Efficacy and safety of umbilical cord-derived mesenchymal stem cell-conditioned media for preventing and treating skin aging

Abstract

BACKGROUND

Research has been increasingly conducted on the connection between mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) and aging. However, most studies have focused on adipose-derived MSC-CM (ADMSC-CM), resulting in a research bias. We hypothesized that umbilical cord-derived MSCs, being younger than adipose-derived MSCs, would be more suitable for overcoming aging-related processes.

AIM

To assess the efficacy and safety of umbilical cord-derived MSC-CM (UCMSC-CM) for preventing and treating skin aging.

METHODS

In vitro and in vivo studies were conducted to compare UCMSC-CM with ADMSC-CM, the most studied active aging-preventive conditioned medium to date. Additionally, the most effective delivery method of UCMSC-CM for aged skin was identified.

RESULTS

UCMSC-CM had a higher content of effective factors, stimulated higher proliferation of fibroblasts, and strongly inhibited melanin production in B16F1 cells. In aged mice, UCMSC-CM application increased skin thickness, the number of Ki-67-positive cells, and the area of collagen deposition. UCMSC-CM was more effective than ADMSC-CM in preventing and treating skin aging. Additionally, a safety evaluation of UCMSC-CM performed in various animal models indicated that it was safe even when used directly on the skin.

CONCLUSION

UCMSC-CM is effective and safe for preventing and treating skin aging.

Key Words: Skin aging; Mesenchymal stem cell; Umbilical cord-derived mesenchymal stem cell; Mesenchymal stem cell-conditioned medium; Treating skin

Core Tip: Umbilical cord-derived mesenchymal stem cell-conditioned medium (UCMSC-CM) contains higher levels of effective factors than adipose-derived mesenchymal stem cell-conditioned medium, enhancing skin fibroblast proliferation and inhibiting melanin production in vitro. It promotes skin regeneration more effectively than adipose-derived mesenchymal stem cell-conditioned medium in aged skin in vivo. Subcutaneous injection of UCMSC-CM was the most effective method for promoting skin regeneration in aged skin in vivo. A non-clinical safety evaluation indicated that UCMSC-CM is a safe substance. These findings highlight UCMSC-CM as a promising and effective treatment for skin aging.

INTRODUCTION

Skin aging and rejuvenation have attracted attention as social issues due to the constant pursuit of youth and beauty[1]. Skin aging, which manifests as wrinkles, loss of elasticity, discoloration, and pigmentation, is a visible sign of aging. Thus, numerous studies have attempted to overcome these changes[2]. Skin aging is caused by intrinsic and extrinsic factors[3]. Aging due to intrinsic factors is a natural consequence of time[3]. The skin has an antioxidant system that protects the cells by removing free radicals, which are strongly and positively correlated with skin aging[4,5]. However, the antioxidant system weakens with age and is further weakened by extrinsic factors such as ultraviolet rays[6], which can accelerate the aging process[3]. Various methods have been developed to prevent, suppress, or treat skin aging. The progression of aging can be suppressed by blocking ultraviolet rays with, for example, sunscreen and protective clothing[3,7]. Therapeutic methods based on the removal of free radicals from the body with antioxidants have been developed as supplements to treatments[2]. In addition, local injections of compounds such as botulinum toxin and fillers, chemical peels, dermabrasion, laser resurfacing, and plastic surgery can provide temporary or long-term improvements in the appearance of aging[8-10].

As society ages and becomes wealthier, there is a growing interest in the prevention and treatment of skin aging, leading to increased demand for anti-aging products[1]. In line with this trend, various substances that affect the physiological activity of skin cells, such as vitamins, hydroxy acids, peptides, and growth factors, have been developed. Technologies such as cell culture and nanotechnology are being incorporated for more efficient delivery of these substances to the skin[11,12]. Media conditioned with cultured stem cells have garnered attention for the prevention and treatment of skin aging[13-15]. Mesenchymal stem cell (MSC)-conditioned medium contains growth factors such as epidermal growth factor, vascular endothelial growth factor (VEGF), and fibroblast growth factor[15], which are effective at preventing and treating skin aging by promoting the growth of skin fibroblasts, increasing extracellular matrix biosynthesis (e.g., collagen and elastin), and exerting antioxidant effects[16-20]. Given its potential, MSC-conditioned medium (MSC-CM) has received increasing attention, with several active studies and products being developed. However, our understanding of its safety and effectiveness remains insufficient[12,14].

