ADR-12708; No of Pages 8

Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

journal homepage: www.elsevier.com/locate/addr

Stem cell treatment of erectile dysfunction☆,☆☆

Amjad Alwaal a,b,⁎, Uwais B. Zaid a, Ching-Shwun Lin a, Tom F. Lue a
a
Department of Urology, University of California, San Francisco, CA, USA
b
King Abdul Aziz University, Jeddah, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history: Erectile Dysfunction (ED) is a common disease that typically affects older men. While oral type-5 phosphodieserase
Accepted 8 November 2014 inhibitors (PDE5Is) represent a successful first-line therapy, many patients do not respond to this treatment leading
Available online xxxx researchers to look for alternative treatment modalities. Stem cell (SC) therapy is a promising new frontier for the
treatment of those patients and many studies demonstrated its therapeutic effects. In this article, using a Medline
Keywords:
database search of all relevant articles, we present a summary of the scientific principles behind SCs and their
Erectile dysfunction
Stem cell therapy
use for treatment of ED. We discuss specifically the different types of SCs used in ED, the methods of delivery tested,
and the methods attempted to enhance SC therapy effect. In addition, we review the current preclinical literature on
SC therapy for ED and present a summary of its findings in addition to the single clinical trial published.
© 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.1. ED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.2. Mechanism of erection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.3. Aging and ED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.4. Metabolic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.5. Prostate cancer therapy-related ED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1.6. Peyronie's disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2. Types of stem cells used in ED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.1. ESC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.2. Endothelial progenitor stem cells (EPSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.3. Bone marrow-derived stem cell (BMSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.4. Skeletal muscle-derived stem cell (SKMSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.5. Neural crest SCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.6. Adipose tissue-derived stem cell (ADSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.7. Testis SC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
2.8. Human urine SC (USC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
3. Methods of SC delivery for ED treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
4. Methods to enhance the therapeutic effects of SCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
5. Assessment of outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
6. Clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
7. Immunocompatibilty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
8. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Regenerative Medicine Strategies in Urology”
☆☆ Author disclosure statement: No competing financial interests exist.
⁎ Corresponding author at: University of California, San Francisco, Box 0738, 1600 Divisadero St., MZ Bldg A, Room A609, San Francisco, CA 94143, USA. Tel.: +1 415 885 7748; fax: +1
415 885 7443.
E-mail address: amjadwal@yahoo.com (A. Alwaal).

