Exosomes Derived from Human Induced Pluripotent Stem Cells Ameliorate the Aging of Skin Fibroblasts

Abstract

Stem cells and their paracrine factors have emerged as a resource for regenerative medicine. Many studies have shown the beneficial effects of paracrine factors secreted from adult stem cells, such as exosomes, on skin aging. However, to date, few reports have demonstrated the use of exosomes derived from human pluripotent stem cells for the treatment of skin aging. In this study, we collected exosomes from the conditioned medium of human induced pluripotent stem cells (iPSCs) and investigated the effect on aged human dermal fibroblasts (HDFs). Cell proliferation and viability were determined by an MTT assay and cell migration capacity was shown by a scratch wound assay and a transwell migration assay. To induce photoaging and natural senescence, HDFs were irradiated by UVB (315 nm) and subcultured for over 30 passages, respectively. The expression level of certain mRNAs was evaluated by quantitative real-time PCR (qPCR). Senescence-associated-β-galactosidase (SA-β-Gal) activity was assessed as a marker of natural senescence. As a result, we found that exosomes derived from human iPSCs (iPSCs-Exo) stimulated the proliferation and migration of HDFs under normal conditions. Pretreatment with iPSCs-Exo inhibited the damages of HDFs and overexpression of matrix-degrading enzymes (MMP-1/3) caused by UVB irradiation. The iPSCs-Exo also increased the expression level of collagen type I in the photo-aged HDFs. In addition, we demonstrated that iPSCs-Exo significantly reduced the expression level of SA-β-Gal and MMP-1/3 and restored the collagen type I expression in senescent HDFs. Taken together, it is anticipated that these results suggest a therapeutic potential of iPSCs-Exo for the treatment of skin aging.

Keywords: exosomes; human induced pluripotent stem cells (iPSCs); photoaging; senescence; skin regeneration.

Figure 1

Characterization of iPSC-Exo. (A) Nanoparticle tracking analysis (NTA) result of iPSC-Exo. Mean diameter of iPSC-Exo was 85.8 nm. (B) The transmission electron microscopy (TEM) image of iPSC-Exo morphology (scale bar = 100 nm). (C) Dynamic light scattering (DLS) data for iPSC-Exo. The mean value for zeta potential of iPSC-Exo was −15.6 mV.

Figure 2

Effect of iPSC-Exo on the proliferation of human dermal fibroblasts (HDFs). Serum-starved HDFs were treated with iPSC-Exo for 48 h. The population of live HDFs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The total population of the iPSC-Exo-untreated group was regarded as 100%, and the populations of other groups were estimated as relative values. * p < 0.05, ** p < 0.01 compared to the iPSC-Exo-untreated group. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 3

iPSC-Exo promoted the migration of HDFs. (A) Scratch wound assay for HDFs treated with iPSC-Exo. Serum-starved HDFs were scratched and simultaneously treated with 20 × 10⁸ particles/mL iPSC-Exo. The representative image indicates more rapid migration of the iPSC-Exo-treated HDFs into the scratched area. (B) Representative image of a transwell assay for migration of HDFs. HDFs attached to the upper side of the transwell membrane migrated to the lower side in serum-free medium without or with iPSC-Exo for 24 h. The cells attached to the bottom of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. (C) Quantitative analysis of a transwell assay. Transwell membrane with the stained HDFs was cut out and immersed in a 50% acetic acid solution to dissolve the crystal violet. The amount of stained crystal violet was determined as absorbance at 560 nm. *** p < 0.001 compared to the iPSC-Exo-untreated group. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 3

iPSC-Exo promoted the migration of HDFs. (A) Scratch wound assay for HDFs treated with iPSC-Exo. Serum-starved HDFs were scratched and simultaneously treated with 20 × 10⁸ particles/mL iPSC-Exo. The representative image indicates more rapid migration of the iPSC-Exo-treated HDFs into the scratched area. (B) Representative image of a transwell assay for migration of HDFs. HDFs attached to the upper side of the transwell membrane migrated to the lower side in serum-free medium without or with iPSC-Exo for 24 h. The cells attached to the bottom of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. (C) Quantitative analysis of a transwell assay. Transwell membrane with the stained HDFs was cut out and immersed in a 50% acetic acid solution to dissolve the crystal violet. The amount of stained crystal violet was determined as absorbance at 560 nm. *** p < 0.001 compared to the iPSC-Exo-untreated group. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 4

Protective effect of iPSC-Exo on UVB-induced cell damage. HDFs were treated with 20 × 10⁸ particles/mL iPSC-Exo for 24 h in serum-free DMEM/F12 and simultaneously irradiated with UVB (315 nm). After further incubation for the indicated time interval in serum-containing growth medium, the populations of viable cells were determined by MTT assay. * p < 0.05, ** p < 0.01 compared to the UVB-irradiated and iPSC-Exo-untreated group. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 5

iPSC-Exo restored the altered expression of specific dermal markers in UVB-irradiated HDFs. After treatment with 20 × 10⁸ particles/mL iPSC-Exo for 24 h in serum-free DMEM/F12, HDFs were harvested after 48 h. The mRNA expression levels of collagen type I (A); MMP-1 (B); and MMP-3 (C) were quantified by quantitative real-time RT-PCR. * p < 0.05, ** p < 0.01. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 6

Effect of iPSC-Exo on the expression of SA-β-Gal in senescent HDFs. The passage numbers of control and senescent HDFs were 5 and 31, respectively. (A) SA-β-Gal-positive cells were shown in blue when observed under optical microscopy. (B) According to the standard shown in the figure, cells were classified into three categories: unstained, weak, and strong, and the number of cells in each group was expressed as a percentage. * p < 0.05, ** p < 0.01 compared to the iPSC-Exo-untreated senescent group. Statistical analysis was performed with three different images.

Figure 7

iPSC-Exo restored the altered expression of specific dermal markers in senescent HDFs. After treatment with 20 × 10⁸ particles/mL iPSC-Exo for 24 h in serum-free DMEM/F12, HDFs were harvested after 48 h. The mRNA expression levels of collagen type I (A); MMP-1 (B); and MMP-3 (C) were quantified by quantitative real-time RT-PCR. ** p < 0.01, *** p < 0.001. Error bars indicate standard deviations of triplicate samples in a single representative experiment.

Figure 7

iPSC-Exo restored the altered expression of specific dermal markers in senescent HDFs. After treatment with 20 × 10⁸ particles/mL iPSC-Exo for 24 h in serum-free DMEM/F12, HDFs were harvested after 48 h. The mRNA expression levels of collagen type I (A); MMP-1 (B); and MMP-3 (C) were quantified by quantitative real-time RT-PCR. ** p < 0.01, *** p < 0.001. Error bars indicate standard deviations of triplicate samples in a single representative experiment.