Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts

Adult skin stem cells are recognized as potential therapeutics to rejuvenate aged skin. We previously demonstrated that human dermal stem/progenitor cells (hDSPCs) with multipotent capacity could be enriched from human dermal fibroblasts using collagen type IV. However, the effects of hDSPCs on cellular senescence remain to be elucidated. In the present study, we investigated whether conditioned medium (CM) collected from hDSPC cultures (hDSPC-CM) exhibits beneficial effects on senescent fibroblasts. We found that hDSPC-CM promoted proliferation and decreased the expression level of senescence-associated β-galactosidase in senescent fibroblasts. In addition, p53 phosphorylation and p21 expression were significantly reduced in senescent fibroblasts treated with hDSPC-CM. hDSPC-CM restored the expression levels of collagen type I, collagen type III, and tissue inhibitor of metalloproteinase, and antagonized the increase of matrix metalloproteinase 1 expression. Finally, we demonstrated that hDSPC-CM significantly reduced reactive oxygen species levels by specifically up-regulating the expression level of superoxide dismutase 2. Taken together, these data suggest that hDSPC-CM can be applied as a potential therapeutic agent for improving human aged skin.


Introduction
Normal somatic cells cultured in vitro display a limited capacity to divide, and thus eventually become senescent [1]. Various harmful signals, including oxidative stress, DNA damage, and telomere shortening, have been reported to promote cellular senescence [2,3]. This cellular status is characterized by growth arrest, enlarged cell size, increased expression of senescence-associated β-galactosidase (SA-β-Gal), and accumulation of the tumor suppressors p53 and p21, which participate in cell cycle arrest [4,5]. Cellular senescence is closely related to the aging process; the number of senescent cells gradually increases with age, and this accumulation contributes to tissue aging and the development of age-related diseases [6].
In the present study, we expanded on these findings and examined whether hDSPC-CM exerts beneficial effects on the cellular senescence that occurs in aging skin. We found that hDSPC-CM significantly increased proliferation, and reduced SA-β-gal expression, p53 phosphorylation, and reactive oxygen species (ROS) levels in senescent fibroblasts. These data suggest that hDSPC-CM is a potential stem cell-based therapeutic agent to prevent aging in the skin.

hDSPC-CM Increased Cell Proliferation in Senescent Fibroblasts
We previously demonstrated that hDSPC-CM could restore UVA-induced damage in HDFs [19]. In the current study, we investigated whether hDSPC-CM would similarly reverse cellular senescence. First, we evaluated the effects of hDSPC-CM on the proliferation of senescent fibroblasts. We observed increased proliferation in senescent fibroblasts treated with hDSPC-CM compared with cells treated with non-hDSPC-CM ( Figure 1A). A cell proliferation assay further revealed that hDSPC-CM promoted the proliferation of senescent fibroblasts in a concentration-dependent manner, whereas non-hDSPC-CM did not ( Figure 1B). As expected, senescent fibroblasts expressed lower levels of the cell proliferation marker Ki67 compared with control fibroblasts (data not shown). However, treatment with hDSPC-CM, but not non-hDSPC-CM, significantly enhanced Ki67 expression in senescent fibroblasts ( Figure 1C,D).

hDSPC-CM Inhibited Senescence-Related Signaling in Fibroblasts
Next, we investigated whether hDSPC-CM could reverse cellular senescence. SA-β-Gal is a well-known marker of cellular senescence in various cells, including fibroblasts [20,21]. We observed higher SA-β-Gal expression in senescent fibroblasts compared to young fibroblasts (Figure 2A, upper panel). However, hDSPC-CM treatment significantly decreased the number of SA-β-Gal-positive cells relative to fresh medium (0% CM) or non-hDSPC-CM treatment (Figure 2A, lower panel and 2B).
The tumor suppressor p53 regulates cell cycle arrest, apoptosis, and cellular senescence [2][3][4][5]. We thus investigated whether hDSPC-CM could inhibit the p53 signaling pathway. Treatment with hDSPC-CM for 72 h reduced p53 phosphorylation. The expression level of p21, which is regulated by p53, was also inhibited by hDSPC-CM treatment ( Figure 2C,D). In summary, our results suggest that hDSPC-CM could contribute to reduce cellular senescence by suppressing p53 signaling.

hDSPC-CM Enhanced HDF-Specific Markers
We next investigated whether hDSPC-CM affects the expression levels of fibroblast-specific markers in senescent fibroblasts. The transcript levels of collagen type 1 alpha 1 (COL1A1), collagen type 3 alpha 1 (COL3A1), and tissue inhibitor of metalloproteinase (TIMP1), which are important components of the skin dermis, were significantly reduced in senescent fibroblasts compared with normal fibroblasts, and hDSPC-CM treatment significantly increased the expression levels of all three markers ( Figure 3A,B and D). Conversely, matrix metalloproteinase 1 (MMP1) transcripts were increased in senescent fibroblasts compared with normal fibroblasts, and hDSPC-CM treatment significantly reduced MMP1 expression ( Figure 3C).