In this study, we used umbilical cord-derived MSC-CM (UCMSC-CM) and evaluated its anti-aging effects on aged mouse skin. As cells age, they produce substances related to aging that have the potential to accelerate aging in other cells[21-24]. Therefore, the anti-aging effects of conditioned medium derived from younger MSCs may be more effective. Based on this, we hypothesized that UCMSC-CM may be more beneficial for aged skin than adipose-derived MSC (ADMSC)-CM (ADMSC-CM), the most studied medium to date. We compared the effective factors present in ADMSC-CM and UCMSC-CM and confirmed the effects of UCMSC-CM on skin fibroblast growth and melanin production in B16F1 cells. To evaluate its anti-aging effects in vivo, UCMSC-CM was applied to the aged skin of mice. Furthermore, in collaboration with external institutions, we evaluated the safety of UCMSC-CM in various animal models.

MATERIALS AND METHODS

Adipose tissue and umbilical cord procurement

The adipose tissue was donated by a researcher in our laboratory, and the umbilical cord was donated by the Obstetrics and Gynecology Department of Lynn Woman’s Hospital (Seoul, South Korea). Consent for the donation of the umbilical cord was obtained from the donor’s mother. The safety of the adipose tissue and umbilical cord was confirmed through the medical history and blood and urine tests of the donor and mother, respectively.

Isolation and culture of ADMSCs

Adipose tissue (40 mL) was placed in a 50-mL conical tube and centrifuged at 900 × g for 3 minutes. Then, only the supernatant (yellow) was transferred to a new 50-mL conical tube, and 0.75% collagenase type I (10% v/v) (Gibco, 17100017, NY, United States) was added to the adipose tissue in a new 50-mL conical tube and mixed. The mixed solution was incubated in a 37 °C, 5% CO₂ incubator for 30 minutes. The mixture was shaken vigorously for 5 minutes. The mixed solution was centrifuged at 900 × g for 3 minutes, and the supernatant was removed and washed three times with 1 × phosphate-buffered saline (PBS). Ten milliliters of 1 × PBS and 5 mL of lymphoprep (STEMCELL Technologies, 07801, Canada) were carefully added and centrifuged at 900 × g for 30 minutes. Afterward, among the three divided layers, only the middle layer (white) was collected with a pipette, transferred to a new conical tube, washed with 1 × PBS, and cultured in MSC culture medium [DMEM-F12 (Biowest, L0092-500, France) 89% + fetal bovine serum (FBS) 10% (Biowest, S1480, France) + antibiotic/antimycotic solution (Welgene, Ls203-01, South Korea) 1%] in a 37 °C, 5% CO₂ incubator. The medium was replaced every 3 days. Subculturing was performed using 0.5% trypsin-EDTA (ThermoFisher Scientific, 25200056, MA, United States) at 80% confluence.

Isolation and culture of UCMSCs

UCMSCs were isolated from donated umbilical cords. Umbilical cords were disinfected with 70% ethanol and washed with 1 × PBS. The three vessels of the umbilical cord were removed, and the umbilical cord was cut into small pieces using surgical scissors. The cut tissues were placed in a 50-mL conical tube containing 0.75% collagenase type I with DMEM-F12, further minced with surgical scissors, and incubated in a 37 °C, 5% CO₂ incubator for 1 hour. The mixed solution containing umbilical cord tissue was centrifuged at 900 × g for 3 minutes, and the supernatant was removed and washed three times with 1 × PBS. Afterward, MSC-CM was added and cultured in a 37 °C, 5% CO₂ incubator. After approximately 2 weeks, stem cells attached to the bottom of the culture dish were observed. After 1-2 weeks, the cells were harvested using 0.5% trypsin-EDTA. During this period, the medium was replaced every 3 days. The cells were sub-cultured in 0.5% trypsin-EDTA at 80% confluence.

Quality evaluation of ADMSCs and UCMSCs

The expression level of MSC-specific proteins [CD73 (eBioscience, 11-0739-42, CA, United States) ≥ 70%, CD90 (BD Biosciences, 555595, NJ, United States) ≥ 90%, and CD105 (eBioscience, E01423-1633, CA, United States) ≥ 90%] were confirmed in the isolated ADMSCs and UCMSCs (Supplementary material). The expression levels of MSC-specific proteins were measured using CyFlow^® Cube 6 (Sysmex, IL, United States) and FCS Express 5 software (De Novo Software, CA, United States).

