The experimental protocols for the isolation of hOOM were approved by the Ethics Committee of Konkuk University Medical Center (IRB number: KUMC 2019-05-043) and conformed to the principles outlined in the Declaration of Helsinki. For the preparation of hOOM-SCs donors who underwent eyelid blepharoplasty were enrolled in this study (Supplementary Table S1). Stem cells were isolated from unused tissue as described in a previous study (4), and adipose-derived (AD)-MSCs were cultured using α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Peak Serum Inc.) and 1% penicillin/streptomycin (Gibco). TS-bFGF, which was manufactured as described previously (6), was subsequently used after being dissolved in 1X phosphate-buffered saline (Gibco) at the required concentration for all experiments and stored at −20℃. In addition, WT-bFGF (Peprotech) dissolved in 1X PBS was used for all experiments.

Cell growth kinetics were assessed based on the population doubling time and cumulative cell numbers. Cells were seeded at a density of 1×10⁵ cells in a 60 mm culture dish (SPL Life Sciences) and incubated at 37℃ in a 5% CO₂ atmosphere until they reached 80%∼90% confluence. After each passage (P), the cells were harvested, and the cell number was determined using a NucleoCounter NC-250^TM (ChemoMetec), as recommended. Cell population doubling was calculated using the following equation:

where population doublings (PD) represents population doubling, Nt corresponds to the cell number after trypsinization or collection, and N0 indicates the number of initially seeded cells.

The cellular DNA content was quantified using the recommended protocol of the NC-250^TM to analyze the G0/G1, S, and G2/M cell cycle phases.

The cells were incubated with primary antibodies anti-CD73 (41-0200; Invitrogen), anti-CD90 (AF2067; R&D Systems, Inc.), anti-CD34 (553731; BD Biosciences), and anti-CD45 (14-0451-82; Invitrogen) followed by secondary antibodies (Alexa488, anti-mouse; Invitrogen or sheep IgG PE-conjugated F0126; R&D Systems) at the recommended concentrations. After washing, antibody fluorescence was measured using a CytoFLEX flow cytometer and analyzed with the CytExpert program.

hOOM-SCs were seeded in 6-well plates (SPL) and cultured in α-MEM until confluence, and 10 μg/mL mitomycin C (Sigma-Aldrich) was added for 2 hours at 37℃ to stop cell proliferation, and subsequently, the cells were washed with serum-free α-MEM and scratched with a 1 mL pipette tip. After washing twice with 1X PBS and adding culture medium, the time-dependent size of the wound closure (in triplicate) was determined using TScratch software (Tobias Gebäck, 2009).

hOOM-SCs were seeded at 100 cells in a 60 mm dish (SPL) in α-MEM with 10% FBS and 1% P/S. After culturing for 14 days with regular replacement of the culture medium every 2∼3 days, the colonies were stained with crystal violet (Sigma-Aldrich), and the number of colonies was manually counted.

Total RNA was isolated from each group of hOOM-SCs using Labozol Reagent (Cosmo Genetech), according to the manufacturer’s instructions, and purified RNA was quantified using a NanoDrop spectrophotometer (NanoPhotometer^Ⓡ N50, IMPLEN). Synthesis of cDNA was performed using 2 μg of total RNA with the M-MuLV reverse transcription kit (Labopass) and oligo-dT primers. The polymerase chain reaction (PCR) mixture was prepared using EzAmp^TM qPCR 2X Master Mix (ELPIS-BIOTECH) and run on a Quant Studio^TM 3 Real-Time PCR System (Thermo Fisher Scientific). The results were normalized using GAPDH expression as an internal control, and the fold change in gene expression was calculated using the comparative cycle time (Δct) method. Primers used are listed in Table 1.

