates these effects. Under our assay conditions, these findings support ApoA4 as a multifunctional mediator contributing to skin-rejuvenation activity within the WJ-MSC secretome.

3.8. Clinical study on WJ-MSC secretome (exploratory single-arm pilot)

To explore the clinical relevance of the in-vitro findings, we conducted an exploratory, single-arm study of topical WJ-MSC secretome in 21 healthy female participants (mean age, 46.95 years; range, 23–63 years). Based on pragmatic considerations and in-vitro performance, the clinical evaluation focused on WJ-MSC secretome. Within-subject analyses over one week showed increases from baseline in an instrument-derived hydration index (Δ = +8.82 units; +35.11 % from baseline; paired t(20) = 16.58, p < 0.0001; 95 % CI, 7.71–9.93; effect size Cohen's d (paired) ≈ 3.62; partial η² ≈ 0.93) and elasticity (Δ = +3.28 percentage points; paired t(20) = 43.84, p < 0.0001; 95 % CI, 3.13–3.44; d (paired) ≈ 9.57; partial η² ≈ 0.99) (Fig. 7A and B). Brightness showed a small change (Δ = +2.36 percentage points; paired t(20) = 5.083, p < 0.0001; 95 % CI, 1.39–3.33; d (paired) ≈ 1.11; partial η² ≈ 0.56) (Fig. 7C). Safety. No treatment-related adverse events were observed in the pilot. In a separate occlusive patch test, topical WJ-MSC secretome was well tolerated: among 33 volunteers, no irritation was observed at 30 min, 24 h, or 48 h post-removal (ICDRG scores all 0; mean skin-reaction score 0.00), yielding a non-irritant classification (Table 2). These preliminary, exploratory observations suggest potential short-term improvements in selected skin parameters following topical WJ-MSC secretome and warrant confirmation in adequately powered, controlled trials with longer follow-up.

Fig. 7.

Exploratory clinical assessment of topical WJ-MSC secretome.

Single-arm pilot in 21 healthy female participants over one week. (A) Hydration: instrument-derived moisture index. (B) Elasticity: biomechanical assessment. (C) Brightness: standardized facial imaging. Data are mean ± SEM; paired tests (paired t-test or Wilcoxon, per Methods) were used for pre-vs post-comparisons; significance thresholds as indicated (∗∗∗∗p < 0.0001).

Table 2.

Primary skin irritation after occlusive patch testing (ICDRG grading) (N = 33)

No treatment-related adverse events were observed in the pilot; a separate Finn-Chambers occlusive patch test in 33 volunteers showed no irritation at 30 min, 24 h, or 48 h (ICDRG scores all 0).

Skin reaction	30 min post-removal	24 h post-removal	48 h post-removal	Mean skin reaction score
Erythema (redness)	0.0	0.0	0.0	0.00
Edema (swelling)	0.0	0.0	0.0
Scaling (desquamation)	0.0	0.0	0.0
Pruritus (itching)	0.0	0.0	0.0
Tenderness/Pain	0.0	0.0	0.0
Burning sensation	0.0	0.0	0.0
Tightness	0.0	0.0	0.0
Stinging	0.0	0.0	0.0

4. Discussion

The WJ-MSC secretome, obtained through master cell banking of umbilical-cord (Wharton's jelly) tissue, showed greater regenerative activity than secretomes derived from adult sources (AD-MSCs and BM-MSCs) under the conditions tested [11]. Despite minimal differences in surface markers and tri-lineage differentiation among MSC sources, WJ-MSCs exhibited enhanced pro-regenerative effects [29]. These advantages likely reflect their ontogenetically younger origin, which is reported to yield secretomes and extracellular vesicles (EVs) enriched in trophic factors and microRNAs that promote cell survival, matrix remodeling, and tissue repair [11,29,30].

WJ-MSCs also displayed stronger immunomodulatory and antioxidant activities than AD- and BM-derived MSCs [31]. In addition, their derivation from umbilical cords-an ethically straightforward, non-invasive source-facilitates consistent, GMP-grade production via master cell banking and minimizes batch-to-batch variability [32,33]. Together, these attributes support WJ-MSCs as an attractive and scalable source for regenerative therapeutics [34].

Secretome profiling identified ApoA4 and SERPINH1 as highly expressed and functionally active mediators within the WJ-MSC secretome. ApoA4 exerted antioxidative and anti-inflammatory effects, including reductions in intracellular ROS and pro-inflammatory cytokine expression [23]. SERPINH1, a collagen-specific chaperone, enhanced ECM remodeling, collagen biosynthesis, and hydration-related proteins (HAS2 and AQP3), thereby supporting skin structural integrity [28,35]. Importantly, secretome experiments incorporated a vehicle-conditioned medium baseline (α-MEM + 5 % hPL, incubated cell-free for 48 h and processed identically), supporting attribution of bioactivity primarily to MSC-secreted factors rather than base medium/hPL components [36].

Although SERPINH1 (HSP47) is canonically an ER-resident, collagen-specific chaperone, our functional data (Fig. 5A–G) indicate non-canonical extracellular activity relevant to skin rejuvenation [37]. We consider three, not mutually exclusive, mechanisms. First, WJ-MSCs may release SERPINH1 via EVs or other unconventional secretion pathways, enabling transfer to recipient skin cells [38]. Second, exogenous SERPINH1 may be internalized by keratinocytes and fibroblasts and thereby augment intracellular collagen folding/processing, resulting in increased ECM deposition and improved wound closure [37]. Third, SERPINH1 may bind procollagen/collagen in the pericellular space or ECM, facilitating fibrillogenesis and fibril stability [38]. Consistent with these possibilities, exogenous SERPINH1 increased cell-associated SERPINH1 and enhanced COL1A1/fibronectin and hydration-related proteins (HAS2/AQP3), whereas SERPINH1 knockdown attenuated TGF-β/Smad signaling and ECM markers (Fig. 5). Future studies separating EV from soluble fractions and blocking cellular uptake will delineate the predominant mode of action.