Production of the MSC-CM

The MSC-CM production process is summarized in Supplementary material. After seeding 3 × 10⁷ ADMSCs or UCMSCs into a 10-chamber plate (Corning, 3271, NY, United States), the cells were cultured in a 37 °C, 5% CO₂ incubator for 1 week with 1000 mL of MSC culture medium. The medium was replaced with the same medium on day 4. After 1 week, the cells were washed twice with PBS and cultured in 1000 mL of DMEM-F12 without phenol red (Gibco, 21041025, NY, United States) in an incubator at 37 °C and 5% CO₂ for another week. The medium was not changed during this period. After 1 week, the cultured medium was harvested by filtering through a 0.22-μM 1000-mL bottle-top vacuum filter (Corning, 431174, NY, United States) and stored at 2-6 °C for 1 day.

Measurement of the total protein concentration of MSC-CM

To measure the total protein concentration in MSC-CM, albumin standard (Thermo Scientific, 23210, MA, United States) was prepared with two-fold serial dilutions from 80 μg/mL to 5 μg/mL with distilled water (DW), and DW was used as a blank. Then 350 μL of standard, blank, and MSC-CM samples were placed in a 1.5-mL tube, and 350 μL of Bradford reagent was added and mixed. Each mixed solution was divided into three 200-μL wells in a 96-well plate, and absorbance was measured at 595 nm. Normalized values were calculated by subtracting the absorbance of the blank sample from the measured absorbance of each sample. A standard curve for the absorbance and protein concentration was created using the measured values of the standard samples, and the concentration of MSC-CM was calculated by substituting the absorbance of MSC-CM. The protein concentration was measured in ppm.

Measurement of the number of effective factors in MSC-CM

Factors affecting the MSC-CM were analyzed using an enzyme-linked immunosorbent assay kit (R&D Systems, MN, United States). Each factor was analyzed according to the kit protocol [basic fibroblast growth factor (bFGF): PDFB50, VEGF: PDVE00, hepatocyte growth factor (HGF): PDHG00B, keratinocyte growth factor (KGF): DKG00, platelet-derived growth factor (PDGF)-AA: DY221, transforming growth factor-beta 1 (TGF-β1): PDB100C, fibrin: DFBN10, and collagen type I: DY6220-05]. Enzyme-linked immunosorbent assay was performed using a Multiskan Spectrum (Thermo Scientific, MA, United States) plate reader.

Evaluation of fibroblast growth

The fibroblasts used in this study were obtained from the skin of a man in his fifties. For fibroblast growth assays, 1 × 10⁵ cells were seeded in a 100-mm TC-treated culture dish (Corning, 430167, NY, United States) and cultured for 3 days in an incubator at 37 °C and 5% CO₂. At this time, cells were cultured in a fibroblast culture medium [MEM (Gibco, 11095080, NY, United States) 88% + FBS 10% + sodium pyruvate 1% (Gibco, 11360070, NY, United States) + antibiotic/antimycotic solution 1%], and, depending on the group, 30 ppm of ADMSC-CM or UCMSC-CM was added at 10% of the total volume of the medium. The doubling time of fibroblasts was calculated by measuring the number of cells 72 hours after seeding.

Comparison of cell amount with and without MSC-CM

Fibroblasts were cultured for 1 month in a fibroblast culture medium containing 10% ADMSC-CM or UCMSC-CM. Each adapted fibroblast (cultured in fibroblast culture medium, cultured in fibroblast culture medium with 10% ADMSC-CM, or cultured in fibroblast culture medium with 10% UCMSC-CM) was seeded at 1.6 × 10⁴ cells per well of a 96-well plate and cultured in an incubator at 37 °C and 5% CO₂. After 18 hours, the medium was removed, and the cells were washed with PBS. After adding 100 μL of each type of medium per well according to each condition, 10 μL of EZ-CYTOX (DoGenBio, EZ-3000, South Korea) was additionally added and cultured in an incubator at 37 °C and 5% CO₂. After 6 hours, absorbance was measured at 450 nm using a Multiskan Spectrum spectrophotometer. Three types of blank were used (fibroblast culture medium, fibroblast culture medium with 10% ADMSC-CM, and fibroblast culture medium with 10% UCMSC-CM).