http://dx.doi.org/10.1016/j.addr.2014.11.012
0169-409X/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: A. Alwaal, et al., Stem cell treatment of erectile dysfunction, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/
j.addr.2014.11.012
2 A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx
1. Introduction
1.1. ED
It is an important health problem that significantly impacts the
patient's quality of life and can have a detrimental effect on his well-
being and relationship with his partner [1]. ED is defined as the inability
to achieve or maintain a penile erection satisfactory for sexual inter-
course [2]. ED is estimated to affect 20% of men above 40 years of age,
with the incidence increasing with increasing age [3], in addition to
other factors such as better diagnosis and increased awareness of the
disease [4]. Men between the age of 61–70 years are twice as likely to
be affected than men aging 51–60 years [5], with 67% of men at age
70 years affected with the disease [6]. Another important reason for
the rise in ED is the rapidly growing prevalence of diabetes mellitus
(DM) [7], which is a major risk factor for ED [4]. Several treatment
modalities exist for ED, including oral PDE5Is, intraurethral alprostadil
suppository, intracorporal (IC) injection of erectogenic medications,
vacuum device, and penile implant. Among these modalities, PDE5Is re-
main the most widely used and have great success rates, mainly due to
their ease of administration as oral medications and proven efficacy.
However, despite such successes, there are several limitations to their
use. For example, they are contraindicated in some patients who take
nitrates due to the risk of developing severe hypotension; some men
cannot tolerate their side effects; they are only partially effective in cer-
tain patients; and they have a considerable financial cost [8]. In addition,
they provide only symptomatic relief of ED and do not offer a cure
for the disease. Therefore, there is a growing interest in developing
therapies that offer a cure for the disease, including gene therapy and
SC therapy [9,10].
1.2. Mechanism of erection
Penile erection occurs through a nitric oxide (NO)-mediated mecha-
nism. NO, the main neurotransmitter involved in erection, is released by
endothelial cells and non-adrenergic non-cholinergic nerve terminals in
the cavernosal tissue. It causes relaxation of the cavernosal smooth
muscles through a cGMP-mediated reduction of intracellular calcium,
after which cGMP gets degraded by PDE5 [11]. Once the lacunar spaces
are filled with blood, they compress the subtunical venules, resulting in
erection. Detumescence occurs when adrenergic receptors are activat-
ed, inducing contraction of the cavernosal smooth muscles with resul-
tant diminished blood in-flow and reduced size of the lacunar spaces,
which in turn allows more venous out-flow through the subtunical ve-
nules [12].
1.3. Aging and ED
Different diseases and conditions cause ED through different mech-
anisms. Aging results in ED through increased penile vascular tone [13]
and inactivation of endothelial nitric oxide synthase (eNOS), the latter
of which is accomplished by decreased phosphorylation of eNOS
positive regulatory site and increased phosphorylation of its negative
regulatory site [14]. In addition, it reduces nNOS nerve fibers, thereby
diminishing the erectile response to cavernosal nerve stimulation and
reducing penile reflexes [15]. Aging also decreases NO bioavailability
by increasing reactive oxygen species (ROS). When ROS are in excess,
superoxide anion interacts with NO to cause endothelial dysfunction
[16,17]. Aging was also found to be associated with abundance of colla-
gen fibers, reduction of smooth muscle cells, and degenerative changes
in elastic fibers [18].
1.4. Metabolic syndrome
The prevalence of DM in the US is 8.3% of the population, with 26.9%
of the population 65 years or older affected with DM in 2010 [19].
Please cite this article as: A. Alwaal, et al., Stem cell treatment of erectile dysfunction, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/
j.addr.2014.11.012
Diabetics are 3 times more likely than non-diabetics to have ED [5],
with a prevalence of 50–75% [6]. ED affects diabetics 10–15 years earlier
than non-diabetics [5], and PDE5Is are less efficacious in this population
[20]. The mechanism by which DM causes ED is multifactorial and there
are different findings and proposed mechanisms for its occurrence. DM
was found to diminish the amount of NO-releasing penile nerves [21,
22], cavernosal endothelial cells, and cavernosal smooth muscle content
[22,23]. Ning et al. [24,25] recently demonstrated that hyperglycemia
induced mitochondrial fragmentation and cellular apoptosis of endo-
thelial cells. Hyperglycemia is also associated with smooth muscle dys-
function through oxidation of low-density lipoproteins and excessive
production of oxygen free radicals. DM also impairs vascular endothelial
growth factor (VEGF) signaling and upregulates the RhoA/RhoKinase
pathway [26–28]. It also reduces the expression and activity of neuronal
nitric oxide synthase (nNOS) [29–31]. Given the penile tissue damage
associated with DM and the negative impact of ED on the quality of
life in diabetics [32], SC therapy holds a great promise to treat DM-
associated ED due to SCs' regenerative ability and their potential to re-
store cavernosal endothelial and smooth muscle cells. Hyperlipidemia
is another metabolic factor responsible for ED [33]. It was found that
for every 1 mmol/L increase in serum total cholesterol there was a
32% increased risk of ED, and for every 1 mmol/L increase in high-
density lipoprotein (HDL) there was a marked reduction in the risk of
ED [34]. Hyperlipidemia causes ED through neuronal and endothelial
dysfunction, leading to a reduction in cavernosal NO levels [35–37].
1.5. Prostate cancer therapy-related ED
Prostate cancer is the most commonly diagnosed cancer in US men
[38]. Earlier detection and treatment have allowed the widespread use
of localized treatment options in about 80% prostate cancer patients
[38], including radical prostatectomy (RP) and radiation therapy (RT)
[39]. However, these treatment options carry a significant risk of post-
treatment ED [40]; for example, RP is associated with 60.8–93.9% risk
of ED [41]. The most widely accepted mechanism for post-RP ED is inju-
ry to the cavernosal nerve fibers that run along the posterolateral aspect
of the prostate. Nerve-sparing RP has been introduced in order to pre-
serve the cavernosal nerves. However, despite the continued refine-
ment in the surgical technique, nerve-sparing RP is still associated
with nearly 20% risk of ED at 24 months post-op [42]. This is likely
due to neurapraxia, in which stretching, heating, or trauma to the
cavernosal nerve fibers during surgery induce Wallerian degeneration
[43]. Alternatively, NO-releasing nerves stop producing NO during the
period of neurapraxia, resulting in cavernosal smooth muscle apoptosis
and fibrosis. In support of this theory is the reduction of smooth muscle
content and subsequent fibrosis in post-RP penile tissue [43,44]. RT has
been much less studied than RP, but generally from the limited data
published, it is likely that post-RT ED follows the same mechanism.
That is, cavernosal nerve injury followed by smooth muscle degenera-
tion [23,45]. PDE5Is are being prescribed routinely in many centers
post-RP as part of the “penile rehabilitation protocol”. The exact mech-
anism for their effect is not known, as PDE5 sits downstream of NO,
which cannot be produced by damaged nerves post-RP. Thus, the
exact mechanism for PDE5Is' effects in post-RP ED patients remains to
be proven, and so does their true effect in this population [44,46].
1.6. Peyronie's disease (PD)
PD affects 3% of men, resulting in pain and penile deformities such as
curvature, indentation, and shortening [47]. Although further research
is needed, it is thought that PD causes ED directly. Among several
mechanisms that have been proposed to describe this effect [48],
veno-occlusive dysfunction resulting from the PD plaque is the most
commonly accepted although further research is needed to prove this
theory [49]. The severity of PD occasionally necessitates incising or ex-
cising the plaque followed by patch grafting the tunica albuginea (TA)
 A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx 3

