Cell cultures
Human MSCs from adipose tissue (C-12977) and dedicated medium (C-28009), normal human dermal fibroblasts (NHDFs) from juvenile foreskin (C-12300) and dedicated medium (C-23020), and normal human epidermal keratinocytes from juvenile foreskin (single donor, C-12001) and dedicated medium (C-20111) were all purchased from Promo Cell. All media were supplemented with 10% fetal bovine serum (Carson, CA, USA), penicillin (100 U ml-1), streptomycin (100 µg ml-1), and kanamycin (10 µg ml-1). Cells were cultured in an incubator at 37°C under 5% CO2 and used in the experiments.
Biochemical reagents
H2O2 and other reagents were purchased from Wako Pure Chemical Industry Co., Ltd.
Extracting, quantifying, and adding exosomes
MSCs and fibroblasts were cultured for 7 days in culture flasks, and culture supernatant was collected from the cells when they reached approximately 80% confluency, with cellular debris removed via centrifugation (12,000 rpm, 15 min). The supernatant was stored at 4°C until exosome isolation. A MiRCURY Exosome Isolation Kit (Product #: 300102; manufactured by EXIQON) was used to isolate exosomes present in the culture medium. Isolated exosome levels were measured using CD9/CD63 enzyme-linked immunosorbent assay (ELISA) kits (for human exosome assays). Isolated exosomes were added to the culture medium at a 1/100 volume (final level, 100 pg ml-1) and incubated for 4 h. These EX-containing media were named MSC-derived exosomes (MSC-Ex) and NHDF-derived exosomes (NHDF-Ex).
Oxidative stress treatment and exosome exposure in cells
NHDFs were incubated for 2 h in medium supplemented with 0.2 mM H2O2, washed with PBS, and then incubated in conventional medium [28]. Conversely, the preventive effects of exosomes were investigated by incubating NHDFs with MSC-Ex for 6 h, washing the cells with PBS, and then treating them with 0.2 mM H2O2 for 2 h in the same manner. Cells or culture medium was collected at various times after incubation. Total RNA was extracted from cells after exosome and H2O2 treatment, and culture supernatants were used for hyaluronan measurements. In addition, measurements of p21 mRNA expression were also performed after 16 h of exosome exposure and 2 h of H2O2 stress.
Wound-healing effects
Wound-healing effects were examined using a CytoSelect 24-well Wound-Healing Assay Kit (Cell Biolabs, Inc. San Diego, CA, USA). In the assay, a wound field was created near the center of NHDF cultures for 48 h using an insert placed in the 24-well plate. Thereafter, the cells were cultured for 48 h in culture medium containing various exosomes and then stained with DAPI to observe their growth via fluorescence microscopy, and images were acquired. The effect was measured as the percent closure (migrated cell surface area/total surface area × 100).
Cell growth-promoting effect
NHDFs were cultured in the presence of exosomes and oxidative stress molecules, and the cells and culture medium were recovered at various times. The total number of cells and the number of surviving cells were counted using Countess cell counting chamber slides (Thermo Fisher Scientific K. K. Tokyo, JAPAN) and a CytoTox-ONE Assay Kit (Promega, Madison, WI, USA), respectively (n = 3/group).
Quantitation of mRNA by quantitative RT-PCR (qRT-PCR)
The mRNA levels of AQP-1, AQP-3[29], SIRT-1 [30], p53 [31], and p21 [32] in NHDFs were measured using the one-step qRT-PCR method. The mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also measured as an internal control [33].
qRT-PCR was performed in the final volume of 20 μl of solution containing 10 μl of 2× Luna Universal One-Step Reaction Mix, 1 μl of Luna WarmStart® RT Enzyme Mix, 2 μl of total RNA solution, 1.6 μl of the primer pair mix (0.4 μM of each primer), and 7.3 μl of H2O under the following conditions: 55°C for 10 min for reverse transcription and 95°C for 1 min for initial denaturation, followed by 45 cycles of 95°C for 10 s and 60°C for 30 s. The relative gene expression was calculated using the ΔΔCt method [34], and GAPDH gene expression was used for normalization. The primers used in qRT-PCR are shown in Table I. Significant differences were judged when ΔΔCt > 2.
Hyaluronan production (ELISA) related to skin-moisturizing effects
Hyaluronan levels were assessed using an ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.
Effects of oxidative stress on the induction of cellular senescence
Various exosomes were continuously added to NHDFs under oxidative stress, and cellular senescence was examined microscopically using a SPiDER-βGal kit (SG03; Dojindo, Kumamoto, Japan).
The abundance of senescent cells was evaluated by quantifying the amount of fluorescence in the acquired images. Fluorescent ROS levels were quantified using ImageJ software. Data were shown as the fluorescence intensity (excitation, 488 nm; emission, 590 nm).
Measurement of intracellular ROS production
Intracellular ROS levels were measured using the fluorescent probe CM-H2DCFDA (Molecular Probes Inc., Eugene, OR, USA). NHDFs were washed with PBS and then incubated with 1 µM fluorescent probe at 37°C for 60 min. Intracellular ROS levels were assessed according to the fluorescence intensity using a microplate reader (SYNERGY/HT, BioTek, Japan).
Statistical analysis
All results are presented as the mean ± SD. Statistical significance was determined using one-way analysis of variance, and differences were considered statistically significant when P < 0.05.