Melanin production inhibition test

B16F1 cells (ATCC, CRL-6323, VA, United States) were seeded at 1 × 10⁵ cells/well in a 6-well clear TC-treated multiple-well plate (Corning, 3516, NY, United States) and incubated for 24 hours in an incubator at 37 °C and 5% CO₂. B16F1 cells were cultured in 2 mL B16F1 culture medium [FluoroBrite^TM DMEM 89% (Gibco, A1896701, NY, United States) + FBS 10% + antibiotic-antimycotic solution 1%] per well. Subsequently, 10% of 30 ppm ADMSC-CM or UCMSC-CM was added depending on the treatment group. After washing twice with PBS, the B16F1 culture medium (with the addition of ADMSC-CM or UCMSC-CM according to the group) was incubated with alpha-melanocyte-stimulating hormone and cultured in an incubator at 37 °C and 5% CO₂ for 2 days. Each culture medium from the group was transferred to a 15-mL tube and washed twice with PBS. After harvesting the cells using 0.25% trypsin-EDTA, the number of cells was counted, and the supernatant was removed after centrifugation (900 × g for 3 minutes) to obtain a cell pellet. After adding 2 mL of 1 N sodium hydroxide solution to the pellet and gentle mixing, the mixed solutions were incubated at 60 °C for 5 minutes. The solutions were centrifuged (900 × g for 3 minutes), and the supernatants were collected and combined with a previously collected culture medium. The cell lysate solution was mixed with the culture medium to calculate the total amount of melanin produced by the B16F1 cells. The absorbance of the mixed solution was measured at 490 nm, and melanin was used as the measurement standard. The amount of melanin synthesized per cell was calculated from the measured absorbance values.

Murine model and in vivo efficacy evaluation

All the mice were housed in a pathogen-free environment until the end of the test. Four-week-old male BALB/c mice were used for efficacy evaluation at 96 weeks of age. Five treatment groups were established for the in vivo efficacy evaluation: DW application [control (n = 3)], ADMSC-CM application [AD-AP (n = 4)], UCMSC-CM application [UC-AP (n = 5)], UCMSC-CM subcutaneous injection [UC-SC (n = 5)], and UCMSC-CM tail-vein injection [UC-IV (n = 4)]. All mice of the control, AD-AP, UC-AP, and UC-SC groups had their back hair removed, and test materials (DW, ADMSC-CM, and UCMSC-CM) were applied or injected daily for 2 weeks at 100 μL per time. The concentration of MSC-CM was 30 ppm, and each mouse in the UC-SC and UC-IV groups was injected under respiratory anesthesia with isoflurane. For the control, AD-AP, and UC-AP groups, the test materials were applied to regions where the fur was removed from the backs of the mice. For the UC-SC group, 25 μL of UCMSC-CM was injected into each of the four back sites (upper left, upper right, lower left, and lower right, Supplementary material). For the UC-IV group, 100 μL of UCMSC-CM was injected into the tail vein each time. After treatment with the test materials for 2 weeks, all mice were allowed to recover in their cages. After 2 weeks, the mice were euthanized with CO₂ gas, and their skin tissues were collected and submerged in a 4% paraformaldehyde solution for fixation. The animal studies were reviewed and approved by the Ethics Committee of the Seoul National University Bundang Hospital Institutional Animal Care and Use Committee in Bundang, South Korea (BA-2207-347-001-01). All animal experiments were performed in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee.

Histopathology and immunohistochemistry

Epidermal and dermal thickness measurement: Slides of each collected skin tissue sample were prepared, and hematoxylin-eosin staining was performed. The stained slides were imaged using a digital camera (DS-Ri2, Nikon, Japan) connected to an optical microscope (Eclipse Ni, Nikon). Three slides were prepared for each tissue, and images were captured randomly. The epidermal and dermal thicknesses were measured randomly at three points in each image using an image analysis program (NIS-Element BR; Basic Research software, Nikon), and statistical analyses were performed.

Count of the number of Ki-67-positive cells: Slides containing skin tissue samples were immunostained with Ki-67 antibody (Abcam, ab16667, United Kingdom). The images were captured using a digital camera (DS-Ri2, Nikon) connected to an optical microscope (Eclipse Ni, Nikon). Three slides were prepared for each tissue, and images were captured randomly. The number of Ki-67-positive cells per unit area was measured using an image analysis program (NIS-Element BR, Basic Research software, Nikon), and statistical analysis was performed.

Measurement of the area of collagen deposition: Masson’s trichrome staining was performed on collected skin tissues. Images of slides containing tissue samples were captured using a digital camera (DS-Ri2, Nikon) connected to an optical microscope (Eclipse Ni, Nikon). Three slides were prepared for each tissue, and images were obtained randomly. The collagen deposition area was measured using an image analysis program (NIS-Element BR, Basic Research Software, Nikon), and statistical analysis was performed.

