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Fang, Xu, Zhang et al. 1431

Aiming to determine the effects of uMSC-Exos on myofibro- showed that the protein and RNA components in the uMSC-
blast formation, we first verified the integration ability of our pu- Exos were degraded thoroughly by the indicated enzyme treat-
rified exosomes by PKH67 assay, using a membrane labeling dye ments (Fig. 3A). In addition, the gel electrophoresis results
(PKH67) that integrates specifically into the membrane bilayer showed that the RNA components carried by the uMSC-Exos
structure on fusion. After staining, washed and ultracentrifuged were mainly small RNAs (,100 base pairs [bp]; Fig. 3A), con-
uMSC-Exos were added to the fibroblasts. Fluorescence micros- sistent with other reports [14]. To determine the structural
copy analysis revealed that cells treated with stained uMSC-Exos integrity of enzyme-treated exosomes, we also performed
showed prominent PKH67 fluorescence located in the cytoplasm NanoSight analysis and showed that intact exosomes were ob-
(Fig. 2B), but the UEFS group showed no obvious fluorescence, in- served after enzyme treatments (Fig. 3B; supplemental online
dicatingthatpurifiedexosomesdohavecellulartransmissionactiv- Fig. 4A, 4B).
ity. Next, we added equal quantities of uMSC-Exos, HEK293T-Exos, Next, we added untreated, proteinase-treated, or RNAse-
UEFS, and PBS to TGF-b-treated cells. Immunofluorescence treated uMSC-Exos into the TGF-b-induced cell models and
analysis showed that uMSC-Exo treatment greatly inhibited the tested their effects onfibroblast differentiation and proliferation.
TGF-b-induced elevation of a-SMA, although no significant ef- The qRT-PCR analysis showed that only the RNase-treated uMSC-
fects were found in the other groups (Fig. 2C). Flow cytometry Exos lost the ability to suppress myofibroblast formation, indi-
analysis showed that by comparing the percentage of a-SMA- cated by the significantly elevated expression of a-SMA in that
negative cells in each group, uMSC-Exo treatment resulted in sig- group (Fig. 3C). Western blot analysis also confirmed this change
nificantly increased amount of a-SMA-negative cells under TGF-b (Fig. 3D). The results of the gel contraction assay also supported
stimulation (Fig. 2D). For qRT-PCR analysis, we also found that the this conclusion, because the RNase-treated uMSC-Exos failed to
mRNA expression of both a-SMA andcollagenIwasgreatly inhibit gel contraction, but the untreated uMSC-Exo group did in-
decreased after uMSC-Exo treatment (Fig. 2E). Therefore, hibit this process (Fig. 3E).
our findings suggest that uMSC-Exos could suppress TGF-b- For cell cycle analysis using flow cytometry, the protease-
induced fibroblast differentiation and possibly even reverse treated uMSC-Exos, but not the RNase-treated uMSC-Exos,
myofibroblast formation. showed a much weakened ability to promote cell cycle progres-
To further verify the influence of uMSC-Exos on the contrac- sion (Fig. 3F), indicating that the proliferation-promoting ability
tion ability of TGF-b-treated fibroblasts, we performed a three- was mainly dependent on the protein components in the
dimensional collagen contraction assay. The cells were treated uMSC-Exos. Taken together, these results indicate that the RNA
with uMSC-Exos, HEK293T-Exos, UEFS, or a contraction inhibitor components of uMSC-Exos play a key role in suppressing myofi-
inthepresenceofTGF-bfor48hours.Theresultsshowedthatthe broblast formation.
uMSC-Exo group exhibited significantly reduced gel contraction
compared with that of the HEK293-Exo and TGF-b only groups,
Identification of a Group of uMSC-Exo-Specific
and the contraction inhibitor-treated group (2,3-butanedione
MicroRNAs by High-Throughput Sequencing
monoxime)showednocontraction(Fig.2F).Collectively,thesedata
indicate that uMSC-Exo treatment abolished the TGF-b-induced Ithaspreviouslybeen shownthatthemaintypeoffunctionalRNA
formation of myofibroblasts and contraction activity. component in exosome is microRNA, which can be efficiently
To fully evaluate the effect of uMSC-Exos on fibroblasts, we transmitted to other cells and functions diversely through exo-
performed cell cycle and cell migration assays. The results dem- some integration [22]. Our gel electrophoresis analysis corre-
onstrated that uMSC-Exo treatment significantly increased the spondingly showed that RNA molecules carried by uMSC-Exos
percentage of cells in the G 2 phase and promoted cell migration; were mostly small fragment RNAs of ,100 bp. We therefore an-
however, the cells treated with HEK293T and UEFS showed no alyzed the global expression of microRNAs in uMSC-Exos via high-
effects (Fig. 2G, 2H). These findings indicate that uMSC-Exos throughput sequencing approaches using HEK293T-Exos as a control.
can also promote fibroblast proliferation and migration ability, We also analyzed the microRNA expression patterns in uMSCs
consistent with previous reports [21]. and HEK-293T cells using existing GEO DataSets (GSE46989 [17]
and GSE56862, all from http://www.ncbi.nih.gov/geo/). We
found that uMSC-Exos had a specific miRNA abundance signature
The Myofibroblast-Suppressing Ability of uMSC-Exos that was quite different from that of HEK293T-Exos and even
Mainly Depends on the RNA Components uMSCs (Fig. 4A, 4B). Among the most abundant 10 miRNAs in
It was previously confirmed that exosomes contain numerous the uMSC-Exos, only miR-21 was also highly expressed in uMSCs
proteins and RNA components [6]. We next explored which (Fig. 4C, 4D). These findings indicated that most of the exosomal
molecules carried by the uMSC-Exos were key for suppressing microRNAs might be actively secreted into exosomes by uMSCs,
myofibroblast formation. We administered equal amounts of which also supports our hypothesis that these microRNAs have
purified uMSC-Exos, together with either proteinase K or RNase biological functions.
A supplemented with 0.05% Triton X-100, for different periods According to these results, using qRT-PCR, we evaluated
of time and tested the efficacy of the enzyme treatments by sil- miRNA and pre-miRNA expression levels in the fibroblasts after
ver staining and gel electrophoresis, respectively. The results treating them with uMSC-Exos or HEK293T-Exos for 48 hours.

(Figure legend continued from previous page.)
populationcellsmeasuredfromthreeindependentexperiments(right).Exo-PROseandExo-RNasegroupswerecomparedwithExogroupindividually.
Dataarepresentedasmean6SD;n=3;pp,p,.01.Abbreviations:BDM,2,3-butanedionemonoxime;Exo,exosome;Exo-PROse,proteinase-treated
uMSC-Exos; Exo-RNase, RNAse-treated uMSC-Exos; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; a-SMA, a-smooth
muscle actin; TGF-b, transforming growth factor-b; uMSC-Exos, umbilical cord-derived mesenchymal stem cell-derived exosomes.

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