ORIGINAL RESEARCH  
Effect of adipose tissue-derived stem cell injection in a rat model of  
urethral fibrosis  
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Premsant Sangkum, MD; Faysal A. Yafi, MD; Hogyoung Kim, PhD; , Mostafa Bouljihad, PhD;  
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Manish Ranjan, PhD; Amrita Datta, PhD; , Sree Harsha Mandava, MD; Suresh C Sikka, PhD;  
Asim B. Abdel-Mageed, PhD; Wayne J.G. Hellstrom, MD  
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Division of Urology, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Department of Urology, Tulane University School of Medicine, New Orleans, LA, United States; Division of Comparative  
Pathology, Tulane National Primate Research Center, Covington, LA, United States  
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Cite as: Can Urol Assoc J 2016;10(5-6):E175-80. http://dx.doi.org/10.5489/cuaj.3435  
Published online May 12, 2016.  
0.9% in 2001, respectively. Various modalities are available  
for the treatment of urethral strictures, including urethral  
dilatation, direct visual internal urethrotomy (DVIU), and  
various urethroplasty techniques. The long-term recurrence-  
free rates after DVIU and urethral dilatation remain quite  
Abstract  
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Introduction: We sought to evaluate the therapeutic effect of adi-  
pose tissue-derived stem cells (ADSCs) in a rat model of urethral  
fibrosis.  
Methods: Eighteen (18) male Sprague-Dawley rats (300350 g)  
were divided into three groups: (1) sham (saline injection); (2)  
urethral fibrosis group (10 μg transforming growth factor beta 1  
poor. Urethroplasty remains the standard of care for urethral  
strictures, but recurrence rates as high as 15.6% have been  
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reported following surgery. Factors predictive of treatment  
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failure are not well-documented. However, long stricture  
length (>45 cm), lichen sclerosus, infectious or iatrogenic  
etiologies, prior urethroplasty, and failed endoscopic therapy  
are risk factors of urethroplasty failure. Current adjunctive  
treatments that aim to improve treatment outcomes include  
(
TGF-β1) injection); and (3) ADSCs group (10 μg TGF-β1 injection  
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plus 2 x 10 ADSCs). Rat ADSCs were harvested from rat inguinal  
fat pads. All study animals were euthanized at two weeks after  
urethral injection. Following euthanasia, rat urethral tissue was  
harvested for histologic evaluation. Type I and III collagen levels  
were quantitated by Western blot analysis.  
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injection of mitomycin c and steroids.  
Results of these  
different treatment options remain inconsistent and, as such,  
none of these modalities have been widely adopted.  
Stem cells have the ability to undergo self-renewal and  
multilineage differentiation, and to form terminally differen-  
tiated cells. Furthermore, a number of animal studies have  
demonstrated that mesenchymal stem cells have antifibrotic  
properties that can reduce fibrosis in the lung, kidney, and  
We sought to evaluate the potential therapeutic  
benefits of adipose-derived stem cell (ADSCs) therapy in  
decreasing fibrotic tissue in a rat model of urethral fibrosis.  
Results: TGF-β1 injection induced significant urethral fibrosis and  
increased collagen type I and III expression (p<0.05). Significant  
decrease in submucosal fibrosis and collagen type I and III expres-  
sion were noted in the ADSCs group compared with the urethral  
fibrosis group (p<0.05). TGF-β1 induced fibrotic changes were  
ameliorated by injection of ADSCs.  
Conclusions: Local injection of ADSCs in a rat model of urethral  
fibrosis significantly decreased collagen type I and III. These find-  
ings suggest that ADSC injection may prevent scar formation and  
potentially serve as an adjunct treatment to increase the success  
rate of primary treatment for urethral stricture disease. Further ani-  
mal and clinical studies are needed to confirm these results.  
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liver.  
Methods  
Study design  
Introduction  
These experiments were performed according to the  
American Guidelines for the Ethical Care of Animals, and  
were approved by the Tulane University Animal Care and  
Use committee. A total of 18 adult male Sprague-Dawley  
rats (300350 g) were purchased from Harlan Laboratories  
(Indianapolis, IN, U.S.) and housed in a regulated environ-  
ment with a 12-hour light and dark cycle in an approved  
Urethral stricture disease is a scarring process of the ure-  
thral mucosa and the surrounding spongy tissue of the  
corpus spongiosum. The reported estimated incidence  
of urethral stricture disease in an older veterans popula-  
tion is 0.6%. The incidence of urethral stricture diagno-  
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ses among Medicare beneficiaries was 1.4% in 1992 and  
CUAJ • May-June 2016 • Volume 10, Issues 5-6  
2016 Canadian Urological Association  
E175  
©
Sangkum et al.  