to maintain the length of the penis [50], and this can further increase the
risk of ED [51]. Several strategies have been proposed for treatment of
PD including the use of long-term PDE5Is as anti-fibrotic strategy [52].
Recent advances in SC research allowed the utilization of porcine
small intestinal submucosa (SIS) with seeded SCs to reduce the risk of
ED [53]. Another recent development is the use of SCs in transforming
growth factor (TGF)-induced PD rat model to study the anti-fibrosis
effect of SCs in PD [54,55]. These are promising new strategies in the
treatment of PD and its associated ED. Table 1 summarizes some of
the ED etiologies and their proposed mechanism.
In this article, we aimed at identifying the different types of SCs in
the treatment of ED as well as their routes of delivery, modifications
(e.g. gene modifications), clinical applications, and possible future use.
Using the terms “stem cell” and “erectile dysfunction”, we performed
a search of the Medline database for all the English language articles
that have been published from 2001 to present. Additional articles
were identified through examination of citations.
2. Types of stem cells used in ED
SCs have the potential to divide indefinitely with the daughter cells
having the ability to either become a SC or differentiate into a special-
ized cell type, such as brain cell, red blood cell, or muscle cell [56,57].
Their self-renewal capacity and ability to regenerate damaged tissues
depend on the various surrounding stimuli and factors [56,57]. They
are classified into (totipotent, pluripotent, multipotent, or unipotent)
depending on their hierarchical differentiation potential [58]. Totipo-
tent SCs, like the zygote and its morula daughter cells, have the greatest
differentiation potential and can divide into any tissue type originating
from the ectoderm, mesoderm, and endoderm tissues [59]. Pluripotent
cells give rise to the three germ cell layers but not to extra-embryonic
tissues. The most widely acknowledged example of pluripotent SC is
the embryonic SC (ESC) derived from the inner cell mass of the blasto-
cyst [60,61]. Multipotent SCs, such as the mesenchymal SC (MSC), are
isolated from the developing germ layer or its developed adult organ
[59]. Unipotent SCs are precursor cells within the developed tissue
and can give rise to only one cell type, such as epithelial cells. They
have limited self-renewal capacity, therefore labeling them as SCs is de-
batable [62].
There have been ethical concerns regarding the use of ESCs, due to
the need to destroy embryos for their isolation, and this has heightened
the interest in adult SCs (ASCs) as alternative SC source [62]. In addition,
induced pluripotent SCs (iPSC) can be generated from somatic cells by
overexpression of ESC-specific transcription factors [63,64]. These cells
share some characteristics with ESCs although the exact differences be-
tween them have not been fully elucidated [65].
The SC niche is a microenvironment that supports SCs in their quies-
cent state and promote SC self-renewal or tissue regeneration upon tis-
sue damage [66]. Niche cells can be classified as epithelial or stromal
depending on their location and proximity to SCs [66]. SC characteristics
within the niche are influenced by many factors, such as cell–cell inter-
action between SCs and their neighboring cells, interaction with sur-
rounding adherent molecules and the extracellular matrix, presence of
cytokines and other growth factors, and oxygen tension and other
physiochemical properties. The SC niche is usually present in the
perivascular space, which allows direct access to the blood stream
when damaged tissues are in need of SCs for their regenerative poten-
tial. SCs in their niche microenvironment were found to be in a state
of relative hypoxia with oxygen tension as low as 2% [67,68], thereby
possibly shaping their unique characteristics and enhancing the produc-
tion of growth factors leading some researchers to replicate this niche
environment in vitro [69].
2.1. ESC
ESCs are pluripotent cells that had been studied only once in ED re-
search. Bochinski et al. was the first study investigating the use of SCs in
ED, and it investigated the effect of ESCs on neurogenic ED induced by
cavernosal nerve injury and demonstrated improved erectile function
when ESCs were injected into the corpora cavernosa or major pelvic
ganglia (MPG) [70]. Given the ethical concerns mentioned earlier, no
further studies had been done using such cells.
2.2. Endothelial progenitor stem cells (EPSCs)
They are bone marrow-derived cells that were discovered in 1997
[71]. When peripheral blood mononuclear cells (PBMCs) are enriched
by selection for CD34 expression, they exhibit mature endothelial cell-
like characteristics and display several endothelial markers such as
CD31, CD34, VEGFR2, Tie-2, and eNOS. In addition, they form vessel-
like structures on matrigel [71]. This discovery has generated promise
for using EPSCs in angiogenic therapy. However, there have been de-
bates over the characterization of these cells with some arguing that
EPSCs can be recognized through their antigenic receptors [72,73]
while others believe that they need to be recognized by using cell be-
havior and growth dynamics in vitro [74]. Generally, when PBMCs are
grown in culture, the late cell outgrowth colonies at 1–3 weeks possess
antigenic properties restricted to endothelial cells and are highly prolific
making this subset of cells the most viable source for angiogenic therapy
[74,75]. Most research done thus far using EPSC has been in heart failure
after myocardial infarction [76]. Low circulating levels of EPSCs have
been found to be an independent predictor for ED [77]. Gou et al. [78],