Measurement of β-galactosidase level per cell: Two serial sections were prepared from the paraffin-embedded skin tissue blocks, and two slides were prepared for each section. One slide was immunostained with β-galactosidase antibody (Proteintech, 15518-1-AP, IL, United States), and the other was immunostained with a Ki-67 antibody. The images were captured using a digital camera (DS-Ri2, Nikon) attached to an optical microscope (Eclipse Ni, Nikon). Five slides were prepared for each tissue, and images were captured randomly. The epidermis region was delineated in each image, and the area of β-galactosidase deposition or the number of Ki-67-positive cells was quantified using an image analysis program (NIS-Element BR, Basic Research software, Nikon), and statistical analysis was performed.

Statistical analysis

One-way analysis of variance was performed to compare three or more groups. The significance of the comparison between the two groups was determined using the following procedure. First, a F-test was performed to determine whether the variances were equal. We then performed an unpaired two-tailed t-test based on the F-test results. Differences between groups were considered significant if the P-value was < 0.05.

Safety evaluation of UCMSC-CM

The safety of UCMSC-CM was evaluated based on 10 assessment items by an external institution, CorestemChemon (South Korea).

RESULTS

Comparison of effective factors in UCMSC-CM and ADMSC-CM

MSC-CM contains various factors that are responsible for the prevention and treatment of skin aging. However, the type and content of these factors may vary depending on the type of MSC and may affect the anti-aging effect on aged skin[21]. We compared the levels of effective factors between ADMSC-CM and UCMSC-CM (Figure 1, Supplementary material). The levels of these effective factors in UCMSC-CM were generally significantly higher than those in ADMSC-CM (bFGF: 5.00 times, VEGF: 2.12 times, HGF: 8.19 times, KGF: 2.41 times, PDGF-AA: 1.35 times, TGF-β1: 2.03 times, fibronectin: 2.59 times, and collagen type I: 6.74 times).

Figure 1 Comparison of effective factors for the prevention and treatment of skin aging in adipose-derived mesenchymal stem cell-conditioned medium and umbilical cord-derived mesenchymal stem cell-conditioned medium. Data were quantified using enzyme-linked immunosorbent assay. bFGF: Basic fibroblast growth factor; VEGF: Vascular endothelial growth factor; HGF: Hepatocyte growth factor; KGF: Keratinocyte growth factor; PDGF-AA: Platelet-derived growth factor-AA; TGF-β1: Transforming growth factor-beta 1; ADMSC-CM: Adipose-derived mesenchymal stem cell-conditioned medium; UCMSC-CM: Umbilical cord-derived mesenchymal stem cell-conditioned medium.

Enhanced proliferation ability of skin fibroblasts by UCMSC-CM

Effective factors enhance the growth of various types of skin cells (e.g., fibroblasts and keratinocytes)[25]. When MSC-CM was added to the culture medium, the proliferative capacity of skin fibroblasts increased (Figure 2A). The enhancement in proliferative ability was greater in fibroblast culture medium supplemented with UCMSC-CM than that supplemented with ADMSC-CM. Consequently, the doubling time of skin fibroblasts cultured with MSC-CM also significantly decreased (control: 17.27 hours, ADMSC-CM: 15.11 hours, and UCMSC-CM: 13.98 hours) (Figure 2B, Supplementary material). In addition, the number of skin fibroblasts cultured with MSC-CM also increased in the WST analysis results (compared to the control, ADMSC-CM: 1.37 times, UCMSC-CM: 2.16 times) (Figure 2C, Supplementary material).

Figure 2 Enhanced proliferation ability of skin fibroblasts by umbilical cord-derived mesenchymal stem cell-conditioned medium. A: Fibroblasts cultured for 72 hours in fibroblast medium containing 10% adipose-derived mesenchymal stem cell (ADMSC-CM) or umbilical cord-derived mesenchymal stem cell-conditioned medium (UCMSC-CM). Scale bar = 250 μm; B: Doubling time, calculated from cell growth curves, of fibroblasts cultured in fibroblast medium with 10% ADMSC-CM or UCMSC-CM; C: WST analysis results of fibroblast culture in fibroblast media with 10% ADMSC-CM or UCMSC-CM. ADMSC-CM: Adipose-derived mesenchymal stem cell-conditioned medium; UCMSC-CM: Umbilical cord-derived mesenchymal stem cell-conditioned medium.

