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Ouyang et al. Stem Cell Research & Therapy (2018) 9:246 Page 4 of 12

MAP. The MPG and cavernous nerve were exposed via Signaling Technology Co., Ltd., #4970,1:1000). After
midline incision. The corpora cavernosum of the penis hybridization of secondary antibodies, the resulting im-
was cannulated with a heparinized (250 U/ml) 25-gauge ages were analyzed using Tanon 5200 (Tanon Science &
butterfly needle via insertion at the crura. The cannula Technology Co., Ltd., Shanghai, China) to determine the
was connected to a BL-420 s Biological Functional Sys- integrated density for each protein band.
tem (Chengdu Taimeng Technology Ltd., Chengdu,
China) for continuous assessment and recording of ICP. Statistical analyses
The stimulus parameters were 1.5 mA, 20 Hz, pulse The results were analyzed using GraphPad Prism (v.5)
width 0.2 ms and duration 50 s [30]. The maximum in- software (GraphPad Software, La Jolla, CA, USA) and
crease in ICP for three stimuli per side for each animal expressed as the mean ± standard deviation. Multiple
was selected for statistical analysis of mean ICP. The groups were compared using one-way analysis of vari-
penis, MPG, and distal cavernous nerve were then har- ance (ANOVA) followed by the Tukey-Kramer test for
vested for histologic analysis and western blotting. post hoc comparisons using SPSS 16.0 software (SPSS
Inc., Chicago, IL, USA). P < 0.05 was considered statisti-
Histologic analysis cally significant.
Penile midshaft tissues were harvested and immediately
fixed for immunofluorescence staining, which was per- Results
formed as previously described [14]. The penile segment Isolation and characterization of MSCs
sections were incubated with primary antibodies to Primary MSCs were isolated from the femurs of
anti-neuronal nitric oxide synthase (Cell Signaling Tech- 4-week-old Sprague-Dawley rats and cultured in vitro.
nology Co., Ltd., Danvers, MA, USA, #4231, 1:200) and MSCs in this study showed typical spindle,
anti-smooth muscle actin (Cell Signaling Technology fibroblast-like morphology, and the colonies were ar-
Co., Ltd., #48938, 1:500). Secondary antibodies included ranged in a circular whirlpool-like fashion (Fig. 1a). To
DyLight 488 and 556-conjugated antibodies (Invitrogen, characterize the MSCs used in this study, we first
Waltham, MA, USA, 1:500). Nuclei were stained with assessed the MSCs in vitro potential to differentiate into
DAPI (0.5 μg/mL; Invitrogen, Carlsbad, CA, USA). Sig- adipocytes and osteocytes. The results showed that the
nals were visualized, and digital images were obtained cells were stained positive with Alizarin Red after osteo-
with a confocal laser scanning microscope (Zeiss LSM genic induction (Fig. 1b), stained positive with Oil Red
710, Oberkochen, Germany). Masson trichrome staining O after adipogenic induction (Fig. 1c) and stained posi-
was used to quantify the ratio between the smooth tive with Alcian blue after chondrogenic induction
muscle and collagen within the corpus cavernosum as (Fig. 1d). We further analyzed the expression of cell sur-
previously described [30]. Image analysis was performed face antigens, as shown in Fig. 1e, the cells expressed
by using Image-Pro Plus 5.1 (Media Cybernetics, Silver markers of MSCs, including CD29, CD44, and CD90,
Spring, MD, USA). but not the hematopoietic or endothelial markers,
CD11b, CD34, and CD45.
TUNEL staining
Apoptotic cells in the penile midshaft tissues were identified Characterization of MSC-Exos
and quantified by using an in situ cell apoptosis kit (Beyo- Transmission electron microscopy (TEM), western blot
time Biotechnology, Shanghai, China) according to the man- analysis, and qNano analysis were performed to identify
ufacturer’s protocols. Images were captured, and apoptotic the purified nanoparticles derived from MSCs. TEM
cell nuclei were characterized by red fluorescence. analysis revealed that MSC-Exos isolated from the su-
pernatants exhibited a cup-shaped morphology (Fig. 2a),
Western blotting similar to previously described exosomes [20]. The ex-
For western blot analysis, tissues and cells were lysed pression of exosomal markers CD63, TSG101, and
with RIPA buffer containing protease inhibitor cocktail, Flotillin-1 were further quantified by western blot ana-
and the protein concentrations of tissue lysates and cell lysis (Fig. 2b). qNano measurement indicated that the di-
lysates were determined by BCA assay. Samples contain- ameters of typical rounded particles mainly ranged from
ing 20 μg of protein were subjected to sodium dodecyl 70 nm to 140 nm (Fig. 2c), which was consistent with
sulfate polyacrylamide gel electrophoresis and trans- the previously reported exosomes size distributions [20].
ferred to a polyvinylidene fluoride membrane. The mem-
brane was blocked with 5% skim milk and incubated at MSC-Exos treatment improves erectile function after CNI
4 °C overnight with primary antibodies against nNOS, The effects of MSC-Exos treatment on the recovery of
anti-smooth muscle actin, caspase-3 (Cell Signaling erectile function are illustrated in Fig. 3. The maximal ICP
Technology Co., Ltd., #9662, 1:1000) or b-actin (Cell (mICP) and total ICP (tICP, area under the curve) were