Table 1
Some etiologies and proposed mechanisms for erectile dysfunction.

Etiology Summary of proposed mechanisms

Aging – Increased penile vascular tone

– Inactivation of eNOS
– Reduction of nNOS nerve fibers
– Reduction of NO bioavailability by increasing reactive oxygen species (ROS)
– endothelial dysfunction
– Abundance of collagen fibers, reduction of smooth muscle cells, and degenerative changes in elastic fibers
DM – Reduction of NO-releasing penile nerves, cavernosal endothelial cells, and cavernosal smooth muscle content
– Mitochondrial fragmentation and cellular apoptosis of endothelial cells
– Smooth muscle dysfunction through oxidation of low-density lipoproteins and excessive production of
oxygen free radicals
– Impairment of VEGF signaling
– Upregulation of the RhoA/RhoKinase pathway
– Reduction of the expression and activity of nNOS
Hyperlipidemia – Neuronal and endothelial dysfunction
– Reduction in cavernosal NO levels
Prostate cancer treatment (RP and RT) – Injury to the CN fibers followed by smooth muscle degeneration
PD – Veno-occlusive dysfunction
– Intracorporal fibrosis

Please cite this article as: A. Alwaal, et al., Stem cell treatment of erectile dysfunction, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/
j.addr.2014.11.012
4 A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx
in the only study using EPSCs in ED, showed an improvement in rat
erectile function in streptozotocin-induced diabetic animals with ED
when they were transplanted with VEGF-transfected EPSCs.
2.3. Bone marrow-derived stem cell (BMSC)
These are adult SCs that are considered the prototype for MSCs [10].
They have pluripotent ability to differentiate into different tissues and
cells [79] and they secrete many cytokines that have trophic effects on
cytoprotection, cell survival, and immunomodulation [80]. They are
characterized by their ease of isolation and ex vivo expansion [81].
MSCs do not elicit an immune response due to their lack of surface co-
stimulatory molecules required for T cell activation, thus eliminating
the need for immunosuppression [82]. Several pre-clinical studies
have shown the beneficial effect of BMSCs on erectile function in differ-
ent rat models such as DM [83–86], cavernous nerve crush injury
[87–90], and aging [81,91].
2.4. Skeletal muscle-derived stem cell (SKMSC)
SKMSCs are another group of pluripotent MSCs. They were exten-
sively studied because of their prolonged proliferation, low immunoge-
nicity, and ability to convert to several cell lineages when implanted
into different organs [92]. They should not be confused with satellite
myoblasts, which are non-pluripotent and have been used to treat
heart disease in clinical trials as they can give rise to cardiomyocytes
[93]. SKMSCs can be easily obtained through muscle biopsy [94] and
have been shown to improve erectile function in a bilateral cavernous
injury ED rat model [95]. Nolazco et al. demonstrated their ability to
transform to SMs in penile tissue in addition to improving erectile func-
tion in aged rat models [96]. Woo et al. also confirmed such findings in a
cavernous nerve injury rat model and showed survival of these cells in
the penile tissue [97].
2.5. Neural crest SCs
Another form of pluriopotent SC is neural crest SC. These cells are
considered the progenitors of the peripheral nervous system, giving
rise to neurons, Schwann cells, and adrenal chromaffin cells [98]. Song
et al. in 2007 demonstrated the ability of these cells to differentiate
into smooth muscle cells and endothelial cells in the rat penis [99]. To
our knowledge, no studies have been conducted to test their therapeu-
tic potential for ED.
2.6. Adipose tissue-derived stem cell (ADSC)
ADSCs are pluripotent SCs that are considered the most widely used
type of SCs in ED research given that they are easy to obtain from abun-
dant tissue sources [10]. There are two populations of SCs derived from
adipose tissue that have been used in ED and non-ED research. The ad-
ipose derived-stromal vascular fraction (AD-SVF) cells, which are de-
rived from the pellet fraction of collagenase-digested adipose tissue
homogenate [100], and ADSCs, which are derived from SVF and are ad-
herent to the culture dish and then further propagated [100]. One gram
of fat yields 250,000 SVF cells, and of those cells only 2% qualify as ADSC
[101]. SVF cells will likely be more commercially available than ADSC
due to the availability of machines for automated isolation of SVF cells
[102]. AD-SVF cells enhance angiogenesis by differentiating into endo-
thelial cells or through paracrine effects by releasing growth factors