Inhibition of melanin production by UCMSC-CM

The effective factors in MSC-CM inhibit melanogenesis by promoting the proteasomal degradation of microphthalmia-associated transcription factors[26]. When MSC-CM was added to the B16F1 culture medium, melanin production decreased (Figure 3A). Additionally, UCMSC-CM demonstrated a significantly stronger inhibitory effect compared to ADMSC-CM (Figure 3B, Supplementary material). Melanin synthesis in B16F1 cells was reduced by 35% in cells exposed to ADMSC-CM and by 63% in cells exposed to UCMSC-CM compared to that in the control.

Figure 3 Inhibition of melanin production by umbilical cord-derived mesenchymal stem cell-conditioned medium. A: B16F1 cells cultured in B16F1 culture media with α-melanocyte-stimulating hormone and 10% adipose-derived mesenchymal stem cell or umbilical cord-derived mesenchymal stem cell-conditioned medium. Scale bar = 250 μm; B: Comparison of the amount of melanin synthesized by B16F1 cells cultured with 10% adipose-derived mesenchymal stem cell or umbilical cord-derived mesenchymal stem cell-conditioned medium. Data were quantified using enzyme-linked immunosorbent assay. ADMSC-CM: Adipose-derived mesenchymal stem cell-conditioned medium; UCMSC-CM: Umbilical cord-derived mesenchymal stem cell-conditioned medium.

Effect of UCMSC-CM on aged skin

To determine the effects of UCMSC-CM on aged mouse skin, mice were divided into five groups according to UC-MSC treatment: Control, AD-AP, UC-AP, UC-SC, and UC-IV. The test substances were administered daily to the mice in each group for two weeks. The mice were monitored for an additional two weeks, and their skin tissues were obtained and analyzed (Figure 4A). Under the same conditions, the MSC-CM groups (AD-AP and UC-AP groups) effectively induced changes in aged skin compared to the control group. Mice treated with MSC-CM exhibited greater epidermal and dermal thickness, a higher number of Ki-67-positive cells, and a large area of collagen deposition compared to the control group (Figure 4B-E, Supplementary material). These effects were more pronounced in UCMSC-CM-treated animals compared to ADMSC-CM-treated animals: Epidermal thickness significantly increased by 93.37% (ADMSC-CM: 12.03%), and dermal thickness significantly increased by 36.18% (ADMSC-CM: 14.08%) compared to the control group. Although the number of Ki-67-positive cells per unit area increased by 59.48% in UCMSC-CM (ADMSC-CM: 45.30%), the collagen deposition area increased by 40.58% in UCMSC-CM (ADMSC-CM: 34.56%) compared to that in the control group. However, there was no significant difference between the UCMSC-CM- and ADMSC-CM-treated groups in terms of the number of Ki-67-positive cells per unit area or collagen deposition area.

Figure 4 In vivo effectiveness test of umbilical cord-derived mesenchymal stem cell-conditioned medium. A: Representative histological images of aged mouse skin stained with hematoxylin-eosin, Ki-67, and Masson’s Trichrome stains. Scale bar = 50 μm; B-E: Quantitative analysis of epidermal thickness (B), dermal thickness (C), number of Ki-67-positive cells per unit area (D), and collagen deposition area (E) according to the type of reagent; F-I: Quantitative analysis of epidermal thickness (F), dermal thickness (G) number of Ki-67-positive cells per unit area (H), and collagen deposition area (I) according to the method used for umbilical cord-derived mesenchymal stem cell-conditioned medium administration; J: Serial sections of aged mouse skin stained with Ki-67 and β-galactosidase. Scale bar = 20 μm; K: Quantification of β-galactosidase expression per epidermal cell. All quantitative data (B-I and K) were obtained from color-based image analysis of stained tissue sections. NS: Not significant; AD-AP: Adipose-derived mesenchymal stem cell-conditioned medium application; UC-AP: Umbilical cord-derived mesenchymal stem cell-conditioned medium application; UC-SC: Umbilical cord-derived mesenchymal stem cell-conditioned medium subcutaneous injection; UC-IV: Umbilical cord-derived mesenchymal stem cell-conditioned medium tail-vein injection; ADMSC-CM: Adipose-derived mesenchymal stem cell-conditioned medium; UCMSC-CM: Umbilical cord-derived mesenchymal stem cell-conditioned medium.

