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Figure1. uMSC-Exossuppressmyofibroblast aggregation and scarformation inafull-thicknessskin defectmousemodel.(A):Upper: Imagesof
indicated cell-transplanted wound at 14th day after initial treatment. Lower: Scar formation of mice in different treatment groups at 25 days
after transplantation. (B): Quantification of wound diameter (upper) and the scar length (lower) of different treatment groups indicated in (A).
pp, p , .01. (C): Representative images of immunohistochemistry showing a-SMA expression in the indicated treatment groups. Scale bar =
500 mm. (D): Representative image of purified exosome particles (left) and the particle size distribution in purified uMSC-Exos (right) as de-
termined by NanoSight. The red arrow indicates exoxomes. Scale bar = 1 mm. (E): Precise particle size distribution of purified uMSC-Exos de-
termined by laser light scattering assessment. The dashed dot line indicates the peak particle size of purified uMSC-Exos. (F): Western blot
analysis identifying purified uMSC-Exos using CD81 and CD63 antibody. (G): Representative immunohistochemistry showing a-SMA expression
in the indicated exosome-treated groups. Phosphate-buffered saline used as control. Scale bar = 500 mm. Abbreviations: Medium, umbilical
cord-derived mesenchymal stem cell culture medium; Mock, phosphate-buffered saline group; N, normal region; SMA, a-smooth muscle actin;
UEFS, umbilical cord-derived mesenchymal stem cell exosome-free supernatant; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-
derived exosomes; W, wound region.

(Pierce Protein Biology; Thermo Fisher Scientific Life Sciences). rehydrated, and incubated with 1 ml of PI (Cell Signaling Technol-
Thepresenceoftheexosomeswassubsequentlyconfirmedbyus- ogy, Danvers, MA, http://www.cellsignal.com). Approximately
4
ing a NanoSight NS300 (Malvern Instruments, Ltd., Malvern, U.K., 1.5 3 10 cells were counted for each test. For quantification
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http://www.malvern.com) and detection of exosomal surface of SMA and p-SMAD2 using cytometry, 5 310 isolated cellsfrom
markers CD81 using Western blot. each sample were collected and fixed in 4% paraformaldehyde.
The cells were washed, permeabilized, and blocked with goat se-
Fluorescence-Activated Cell Sorting and Cell rum before specific antibody incubation. Unconjugated anti-
Cycle Analysis phosphate SMAD2 (at 1:25 dilutions; Abcam, Cambridge, UK,
Flow cytometry analysis were performed as follows. For cell cycle http://www.abcam.com)andanti-SMA(at1:25dilutions;Abcam)
5
analysis, approximately 1 3 10 cells were fixed in 75% alcohol, were incubated with the cells. After washing, Alexa Fluor
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