thereby enhancing erectile function [103,104]. ADSC on the other
hand demonstrated an improvement in erectile function in pre-
clinical trials [30] through direct transformation to endothelial cells,
smooth muscle cells, and neurons [105] as well as through release of
stimulatory cytokines [106] such as VEGF and fibroblast growth factor
(FGF) [104,107]. It is likely that the erectile function improvement effect
seen with ADSCs is due to cytokine release rather than the direct
Please cite this article as: A. Alwaal, et al., Stem cell treatment of erectile dysfunction, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/
j.addr.2014.11.012
transformation to other cell types. This has been suggested through
studies showing improvement in erectile function when ADSC lysate
or ADSC conditioned growth medium was used [104,108]. However,
there are some problems associated with ADSCs that might make SVF
more desirable to use, including the longer process of isolation relative
to SVF, the requirement for certain facilities and personnel, and possible
alteration of cellular characteristics [109].
2.7. Testis SC
Choi et al. in 2013 [110], isolated CD34+/CD73+-double-positive
testicular stromal cells (HTSCs). These cells demonstrated the ability
to proliferate for extended periods of time without forming tumors
in vivo. They gave rise to all three germ cell lines, and more importantly,
restored erections in a CN injury rat model without forming teratomas.
2.8. Human urine SC (USC)
There has been one preclinical trial looking at USCs in the treatment
of ED [111]. USCs are pluripotent SCs that have been shown to differen-
tiate into endothelial and smooth muscle cells. They have the advantage
of being easy to obtain. The study showed a positive response on erectile
function in diabetic rats.
3. Methods of SC delivery for ED treatment
Intracavernosal (IC) SC delivery is by far the most common SC deliv-
ery route for ED treatment, given its already proven success and ease of
administration [10]. In the CN injury rat models, the damage incited is
not in the cavernosa but rather in the axonal fibers, with the neurons lo-
cated in the MPG. By injecting into the corporal tissue, the SCs exert
their regenerative effect on the MPG by either secreting soluble factors
into the blood stream or by migrating to the MPG [10]. Our experiments
have demonstrated that SCs migrate to the MPG after IC injection [112,
113]. SC injection whether immediately at the time of CN injury or 4
weeks after injury had similar positive effects on erectile function
[103]. Injecting SCs directly into the MPG has also been tried and dem-
onstrated regenerative effects, however this route has not been exten-
sively studied because of the technical difficulties involved and
similarly positive results obtained with direct penile injection [70,89].
Application of SCs directly onto injured CNs followed by coverage
with poly(lactic-co-glycolic acid) (PLGA) or hydrogel has also been
studied. While recovery of erectile function was demonstrated, no di-
rect comparison to the IC method was done; therefore, we do not
know the relative efficacy of such approaches [114–117]. Three studies
have been done by injecting SCs into the periprostatic tissue with or
without simultaneous IC injection [90,110,118]. In one of these studies,
all the groups showed similar recovery of erectile function, whether IC,
periprostatic, or both [118]. Recently, Ryu et al. demonstrated that intra-
peritoneal injection of SCs was less effective than IC injection in restor-
ing erectile function in CN injury mouse model [119].
Our group has previously attempted to use adipose-derived acellular
matrix seeded with ADSCs as nerve graft. While there was some recov-
ery of erectile function, the results did not reach a statistical significance
[120]. Further technical refinement is needed, but given the good re-
sponse with IC injection, this may not be necessary. We also previously
suggested that the effect seen with SC injection might be the result of SC
homing to the injury site [121], and we have proven the beneficial effect
of intravenous ADSC injection on erectile function [121]. Tracking of the
injected cells showed that they migrate into the MPG in post-RT ED rats
[121]. This suggests that the intravenous route may be another valuable
method for SC delivery. Recently, Ying et al. demonstrated an improve-
ment in the erectile function of CN injury rat model when autologous
saphenous vein grafts were interposed at the nerve stumps and ADSCs
injected into the vein graft [122].
 A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx 5