Next, to confirm the effects of UCMSC-CM on aged skin, the UCMSC-CM groups (UC-AP, UC-SC, and UC-IV) were compared to the control group (Figure 4F-I, Supplementary material). Although the UC-IV group was not significantly different in all analytical tests, the UC-AP and UC-SC groups showed significant differences compared with the control group. The effect of UCMSC-CM on the aged skin was more pronounced when injected subcutaneously than when applied directly to the skin. Epidermal thickness increased by 305.26% (application: 93.37%), dermal thickness increased by 35.22% (application: 36.18%), the number of Ki-67-positive cells per unit area increased by 87.99% (application: 59.48%), and collagen deposition increased by 47.23% (application: 40.58%) compared to the control group. However, no significant differences were observed between the UC-AP and UC-SC groups, except for epidermal thickness.

To determine whether the changes observed in the UC-AP and UC-SC groups represented fundamental changes at the cellular level, serial sections were stained with β-galactosidase and Ki-67 (Figure 4J). The analysis of β-galactosidase and Ki-67 was restricted to the epidermis to avoid interference from β-galactosidase expression in hair follicle cells (Figure 4K, Supplementary material). In all groups treated with UCMSC-CM, the β-galactosidase level per cell significantly decreased compared to that in the control group, with reductions of 51.8% and 78.52% in the UC-AP and UC-SC groups, respectively.

Safety evaluation of UCMSC-CM

In terms of safety, 10 evaluations were performed to confirm the safety of UCMSC-CM (Table 1). Seven assessments were conducted to confirm skin safety, including the eye mucosa, and three to confirm genotoxicity. The results confirmed that UCMSC-CM exhibited no skin or genotoxic effects.

Table 1 Safety evaluation of umbilical cord-derived mesenchymal stem cell-conditioned medium.

Toxicity	Test	Target	Result
Toxicity for skin	Skin irritation test	Rabbit	Pass
	Eye irritation test	Rabbit	Pass
	Skin sensitization test	Guinea pig	Pass
	Photosensitization test	Guinea pig	Pass
	Phototoxicity test	Guinea pig	Pass
	Single dose toxicity	Rat	Pass
	Repeated dose toxicity	Rat	Pass
Genotoxicity	Micronucleus test	Mouse	Pass
	Bacterial reverse mutation test	Escherichia coli (WP2 uvrA), Salmonella typhimurium (TA98, TA100, TA1535, and TA1537)	Pass
	Chromosomal abnormality test	Chinese hamster lung cell	Pass

DISCUSSION

MSC-CM contains effective factors, such as bFGF, VEGF, HGF, KGF, PDGF-AA, TGF-β1, fibronectin, and collagen type I, that have beneficial effects on aging skin. These factors stimulate skin regenerative functions by influencing the proliferation, differentiation, migration, and synthesis of extracellular matrix proteins[27-30]. We hypothesized that these factors might be positively correlated with the anti-aging effect in aged mouse skin. The type and content of active ingredients in MSC-CM depend on both the MSC type and the level of aging[30-32]. As MSCs age, the levels of aging-related factors increase, which can affect surrounding cells. Therefore, conditioned medium derived from younger cells may have a stronger anti-aging effect on the skin of aged mice. Therefore, UCMSC-CM was selected over the more commonly studied ADMSC-CM. As expected, the content of effective factors was higher in UCMSC-CM compared to ADMSC-CM, and the levels of growth factors positively correlated with the enhancement of skin fibroblast proliferation and inhibition of melanin production in B16F1 cells.

Aged mice were used to determine the direct effects of UCMSC-CM on aged skin. As the skin ages, the thickness of the epidermis and dermis[32,33], number of Ki-67-positive cells, which affects regeneration of the epidermal layer[34], and amount of proteins that support the skin such as collagen and elastin, which affects moisture content and elasticity[35-37], decrease. The 96-week-old male BALB/c mice used in this study represent older adults in humans and exhibit aging-related phenotypes. As expected, these phenotypes were confirmed to improve in the skin of aged mice treated with MSC-CM compared to the control group. This effect was more pronounced in the UCMSC-CM-treated group than in the ADMSC-CM-treated group, likely due to the higher content of factors in UCMSC-CM that benefit aged skin.