experimental laboratory. The animals had free access to  
food and water. Animals were randomized into three equal  
groups: (1) sham (saline injection to urethra); (2) urethral  
fibrosis (10 μg transforming growth factor beta 1 (TGF-β1;  
Franklin Lakes, NJ, U.S.) in 50 μL of PBS for 30 minutes in  
the dark at 4°C. The conjugated cells were washed thrice  
followed by analysis on a fluorescence-activated cell sorter  
(FACSCalibur; BD Biosciences, Franklin Lakes, NJ, U.S.).  
Data acquisition and analysis were performed using Cell  
Quest software (Becton Dickinson, Franklin Lakes, NJ, U.S.).  
Aviscera Bioscience, Santa Clara, CA, U.S.) injection); and  
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3) ADSC (10 μg TGF-β1 injection plus 2x10 ADSCs). All  
rats were euthanized at two weeks following normal saline  
or TGF-β1 or TGF-β1 plus ADSCs injection. Urethral tis-  
sues were harvested and divided into two pieces for further  
analysis. Histological assessment of urethral tissues was then  
performed. Type I and III collagen levels were evaluated by  
Western blot analysis.  
Urethral injection procedure  
The urethral injection procedure was performed as previ-  
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ously described. Briefly, each rat was anesthetized with  
100 mg/kg ketamine (NWI Veterinary Supply, Boise, ID,  
U.S.) and 10 mg/kg xylazine (Akorn, Decatur, IL, U.S.) intra-  
peritoneally. The rats were placed in the supine position.  
To facilitate urethral exposure and prevent urethral injury, a  
lubricated urinary catheter (polyethylene tube, 0.61 mm in  
diameter [equal to 1.8 French]) was gently inserted into the  
urethra. A small penoscrotal incision was created and the rat  
urethra was meticulously dissected. In the sham group, 0.05  
ml normal saline was injected into the urethra at the 3 and  
9 o’clock positions with a 30-gauge needle. The other two  
groups were injected with 10 μg of TGF-β1 (0.05 ml) using  
the same technique in order to induce urethral fibrosis. In the  
Adipose tissue-derived stem cell isolation and culture  
ADSCs were harvested from the inguinal fat tissue of a donor  
male Sprague-Dawley rat (300350 g). A lower abdomi-  
nal skin incision was made and the fat pads around both  
sides of the inguinal area and spermatic cord were excised.  
Approximately 1 g of fresh inguinal fat tissue was washed  
three times in Dulbecco’s phosphate-buffered saline (DPBS)  
and minced on ice using a sterile blade into three 1 mm  
pieces. The minced tissue was suspended in 2 mg/ml of  
collagenase type-I (GIBCO, Invitrogen, Carlsbad, CA, U.S.)  
dissolved in DPBS. The tissue/collagenase suspension was  
incubated at 37°C in a shaking water bath for 2.5 hours.  
The tissue suspension was then filtered first through a 70  
μm, followed by 40 μm, cell strainer to remove the tissue  
debris. Following this, mature adipocytes were removed by  
centrifugation (1500 g for 10 minutes). The formed pellet  
was then suspended in DPBS and centrifuged again. The  
resulting stromal vascular fraction pellet was suspended and  
incubated for two minutes in red blood cell lysis solution  
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ADSCs group, 2 x 10 ADSCs were injected into the same  
area immediately after TGF-β1 injection. A non-absorbable  
suture was placed into the corpus cavernosum at the same  
level of the urethral injection to serve as a landmark for  
future site identification. The penile skin was approximated  
with 4-0 interrupted absorbable sutures.  
The rats were euthanized at two weeks following the  
urethral injection. The urethral tissues were harvested and  
stored for further analysis. The same investigator performed  
all injections and sacrifice procedures.  
(
0.15 M ammonium chloride, 10 mM potassium bicarbon-  
ate and 0.1 mM EDTA). ADSCs were washed in 2 ml of  
% bovine serum albumin (Sigma-Aldrich, St. Louis, MO,  
U.S.), suspended in Dulbecco’s modified eagle’s medium  
DMEM)/F12 medium (GIBCO, Invitrogen, Carlsbad, CA,  
Histology  
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The rat urethral specimens were harvested and fixed in  
10% buffered neutral formalin and processed using rou-  
tine histological methods. Haematoxylin and eosin (H&E)  
and Masson’s trichrome (MT) stained urethral cross sections  
(4–6 μm thick) were examined using a digital camera (Leica  
EC3, Leica Microsystems, Heerbrugg, Switzerland) coupled  
with an optical microscope (Leica Model DM 2500; Leica  
Microsystems CMS, Weltzar, Germany). The degree of fibro-  
sis was evaluated and described as minimal, mild, moder-  
(
U.S.) supplemented with 20% fetal bovine serum and 1%  
antibiotic-antimycotic solution (penicillin G, streptomycin,  
and amphotericin B; Mediatech, Herndon, VA, U.S.), and  
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maintained at 37 C in a cell culture incubator with 5% CO .  
