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. 2018 May;80(2):141-153.
doi: 10.18999/nagjms.80.2.141.

＜Editors' Choice＞ Effects of exosomes derived from the induced pluripotent stem cells on skin wound healing

Hitoshi Kobayashi  1 , Katsumi Ebisawa  1 , Miki Kambe  1 , Takatoshi Kasai  2 , Hidetaka Suga  2 , Kae Nakamura  3 , Yuji Narita  4 , Aika Ogata  4 , Yuzuru Kamei  1
Affiliations

＜Editors' Choice＞ Effects of exosomes derived from the induced pluripotent stem cells on skin wound healing

Hitoshi Kobayashi et al. Nagoya J Med Sci. 2018 May.

Abstract

Recently, the effects of stem cell supernatants or exosomes, such as skin wounds, have attracted attention. However, the effects of the induced pluripotent stem (iPS) cell-derived exosomes (iPS-Exos) have not been investigated in detail. Here, we investigated the effects of iPS-Exos on skin wound healing using an animal model. We isolated iPS-Exos from the iPS cell culture media. Control exosomes were isolated from unused iPS cell culture media (M-Exos). We first observed the morphologic characteristics of the isolated exosomes and examined the expression of surface antigens. The effects of these exosomes on the migratory response and proliferation of fibroblasts were analyzed as well. Additionally, using a diabetic ulcer model, the effects of iPS-Exos and M-Exos on skin wound healing were investigated. Transmission electron microscope analysis demonstrated that the size of iPS-Exos (120 ± 25 nm) was significantly larger than that of M-Exos (≤ 100 nm). Flow cytometry analyses showed that iPS-Exos were positive for CD9, CD63, and CD81, whereas they were negative for HLA-ABC and -DR expression. The migratory ability of fibroblasts cocultured with iPS-Exos was shown to be higher than that of the cells cocultured with M-Exos, as demonstrated using scratch assay. Skin wound healing model results showed that the administration of iPS-Exos results in a faster wound closure compared with that observed in the M-Exo group. In conclusion, the results obtained here indicate that iPS-Exos may promote the migration of fibroblasts in vitro and in vivo, suggesting the possibility of using iPS-Exos for the treatment of diabetic ulcer.

Keywords: diabetic ulcer; exosome; induced pluripotent stem cell; wound healing.

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Figures

Fig. 1
Fig. 1
Animal model Excisional wounds were created bilaterally in the dorsal skin of diabetic mice, and silicone splint was placed and affixed. The stents prevented wound contraction.
Fig. 2
Fig. 2
Topical application of induced pluripotent stem cell-derived exosomes (iPS-Exos) to the wound beds The wounds made in the dorsal skin areas were treated with phosphate buffered saline (PBS; untreated group), M-Exo (control group), or iPS-Exo (experimental group). iPS-Exo, M-Exo and PBS were injected at four sites into the subcutaneous layer around the wound.
Fig. 3
Fig. 3
Electron micrographs of induced pluripotent stem cell exosomes (iPS-Exos) Representative images of vesicles with approximately 100-nm diameters in iPS-Exos samples, which were not observed in M-Exos (control group) samples. Scale bars, 200 nm.
Fig. 4
Fig. 4
Surface antigen expression on the isolated exosomes Representative analysis showing surface expression of CD9, CD63, CD81, HLA-ABC, and HLA-DR in the induced pluripotent stem cell-derived exosomes (iPS-Exo) (A) and control sample exosomes (M-Exo) (B) coupled to aldehyde-sulfate latex beads. FITC-conjugated isotype controls and PE-conjugated isotype controls are included for comparison.
Fig. 5
Fig. 5
Scratch assay results (A) Effect of induced pluripotent stem cell-derived exosomes (iPS-Exos) on cell migration of diabetic mouse fibroblasts. Representative phase contrast images of cells migrating into wounded area at 0, 12, and 24 h in an in vitro scratch assay are presented. Bars, 300 μm. (B) Migration rates of diabetic mouse fibroblasts into scratched areas after 24-h incubation with 10, 50, 100, or 200 μg/ml iPS-Exo. (C) Cell migration time course. Cell migration was quantified using relative would density (RWD). Error bars represent standard deviations (SD).
Fig. 6
Fig. 6
Proliferation assay results Effect of induced pluripotent stem cell-derived exosomes (iPS-Exos) on the proliferation of diabetic mouse fibroblasts. Proliferation rates of diabetic mouse fibroblasts treated with 100 μg/ml iPS-Exo for 48 h. Error bars, standard deviations (SD).
Fig. 7
Fig. 7
Wound closure in db/db mice (A) Representative images of wounds treated with phosphate-buffered saline (PBS), control exosomes (M-Exos), or induced pluripotent stem cell-derived exosomes (iPS-Exos) at day 0, 1, 3, 5, 7, 10, 14, 18, 21, and 28. (B) Quantification of PBS, M-Exo, or iPS-Exo treatment effects at day 0, 1, 3, 5, 7, 10, 14, 18, 21, and 28. *P < 0.05, until day 7. (C) The rates of wound healing following different treatments. P < 0.05, compared with the controls. (D) Representative images of wound healing in the PBS, M-Exos, and iPS-Exos group.
Fig. 8
Fig. 8
Immunofluorescence analyses of vessel formation (A) Blood vessel numbers were determined in two areas at wound edges and three areas near the center of wounds. (B) Representative hematoxylin & eosin staining images. (C) Representative CD31 and α-SMA co-staining images, showing newly-formed vessels at wound sites. (D) Quantification of average vessel density in each group. * P < 0.05.
Fig. 9
Fig. 9
Immunofluorescence analyses of neurofilaments (A) The number of neurofilament heavy polypeptide-positive cells determined in two areas at the wound edge and in three areas near the wound center. (B) Quantification of neurofilament heavy polypeptide expression. * P < 0.05.
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