There is a limit to the effective factors in MSC-CM delivered inside the skin that pass through the skin barrier[38]. Therefore, we treated UCMSC-CM in three ways and compared their effects to find a method to deliver effective factors to the skin UCMSC-CM. To confirm this, UCMSC-CM was administered via: (1) Skin application (UC-AP); (2) Subcutaneous injection (UC-SC); or (3) Tail vein injection (UC-IV). Skin tissues were obtained from aged mice in each group, and changes in epidermal thickness, dermal thickness, number of Ki-67-positive cells, and area of collagen deposition were analyzed. Compared with the control group, although there was no difference in the UC-IV group, significant changes were observed in the UC-AP and UC-SC groups. The UC-SC group showed greater changes than the UC-AP group in all analyzed parameters, except dermal thickness. However, the two groups showed no significant differences in any of the analyzed parameters, except for epidermal thickness. Epidermal thickness in the UC-SC group was significantly greater than that in the UC-AP group. Although there was no significant difference, the number of Ki-67-positive cells and the collagen deposition area increased. Based on these results, subcutaneous injection of UCMSC-CM may be more effective than its direct application to aged skin. However, subcutaneous injection is accompanied by localized pain, and in some countries, there are legal restrictions on who can administer injections, which is why a microneedle-based delivery method can also be considered as an alternative. Although the microneedle delivery method does not deliver UCMSC-CM as deeply as direct injection, it offers the advantages of more effective targeted delivery and higher bioavailability. Additionally, it reduces localized pain and improves convenience[39-41].

We confirmed that epidermal thickness, dermal thickness, number of Ki-67-positive cells, and collagen deposition area were significantly increased in the AD-AP, UC-AP, and UC-SC groups treated with MSC-CM compared to those in the control group (Figure 4B-E and G-J; Supplementary material). Based on these results, we hypothesized that UCMSC-CM would rejuvenate aged skin. Therefore, we expected that the β-galactosidase level per cell, a representative aging indicator, would decrease in these groups compared with that in the control[42]. To conduct a more precise quantitative analysis of β-galactosidase-positive areas and Ki-67-positive cells, we performed serial sectioning of paraffin-embedded tissue blocks and stained them individually. The analysis was restricted to the epidermis to minimize errors caused by β-galactosidase expression in the hair follicle cells. This quantitative analysis revealed a significant reduction in β-galactosidase levels per cell in the UC-AP (51.8%) and UC-SC (78.52%) groups compared to those in the control group. These findings suggest that UCMSC-CM treatment not only induces phenotypic changes in aged skin but also promotes cellular rejuvenation.

Although UCMSC-CM has anti-aging effects, it is difficult to develop a method for its use as an anti-aging formula without confirming its safety. We conducted 10 safety evaluations (seven for skin toxicity and three for genotoxicity) at a safety evaluation institution and confirmed that UCMSC-CM was safe for use on the skin. Although this evaluation confirmed the safety of UCMSC-CM in various animal models, clinical studies in humans are required to ensure its safe use.

Based on the results of our study, we propose UCMSC-CM as an effective and safe anti-aging material for aged skin. When UCMSC-CM was applied to the aged skin, increases in epidermal and dermal thickness, cell proliferation, and collagen deposition were observed. However, the extent of these effects varies depending on the delivery method used. Subcutaneous injection is more effective than direct skin application for delivering the active ingredients of UCMSC-CM. Although mouse models are widely used in aging research due to their experimental accessibility and genetic similarity to humans, caution must be taken when extrapolating findings from mice to humans. Considerable interspecific differences exist in skin architecture, immune response, and metabolic stability, which can influence aging phenotypes[43,44]. For example, transcriptomic analyses have revealed the opposite age-related gene expression patterns in immune-related genes, such as Clec7a and Lyz1/2, between human and mouse skin[45,46]. Furthermore, the structural simplicity and thinner dermal layers of murine skin, along with differences in skin appendages, limit its ability to fully replicate human aging processes[44,47]. The sex-specific immune aging patterns that have been observed in humans are also largely absent in mouse models[45,46]. These discrepancies highlight the need for careful interpretation of data derived from mouse models in the context of human skin aging. To address this limitation, future research may benefit from the use of complementary models, such as three-dimensional human skin organoids, which more accurately reflect the structural and cellular complexity of aged human skin[48]. Furthermore, clinical studies involving human subjects are needed to confirm the safety and anti-aging effects of UCMSC-CM.

CONCLUSION

UCMSC-CM demonstrated both efficacy and safety in the prevention and treatment of skin aging, with the most pronounced improvements observed after subcutaneous injection. However, the efficacy and safety profiles established in animal models may not fully translate to humans. Therefore, additional clinical trials in humans are required to confirm the therapeutic potential and safety of UCMSC-CM.

ACKNOWLEDGEMENTS

We would like to thank CorestemChemon for conducting the safety evaluation of umbilical cord-derived mesenchymal stem cell-conditioned medium.