Exosomes Secreted from Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Accelerate Skin Cell Proliferation

Abstract

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton's jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

Keywords: MSCs; exosome; iPSCs; wound healing.

Figure A1

Characterization of iPSCs. (a) Light microscopic image and flow cytometry analysis of iPSCs. iPSCs were negative with typical MSC markers (CD73, CD90 and CD105) and endothelial/hematopoietic marker (CD34), while positive with pluripotency marker SSEA-4. (b) Immunofluorescence analysis of iPSCs with pluripotency markers. iPSCs were positive with SOX2, TRA-1-60, OCT-4 and SSEA-4. Scale bars are 200 μm. Nuclei were stained with DAPI for counterstaining.

Figure 1

Comparison of the morphology and cell surface marker profile between human Wharton’s jelly MSCs and iMSCs. Both types of cells showed a typical morphology of MSC, with spindle- or fibroblast-like appearance. Flow cytometry analysis showed that both types of cells were positive for MSC markers CD73, CD90, and CD105, but negative for CD34 and SSEA-4. Scale bars are 200 µm.

Figure 2

Characterization of exosomes derived from MSCs and iMSCs. (a) Nanoparticle analysis of MSC-exo and iMSC-exo. The mean diameter was 167 and 147 nm for MSC-exo and iMSC-exo, respectively. (b) TEM analysis of exosomes. Scale bars are 200 nm. (c) Immunoblotting for CD63 and CD9 in exosomes. (d) Verification of the uptake of exosomes in skin cells. MSC-exo or iMSC-exo (20 µg/mL) were stained with PKH26^® (red) and incubated with HaCaT and HDFs for 24 h. Before analysis, cells were counterstained with CellTracker^® (green). Scale bars are 20 µm. (e) Confocal images of mouse skin tissues treated with MSC-exo or iMSC-exo. A total of 30 µg of PKH26-labeled (red) exosomes were injected into the dorsal skin and tissues were collected after 24 h. Saline (vehicle) was used as negative control. Nuclei were stained with DAPI (blue) for counterstaining. Green dotted lines delineate epidermal-dermal junction. Scale bars are 200 µm.

Figure 3

Growth kinetics, cell cycle, and survival analyses of skin cells treated with exosomes. Exosomes collected from MSCs (MSC-exo) or iMSCs (iMSC-exo) were incubated with HaCaT (left) or HDFs (right). (a) Growth profile was measured in exosome-treated cells at designated study points. Negative control (NC) is cells from serum-free culture. Culture with serum (10%) was used as positive control (PC). (b) At 48 h of treatment, the percentage of cells in each cycle was measured by flow cytometry. Cells cultured in serum (10%) were used as positive control. (c) Cell proliferation analysis by MTT assay. At 48 h of exosome treatment, the absorbance of final precipitates was measured at a wavelength of 570nm, and normalized against the value obtained from serum-free negative control (NC). All data are expressed mean ± standard deviation (SD) from three replications. * p < 0.05, ** p < 0.01, and *** p < 0.005.

Figure 4

Wound scratch assay of skin cells treated with exosomes. (a) Relative wound area changes by exosome treatment. MSC-exo or iMSC-exo were co-incubated with HaCaT (left) or HDFs (right), and the wound area at designated study points was normalized against that obtained at 0 h. NC, negative control (serum-free culture). * p < 0.05, ** p < 0.01. (b) Light microscopy images of wound scratch assay at designated study points. The wound area of HaCaT was calculated using inherent protocol in ImageJ software, while that of the HDFs was manually delineated and subjected to ImageJ software analysis. NC, negative control (serum-free culture). Scale bars are 200 µm.

Figure 5

Comparison of the relative soluble protein and mRNA expression following exosome treatment. (a) A total of 20 µg/mL of MSC-exo or iMSC-exo were incubated with HaCaT and HDFs for 48 h, and the concentration of fibronectin and collagen was measured using a Human Magnetic Luminex^® Screening Assay and SirCol assay kits, respectively. (b) Exosome-treated HaCaT and HDFs were subjected to qRT-PCR analysis and the expression of each gene was normalized against the expression detected in the non-treated negative control (NC). Cells cultured with serum (10%) were used as positive control (PC). Negative control (NC) was cells cultured without serum. All data are expressed as mean ± standard deviation (SD) from three replicates. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 6

Immunoblotting for the detection of phosphorylated ERK1/2 in HaCaT and HDFs. (a) Cells were cultured with MSC-exo or iMSC-exo for 1 h and then the cell extracts were analyzed by immunoblotting for total ERK1/2 and phosphorylated ERK1/2 (Thr202/Tyr204). Beta-actin was used as the loading control. NC and PC are cells cultured without and with serum, respectively. (b) Densitometric analysis of the relative level of phosphorylated ERK1/2 (Thr202/Tyr204) against their total levels in HaCaT and HDFs. The value was normalized against that observed for negative control (NC, no serum). All data are expressed as mean ± standard deviation (SD) from three replicates. * p < 0.05 against NC. HDFs indicate human postnatal dermal fibroblasts.