To determine the level of intracellular accumulation of ROS, the CellROX Green reagent (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. The hOOM-SCs were cultured in α-MEM until they reached confluence. Subsequently, the cells were thoroughly washed and then incubated in culture medium containing 5 μM CellROX reagent for 30 minutes at 37℃. Following the incubation period, the solution was discarded, and the cells were washed three times with 1X PBS. Their fluorescence intensity was captured using a fluorescence microscope (Nikon), which was analyzed using ImageJ software (version 1.53c; National Institutes of Health).

Changes in the level of reduced thiols in cells were analyzed using the NC-250^TM following the recommended procedure. A micropipette was used as a representative cell sample from the cell suspension in a 1 mL microcentrifuge tube (SPL). One volume of 10 μL propidium iodide solution (Solution 6, VB-48^TM PI Staining Reagent; ChemoMetec) was added into 190 μL of the cell suspension.

The hOOM-SCs treated with various bFGF were prepared, and the senescence SA-β-Gal assay was performed according to the protocol of Debacq-Chainiaux and colleagues (11). When cells cultured in 6-well cell culture plates (SPL) reached 80% confluence, they were gently washed with 1X PBS at 2∼3 times and fixed for 15 minutes at room temperature with 2% paraformaldehyde (Biosesang) and 0.2% glutaraldehyde. Subsequently, the cells were incubated overnight at 37℃ with freshly prepared SA-β-Gal stain solution. SA-β-Gal-positive cells appeared blue and were observed and counted under a light microscope (Nikon).

Cell size was quantified using the NC-250^TM following the recommended protocol. The cell suspension was mixed to obtain a homogeneous suspension. A micropipette was used as a representative cell sample from the cell suspension in a 1 mL microcentrifuge tube. One volume of 5 μL DAPI Solution (Solution 18; ChemoMetec) was added into 20 volumes of the 100 μL cell suspension sample.

Using the BCA protein assay kit, we measured the isolated protein levels according to the manufacturer’s protocol. We extracted the cellular proteins using a buffer composed of 100 mM Tris-HCl (pH 7.5), 1% Triton X-100 (Sigma-Aldrich), 10 mM NaCl, 10% glycerol (Amresco), 50 mM sodium fluoride (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich), 1 mM p-nitrophenyl phosphate (Sigma-Aldrich), and 1 mM sodium orthovanadate (Sigma-Aldrich). The cell lysates were centrifuged at 16,000 g for 15 minutes at 4℃. The supernatant was carefully transferred into a new E-tube, and protein quantification was performed using a BCA protein assay kit. The proteins were separated by 8%∼12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated overnight at 4℃ with the appropriate primary antibodies: NRF2 (sc-365949), P53 (sc-126), P21 (sc-6246) (Santa Cruz Biotechnology), and anti-β-actin (4970S; CST). Subsequently, the membranes were incubated with secondary antibodies (anti-mouse or -rabbit IgGs) conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 1 hour at room temperature. Protein signals were visualized using an enhanced chemiluminescence kit (Amersham Biosciences) and ChemiDoc^TM Imaging System (17001401; Bio-Rad).

All statistical analyses were performed using GraphPad Prism (version 9.5.1). In all experiments designed in triplicate, adjusted p-values were calculated using the Student’s t-test. In all figures, the p-values are marked with asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Based on previous findings showing superior outcomes with TS-bFGF in pluripotent stem cells (6), this study evaluated its effects on hOOM-SCs. Stem cells from orbicularis oculi muscle surgeries were preserved and cultured with WT-bFGF or TS-bFGF. In 5G1 cells from a 5-year-old donor, TS-bFGF treatment significantly increased cumulative cell numbers compared to the control and WT-bFGF groups. After continuous culture in the control group, differences in cell morphology between passage 9 were compared at 40× and 100× magnifications, but there were no significant changes in cell morphology (Fig. 1A). Additionally, the doubling times of the cells were lower than those of the controls, whereas no significant difference was observed in cell viability (Fig. 1B). Moreover, a relatively higher population of TS-5G1 cells was observed in S phase compared with WT-5G1 and the control group (Fig. 1C), whereas no difference in cell surface phenotypes was observed between different cell treatment groups (Fig. 1D). Similar culture outcomes were observed for hOOM-SCs (lines 18T1, 74U1, and 5S1 derived from donors aged 18, 74, and 5 years, respectively) and AD-MSCs cultured with TS-bFGF (Supplementary Fig. S1), indicating that the effects observed were not specific to the 5G1 cell line. In summary, TS-bFGF showed superior cell culture outcomes compared to WT-bFGF, likely by promoting cell cycle progression, leading to faster cell duplication and higher cumulative cell numbers without altering cellular morphology or surface phenotypes.