Flow cytometry  
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The rat ADSCs (passage 3) were evaluated for viability and  
ate, and severe. All sections were reviewed by the same  
pathologist, who was blinded to the study design.  
stained for flow cytometry analysis, as previously described  
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by Donnenberg. Briefly, 2 x 10 cells were suspended in  
PBS and were incubated with anti-rat monoclonal antibod-  
Western blot  
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ies for CD29, CD45, CD90, and CD105. Also, 2 x 10  
rat ADSCs were suspended in 50 μL PBS with anti-human  
CD90 or anti-human CD105 conjugated with either phy-  
coerythrin or fluorescein isothiocyanate (BD Biosciences,  
Urethral tissue was collected and samples were prepared  
by homogenizing in radioimmunoprecipitation assay lysis  
buffer (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.).  
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CUAJ • May-June 2016 • Volume 10, Issues 5-6  
Effect of ADSCs in rat model of urethral fibrosis  
Results  
Protein contents were assessed using the Bio-Rad protein  
assay (Bio-Rad, Hercules, CA, U.S.) according to manufac-  
tures protocol. Immunoblot analyses were conducted as pre-  
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viously described. 20 μg of protein from each lysate was  
then fractionated by sodium dodecyl sulphate-polyarylamide  
gel electrophoresis (SDS-PAGE) on a 4–20% gradient gel  
and were transferred to a polyvinylidene fluoride mem -  
brane. To ensure equal loading and transfer of samples,  
Ponceau staining was performed and the membrane then  
probed with antibodies to collagen type I and collagen type  
III (Abcam, Cambridge, MA, U.S.), and with α-tubulin (Cell  
Signaling, Beverly, MA, U.S.). Appropriate secondary anti-  
bodies and horseradish peroxidase based chemilumines-  
cence reagents were used to detect the immune complexes  
Characterization of ADSCs  
ADSCs were isolated from the harvested inguinal adipose  
tissue of a donor rat, and were passaged when 80% conflu-  
ence was reached. To ascertain the characteristics of ADSCs,  
flow cytometric analysis was performed. The ADSCs were  
strongly positive for mesenchymal stem cell surface mark-  
ers CD29 (97.48%), CD90 (98.48%), and CD105 (96.16%),  
whereas they were negative (0.00%) for hematopoietic stem  
cell marker CD45 (Figs. 1AD). Furthermore, rat ADSCs  
were also negative for anti-human CD90 (0.77%) as well  
as anti-human CD105 (0.22%), establishing the specificity  
of anti-rat antibodies. The last panel revealed the unstained  
rat ADSCs isotypes (Fig. 1G).  
(
Pierce, Rockford, IL, U.S.).  
Statistical analysis  
Statistical analysis was performed with Prism 5.0 (GraphPad  
Software, San Diego, CA, U.S.). All data were expressed as  
means (SD) and the differences between multiple groups  
were compared by one-way analysis of variance, followed  
by the Tukey multiple comparisons test (p<0.05 was con-  
sidered statistically significant).  
Histology  
Comparative microscopic evaluation of representative H&E  
and MT-stained urethral cross-sections revealed normal  
urethral structure without submucosal fibrosis in the sham  
group, whereas there was moderate fibrosis with densely  
packed collagenous stroma involving submucosal tissue in  
the positive control group. In contrast, there was only mild  
submucosal urethral fibrosis in the ADSC group (Fig. 2).  
Fig.1. Characterization of adipose tissue-derived stem cells (ADSCs). Flow cytometric analysis of early passage rat ADSCs depicting positive expression for: (A)  
CD29 (97.48%); (B) CD90 (98.48%); (C) CD105 (96.16%); and (D) negative expression for CD45 (0.00%). Rat ADSCs were also negative for anti-human: (E) CD90 (0.77%)  
and (F) CD105 (0.22%), further verifying the specificity of the anti-rat antibodies. (G) Unstained ADSC isotypes are shown.  
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