hDSPC-CM Specifically Increased the Superoxide Dismutase 2 (SOD2) Expression Level among ROS Scavenging Enzymes in Senescent Fibroblasts
ROS scavenging enzymes are responsible for reducing H2O2 levels to maintain cell homeostasis [21][22][23]. Thus, we tested whether hDSPC-CM could increase the expression of ROS scavenging enzymes such as superoxide dismutases (SODs) and catalase. Interestingly, among the ROS scavenging enzymes tested, hDSPC-CM specifically enhanced the expression level of SOD2 in senescent fibroblasts ( Figure 5).

Discussion
The aging process gradually decreases the homeostatic and regenerative potential of all tissues [25][26][27][28]. In particular, photoaging and intrinsic aging decreases the elasticity of the skin, resulting in the formation of wrinkles and impairing wound healing [29][30][31]. These age-related changes might reflect the gradual increase in the proportion of senescent cells in specific tissues. Adult stem cells have been considered as potential therapeutics to slow this aging process. Indeed, we recently demonstrated that hDSPC-CM restored UVA-induced damages to fibroblasts [19].
In the present study, we further demonstrated that hDSPC-CM reversed multiple phenotypes associated with cellular senescence. First, we found that hDSPC-CM significantly enhanced the proliferation of senescent fibroblasts (Figure 1). Second, we found that hDSPC-CM significantly decreased SA-β-gal production, p53 phosphorylation, and the expression level of p21, all of which were generally increased in senescent fibroblasts ( Figure 2). Third, RT-PCR analyses revealed that hDSPC-CM restored the expression of major dermal biomarkers, including COL1A1, COL3A1, and TIMP1, which were typically down-regulated in senescent fibroblasts ( Figure 3). Fourth, we demonstrated that hDSPC-CM significantly decreased H2O2 levels by specifically increasing SOD2 expression (Figures 4 and 5).
Mesenchymal stem cell (MSC) transplantation accelerates the wound-healing process in damaged skin [32][33][34]. Moreover, some MSC transplantation studies have suggested that the therapeutic potential of MSCs might be mediated by secreted growth factors rather than their long-term presence in injured tissues [35,36]. Recent studies demonstrated that CM harvested from stem cell cultures exerts beneficial effects on multiple cellular defects and diseases [37][38][39]. For example, Chen et al. demonstrated that cytokines present in murine bone marrow-derived MSC-CM stimulated macrophages, endothelial migration, and wound healing in BALB/c mice [37]. Similarly, another study showed that the paracrine effects of MSCs accelerated the regeneration of endogenous stem cells and ameliorated obstruction-induced overactive bladders [38]. Kim et al. reported that soluble intracellular adhesion molecule-1 secreted by human umbilical cord blood-derived MSCs reduced the number of amyloid-β plaques, which can cause Alzheimer's disease [39]. In our previous study, cytokine array analysis revealed that hDSPC-CM contains higher levels of multiple growth factors, including basic fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-binding protein-1 and -2, insulin-like growth factor, and vascular endothelial growth factor, compared with non-hDSPC-CM [19]. Thus, we speculate that the beneficial effects of hDSPC-CM on senescent fibroblasts may be mediated by hDSPC-secreted growth factors.
According to previous reports, cell cycle regulators and DNA damage response pathways are strongly implicated in the onset of senescence [1][2][3]5,6,20]. For example, increased p53 phosphorylation forces cells into a state of premature senescence [2][3][4]; as cells enter G0, p21, a well-known target gene of p53, increases in a p53-dependent manner. Thus, we investigated the expression of these proteins and demonstrated that hDSPC-CM significantly down-regulated p53 phosphorylation as well as p21 expression ( Figure 2C).
ROS are also considered to be major factors contributing to the aging process. Metabolic dysfunction and exogenous stress can generate excessive ROS levels [22][23][24]. Our data demonstrated that hDSPC-CM decreased H2O2 levels in senescent fibroblasts (Figure 4). To investigate the underlying mechanism to explain this phenomenon, we further examined the expression of several antioxidant enzymes, including members of the SOD family and catalase. According to previous reports, SOD1 is located in the cytoplasm, SOD2 is sequestered to the mitochondria, and SOD3 is secreted into the extracellular matrix [40,41]. Interestingly, we observed that only SOD2 expression was specifically enhanced in senescent fibroblasts cultured with hDSPC-CM compared with control and non-hDSPC-CM ( Figure 5), implying that increased ROS production by senescent fibroblasts is mainly generated in the mitochondria [40,41].
In conclusion, we suggest that hDSPC-CM ameliorates fibroblast senescence in a paracrine manner ( Figure 6). Thus, the factors secreted by hDSPCs represent potential therapeutics to promote skin regeneration. Figure 6. A schematic representation of the mechanism contributing to the beneficial effects of hDSPC-CM on senescent human dermal fibroblasts. ↑ means increase, whereas ↓ means decrease.
To enrich hDSPCs that are present in only small amounts in fibroblasts, collagen type IV (Sigma-Aldrich, St. Louis, MO, USA)-coated dishes were prepared by coating 150-mm tissue culture dishes with collagen type IV (20 mL, 20 μg/mL) overnight at 4 °C. We observed that >50% of the HDFs adhered to the collagen type IV-coated dishes after 10 min of incubation. Therefore, we gradually reduced the adherence time in 1-min increments. Cells were not to adhere to dishes in less than 4 min. Ultimately, an incubation time of 12 h was selected because most of the cells could adhere to the dishes during this period. Thus, the cells were separated based on their ability to adhere to the plates within 4-5 min (hDSPCs) or within 12 h (non-hDSPCs) at 37 °C. About 5%-10% of the total fibroblasts adhered to the dishes within 4-5 min of incubation [17,18].
For CM treatment, 3 × 10 5 cells were seeded onto 60-mm dishes in the CM, and DMEM with 50% non-hDSPC-CM or 50% hDSPC-CM was added to the cultures for the indicated times. All CMs were changed every 24 h.