To compare the effect of WT-bFGF and TS-bFGF on stemness and cellular wound-healing ability, the expression levels of stemness markers, such as Sox2, Oct4, klf4, L-myc, and Nanog (12), were evaluated in WT-5G1 and TS-5G1 cells. In contrast to the situation in pluripotent stem cells, where TS-bFGF stimulates the expression of stemness markers, these markers in 5G1 cells were not upregulated in response to TS-bFGF, except for a slight increase in the level of Sox2 (Fig. 2A). Sox2 supports the pluripotency and self-renewal of MSCs and enhances the expansion and differentiation of somatic stem cells (13). This suggests that long-term culture of hOOM-SCs with TS-bFGF can enhance cell performance. To further understand the effect of TS-bFGF on stemness, the WT-5G1 and TS-5G1 cells were evaluated for colony formation ability. A significant increase in colony formation ability was observed in TS-5G1 cells compared to the control group (Fig. 2B). Furthermore, we observed that TS-5G1 cells showed better wound healing ability than either the WT-5G1 or control cells (Fig. 2C). Taken together, TS-5G1 cells exhibited greater colony formation and wound healing abilities, suggesting that TS-bFGF enhances stemness and promotes cell proliferation during long-term culture.

Given the increased cell proliferation observed in TS-bFGF-treated 5G1 cells, we investigated its potential anti-senescence effects. WT-5G1 and TS-5G1 cells were analyzed for senescence-related changes during extended culture. First, it was observed that the number of SA-β-Gal stained positive cells decreased in TS-5G1 cells compared with the WT-5G1 or control cells (Fig. 3A), which was supported by similar results using WJ-MSCs (Supplementary Fig. S2A). Next, we observed that the expression levels of p16, p21, and p53, three well-known senescence markers, were significantly lower in TS-5G1 cells than in WT-5G1 or control cells (Fig. 3B). In addition, the TS-5G1 cell line showed a further decrease in p16 (11.1%), p21 (23.7%), and p53 (17.1%) compared to the WT-5G1 cell line. Moreover, oxidative stress levels, quantified by measuring cellular ROS, were significantly decreased in TS-5G1 cells compared to WT-5G1 or control cells (Fig. 3C). Further, based on the protective role of reduced glutathione (GSH), we measured the intracellular GSH levels and found that it was higher in TS-5G1 cells than in WT-5G1 or control cells (Fig. 3D). Interestingly, we did not detect any significant change in cell size, which is known to increase during cellular senescence (Supplementary Fig. S2B). After continuous treatment with TS-bFGF and WT-bFGF in 5G1 cells, the expression of activity-related markers, including NRF2 (related to ROS and GSH production) and the senescence markers P53 and P21, was evaluated. NRF2 levels were higher in the TS-bFGF-treated group compared to the control. Furthermore, the expression of P53 and P21, related to senescence, was observed to be decreased compared to the control group (Fig. 3E). Taken together, these results suggested that TS-bFGF has a positive effect on delaying cellular senescence in stem cells in continuous culture by reducing the levels of various senescence factors.

This study shows that TS-bFGF delays senescence in hOOM-SCs, increasing cell proliferation, SOX2 expression, and reducing ROS. It enhances cell lifespan in long-term culture, offering potential for high-quality stem cells in therapies and applications like cultured meat.