ORIGINAL RESEARCH

Effect of adipose tissue-derived stem cell injection in a rat model of

urethral ﬁbrosis

Premsant Sangkum, MD; Faysal A. Yaﬁ, MD; Hogyoung Kim, PhD; , Mostafa Bouljihad, PhD;

Manish Ranjan, PhD; Amrita Datta, PhD; , Sree Harsha Mandava, MD; Suresh C Sikka, PhD;

Asim B. Abdel-Mageed, PhD; Wayne J.G. Hellstrom, MD

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

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

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 (300‒350 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

reported following surgery. Factors predictive of treatment

failure are not well-documented. However, long stricture

length (>4‒5 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

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.

-10

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.

2-14

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 (300‒350 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-

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

(

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-

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 (300‒350 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

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

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

maintained at 37 C in a cell culture incubator with 5% CO .

Flow cytometry

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

by Donnenberg. Brieﬂy, 2 x 10 cells were suspended in

PBS and were incubated with anti-rat monoclonal antibod-

Western blot

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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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-

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. 1A‒D). 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.

CUAJ • May-June 2016 • Volume 10, Issues 5-6

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Sangkum et al.

Fig. 2. Histological assessments of the rat urethral tissue sections (top-H&E stain, bottom-MT stain, x400) demonstrate normal urothelium surrounded by smooth

muscle cells without ﬁbrosis in the sham group, moderate submucosal ﬁbrosis with disorganized densely packed collagenous stroma in the urethral ﬁbrosis group,

and mild submucosal ﬁbrosis in the ADSC group. Urethral lumen (L), urothelium (U), ﬁbrosis (FB), corpus spongiosum (CSP).

Western blot analysis

sion. Furthermore, there are no obvious ethical concerns

with using ADSCs, compared to embryonic stem cells.¹⁸

The study by Liu et al demonstrated that human ADSCs

have the capacity to differentiate into urothelium-like cells

Representative chemiluminescence images of blotted mem-

branes containing protein extract from all three groups are

shown in Fig. 3. Western blot analysis demonstrated signiﬁ-

cantly increased expression of type I and type III collagen

levels in the urethral ﬁbrosis group as compared to the sham

group. In the ADSC group, there was a signiﬁcant decrease in

collagen type I and III expression (p<0.05) in comparison with

the urethral ﬁbrosis group. Western blot analysis of the expres-

sion of collagen corroborated with the histologic ﬁndings.

when co-cultured with urothelial cells. Furthermore, sev-

eral animal studies determined that the administration of

mesenchymal-derived stem cells signiﬁcantly reduced ﬁbro-

2-14

sis in the lung, kidney, and liver.

There are, however,

no reports of stem cell-based therapy in the clinical man-

agement of urethral stricture disease. Our study is a proof-

of-concept study that demonstrates that local injection of

ADSCs prevents ﬁbrosis induced by TGF-β1 injection.

ADSC injection therapy has been previously studied in a

Discussion

21,22

rat model of Peyronie’s disease.

Local injection of ADSCs

Stem cells are undifferentiated cells that have self-renewal

capabilities and are able to differentiate into mature non-

into the tunica albuginea decreased Peyronie’s-like changes

by decreasing the expression of tissue inhibitors of metal-

loproteinases (TIMPs) and increasing expression of matrix

metalloproteinases (MMPs), which resulted in decreased

9,20

regenerative and effector cells.

Currently, stem cells can

be obtained from embryos, gestational tissue, adult tissue,

and using advanced reprograming techniques. Adult stem

cells can be isolated from various sources, such as bone

marrow, adipose tissue, peripheral blood, connective tissue

1,22

elastosis and fibrosis.

Urethral stricture disease and

Peyronie’s disease have similar ﬁbrotic properties despite

involving different anatomic locations. Both conditions are

characterized by the formation of ﬁbrous tissue with the

of dermis, skeletal muscle, etc. There is an abundance of

stem cells in adipose tissue and this is an ideal stem cell

source because it can be easily obtained by using a mini-

mally invasive approach. ADSCs are autologous stem cells,

which eliminates the risk of rejection and immunosuppres-

23-26

same types of collagen.

These ﬁndings support our obser-

vations that local ADSC injection can decrease ﬁbrosis and

collagen type I and type III expression in rat urethral tissue.

To the best of our knowledge, this is the ﬁrst animal study

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CUAJ • May-June 2016 • Volume 10, Issues 5-6

Effect of ADSCs in rat model of urethral ﬁbrosis

Fig. 3. Representative Western blot demonstrating signiﬁcantly increased collagen type I and type III in the urethral ﬁbrosis group, whereas there is a signiﬁcant

decrease of collagen type I and type III in the ADSC group. Densitometry demonstrating relative density values of collagen proteins expressed as protein expression

relevant to α-tubulin control. Results are expressed as mean ± standard errors (pꢀ0.05 is signiﬁcant, using ꢁraphPad Prism 5.0).

that has investigated the effect of ADSCs-based injection

therapy in a model of urethral ﬁbrosis.

plasty would be more practical and easily translatable to

clinical practice. We hypothesize that additional injections

of autologous ADSCs may improve the outcomes and poten-

tially decrease the recurrence rates after either graft or ﬂap

urethroplasty procedures. Further animal and clinical studies

are warranted to validate this concept.