Immunofluorescence Staining for Ki67
Senescent fibroblasts were incubated with 50% non-hDSPC-CM or hDSPC-CM for 24 h. Following CM treatment, the cells were fixed with 4% formaldehyde (Sigma-Aldrich) and then blocked with 1% bovine serum albumin (Sigma-Aldrich). The cells were then incubated with mouse monoclonal anti-Ki67 antibody (Abcam, Cambridge, MA, USA) for 12 h at 4 °C, followed by Alexa-488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1.5 h at room temperature. The nuclei were stained with 5 mg/mL DAPI (Sigma) for 5 min. Samples were washed with phosphate-buffered saline with Tween-20 after each step. Slide-mounted samples were imaged with an EVOS fluorescence microscope (Advanced Microscopy Group, Mill Creek, WA, USA). Three images per dish were collected, and Ki67-positive cells were counted.

Cell Proliferation Assay
Senescent fibroblasts were seeded onto 96-well culture plates and treated with non-hDSPC-CM or hDSPC-CM at the indicated dilutions for 24 h. Following each CM treatment, 10 μL of Cell Counting Kit-8 (Sigma-Aldrich) was added to each well. After incubating for 1 h, absorbance at 450 nm was measured.

SA-β-Gal Assay
SA-β-Gal activity was detected using a senescence detection kit (Abcam) following the manufacturer's protocol. Briefly, senescent fibroblasts were treated with 50% non-hDSPC-CM or hDSPC-CM for 72 h. The cells were then fixed and incubated at 37 °C with a staining solution containing 1 mg/mL of 5-bromo-3-chloro-4-indolyl β-D-galactoside (X-gal) for 12 h. Five images per dish were collected, and the blue cells were counted.

H2O2 Production Measurements
H2O2 released from senescent HDFs was measured using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Technologies), according to the manufacturer's instructions. Senescent HDFs were treated with non hDSPC-CM or hDSPC-CM. The CM (50 μL) of each sample was harvested and incubated with 100 μL reaction solution containing 100 μM Amplex Red reagent and 0.2 U/mL horseradish peroxidase for 10 min. The fluorescence of each sample was measured at 590 nm emission following excitation at 560 nm using a Gemini XPS microplate reader (Molecular Device, Sunnyvale, CA, USA).

Statistical Analysis
Statistical analysis was performed using SPSS software (IBM Corporation, Armonk, NY, USA). An unpaired Student's t-test or one-way analysis of variance was used as indicated in the figure legends. p-values less than 0.05 were considered statistically significant.

Conclusions
For the first time, we demonstrated that CM derived from hDSPCs exhibits various anti-aging effects on senescent HDFs. hDSPC-CM not only reduced SA-β-gal levels but also decreased p53 phosphorylation and p21 expression in senescent HDFs. In addition, hDSPC-CM restored the expression of a major structural protein, collagen type I, and reduced H2O2 levels in senescent HDFs. In conclusion, we suggest that hDSPC-CM can be clinically used as a potential therapeutic agent for improving human aged skin.