From a translational perspective, the short-term, single-arm human pilot showed within-subject improvements in hydration and elasticity after topical WJ-MSC secretome [39,40]. With appropriate paired analyses now applied, these observations, together with the in-vitro data, suggest potential clinical benefit; however, they remain exploratory and require confirmation in adequately powered, controlled trials with extended follow-up and formal safety monitoring [41]. Several limitations warrant consideration. First, whereas the AD-MSC and BM-MSC secretomes were derived from single-donor lines, the WJ-MSC secretome was generated by pooling conditioned media from three independent WJ-MSC donors in our master cell bank [42]. Consequently, tissue-of-origin effects may be confounded by donor-specific variability, and the asymmetric design (pooled WJ vs single-donor AD/BM) could bias comparisons; thus, results may not fully generalize across donors. Although the sample size was limited, the direction and magnitude of effects were consistent and reproducible across independent experiments (assay-specific n values are provided in the Methods and in Fig. 2B). Second, the molecular cargo of the WJ-MSC secretome (e.g., microRNAs and EV subtypes) was not comprehensively characterized; expanded cargo profiling and validation in longer-term, placebo-controlled studies would better substantiate translational potential. Third, the human component comprised a short-term, single-arm pilot; the observed within-subject improvements should therefore be regarded as exploratory, pending confirmation in controlled trials [43]. Fourth, we used established skin-derived cell lines (HaCaT keratinocytes and HS68 dermal fibroblasts) rather than primary cells; while widely used for early mechanistic work, primary cells may better recapitulate in-vivo physiology [44].

Fifth, the anti-inflammatory screening was performed in LPS-stimulated RAW 264.7 murine macrophages, which are widely used for initial anti-inflammatory evaluation and have been employed in skin-related inflammation models. However, because RAW 264.7 is a murine cell line, species-specific differences should be considered when extrapolating to human cutaneous immune responses [45,46].

In summary, the WJ-MSC secretome demonstrates broad pro-regenerative activity in vitro and preliminary clinical signals in humans, with ApoA4 and SERPINH1 emerging as key mediators. Future work should include multiple donors per source (with per-donor analyses), mechanistic dissection of EV versus soluble compartments and cellular-uptake pathways, and randomized, controlled clinical trials to establish efficacy, durability, and safety.

5. Conclusion

This study shows that the WJ-MSC secretome had greater in-vitro pro-regenerative, antioxidant, and anti-inflammatory activity than adult-source MSC secretomes (AD-MSC and BM-MSC) under the conditions tested, including enhanced dermal-fibroblast proliferation and ECM remodeling, with ApoA4 and SERPINH1 emerging as contributory mediators. In an exploratory, short-term, single-arm study, topical WJ-MSC secretome was associated with within-subject improvements in hydration, elasticity, and brightness; these preliminary observations require confirmation in adequately powered, controlled trials with longer follow-up and formal safety monitoring. Collectively, the findings support the WJ-MSC secretome as a cell-free candidate for skin rejuvenation. Future work should include donor-level replication, molecular refinement with separation of EV versus soluble fractions (including EV-based enhancement), dose/formulation/stability optimization with GMP-grade release criteria, and randomized, comparative trials to establish efficacy, durability, and safety and to inform regulatory evaluation.

Ethics approval and consent to participate

Human umbilical cords were obtained from three healthy donors following normal pregnancies, with written informed consent, in accordance with protocols approved by the Institutional Review Board (IRB) of Samsung Medical Center (IRB No. 2016-07-102-037, approved on August 9, 2022). The clinical study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the IRB of the Korea Institute of Dermatological Sciences (IRB No. KIDSIRB-2024-0664, approved on May 13, 2024). Written informed consent was obtained from all participants prior to enrollment.

Author contributions

Na Eun Lee: Conceptualization, Methodology, Investigation, Data Curation, Formal Analysis, Writing Original Draft.

Jong Ik Hwang: Clinical Investigation, Resources, Patient Recruitment, Visualization, Writing Review & Editing.

Chi Young Bang: Methodology, Validation, Supervision, Project Administration, Writing Review & Editing.

Eun Hee Kim: Data Curation, Formal Analysis, Visualization, Statistical Analysis.

Oh Young Bang: Conceptualization, Supervision, Funding Acquisition, Project Administration, Writing Review & Editing, Correspondence.

Declaration of competing interest

Oh Young Bang, Eun Hee Kim, and Na Eun Lee are affiliated with S&E bio, Inc. Oh Young Bang is the Chief Executive Officer (CEO) of S&E bio, Inc. In the Methods, the WJ-MSC secretome evaluated in this study is referred to by the trade name SNE-Secretome®. S&E bio, Inc. has filed a patent application with the Korean Intellectual Property Office (KIPO) related to the subject matter of this manuscript (Application No. 10-2025-0020820; filed; not yet granted), and Na Eun Lee is listed as an inventor on this application. All other authors declare no competing interests.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

figs1.