In recent years, stem cell-seeded tissue engineering has

been investigated for the surgical treatment of urethral stric-

tures.²The concept of the tissue engineering approach

is using natural or synthetic matrices (scaffolds) to provide

proper position and orientation to promote tissue regen-

7,28

One of the limitations of our study is the animal model

used. There is no universally accepted animal model for

study of urethral stricture disease. The rat model of TGF-β1

induced-urethral ﬁbrosis is a novel animal model of ure-

thral ﬁbrosis. Based on our previously published study, we

observed a number of TGF-β1 receptors in both human and

rat urethras, and there was a signiﬁcant increase in collagen

type I and III expression following TGF-β1 injection in the

same manner as in human urethral stricture specimens.¹⁷

Furthermore, researchers have demonstrated that halofugi-

none, which is a speciﬁc inhibitor of TGF-β1 up-regulation,

can prevent urethral ﬁbrosis formation in rat urethra. ³¹^,³²

As such, we postulate this animal model to be suitable for

our proof-of-concept study to evaluate the effects of ADSC

injections. Our study was focused on the prevention of scar

or ﬁbrosis formation. Therapeutic beneﬁts of ADSC injec-

tion may not be the same in urethral stricture patients who

already have ﬁbrotic scar. However, our ﬁndings suggest

9,29

eration.

Cell-seeding helps promote the regeneration or

reconstruction of new functional tissue. The synthetic matri-

ces or scaffolds are degraded and eventually replaced by

9,30

an extracellular matrix secreted by the invading cells.

ADSCs seeded on a porous silk ﬁbrin scaffold have been suc-

cessfully used to repair a rabbit urethral defect. Histological

examination revealed spontaneous angiogenesis with ure-

thral epithelium and smooth muscle regeneration in the

ADSC-seeded group compared to the simple porous silk

ﬁbrin scaffold or the control group.

Autologous grafts (buccal, bladder mucosa, lingual, etc.)

or ﬂaps have been widely accepted and adopted for use

during urethral reconstruction, particularly in the setting of

recurrent or complex urethral strictures. Our study demon-

strates that ADSC injection therapy signiﬁcantly reduced

tissue fibrosis and collagen expression. Stem cell-based

injection therapy to the area of a graft-augmented urethro-

CUAJ • May-June 2016 • Volume 10, Issues 5-6

E179

Sangkum et al.

2. Alfarano C, Roubeix C, Chaaya R, et al. Intraparenchymal injection of bone marrow mesenchymal stem cells

reduces kidney ꢀbrosis after ischemia-reperfusion in cyclosporine-immunosuppressed rats. Cell Transplant

that ADSC injection potentially decreases or prevents scar

formation and may potentially serve as an adjunct treat-

ment to increase the success rate of primary treatment for

urethral stricture disease patients. Further studies are needed

to validate these ﬁndings and the potential role of ADSCs

injection in urethral stricture patients.

012;21:2009-19. http://dx.doi.org/10.3727/096368912X640448

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Conclusion

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2015;194:820-7. http://dx.doi.org/10.1016/j.juro.2015.02.014

Local injection of ADSCs signiﬁcantly attenuates urethral

tissue ﬁbrosis, speciﬁcally collagen type I and III expres-

sion, in a rat model of urethral ﬁbrosis. This data suggests a

potential role for ADSC injection therapy in the adjunctive

treatment of urethral stricture disease.

18. Kim H, Naura AS, Errami Y, et al. Cordycepin blocks lung injury-associated inꢁammation and promotes

BRCA1-deꢀcient breast cancer cell killing by effectively inhibiting PARP. Mol Med 2011;17:893-900.

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Competing interests: Dr. Hellstrom has been an Advisory Board member for Abbvie, Allergan,

AMS, Astellas, Coloplast, Endo, Lipocine, Pꢀzer, and Repros; a Speakers’ Bureau member for Endo,

Menarini; has received grants/honoraria from Endo/Auxillium; and has participated in clinical trials

for Coloplast and NERI. The remaining authors declare no competing ꢀnancial or personal interests.

001;105:829-41. http://dx.doi.org/10.1016/S0092-8674(01)00409-3

1. Gokce A, Abd Elmageed ZY, Lasker GF, et al. Adipose tissue-derived stem cell therapy for prevention

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2. Castiglione F, Hedlund P, Van der Aa F, et al. Intratunical injection of human adipose tissue-derived stem

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Correspondence: Dr. Wayne Hellstrom, Department of Urology, Tulane University School of Medicine,

New Orleans, LA, United States; whellst@tulane.edu

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CUAJ • May-June 2016 • Volume 10, Issues 5-6