In regard to SC treatment for PD-associated ED, reconstruction of the
TA, with ADSC-seeded SIS resulted in better erectile function than
with unseeded SIS [53]. In another study that simulates PD by using
intratunical TGF injection, less TA fibrosis and better erectile function
were observed when ADSC, as compared to saline, was injected into
the injured TA [54]. Similar results were obtained in a more recent
study [123].
sildenafil to MDSC application did not have any additive effect in
preventing or reversing corporal veno-occlusive disease in the CN injury
model [126]. FGF-transfected USCs had similar effect to USCs alone in
improving erectile function [111].
5. Assessment of outcome

The response to SC ED treatment in preclinical studies involves func-

4. Methods to enhance the therapeutic effects of SCs
In order to boost the therapeutic potential of SC therapy, several pre-
clinical trials attempted to improve SC performance by genetically alter-
ing their intrinsic characteristics or by combining them with scaffolds,
growth factors, medications, etc. Two studies showed an improvement
in erectile function with increased endothelial and smooth muscle
markers when VEGF-transfected ADSCs were injected in diabetic rats
[84,124]. The effect was greater than when either ADSC or VEGF was
used alone [84,124]. In another study, BMSCs were transfected with
KCNMA1, a gene involved in cellular excitement and neurotransmitter
release [85]. The results showed improved erectile function in diabetic
rats [85]. As mentioned earlier, ADSC with PLGA membrane [114,116]
or hydrogel [115] improved angiogenesis and erectile function in CN in-
jury model either alone or in combination with oral udenafil [84,116].
Matrixen was also combined with BMSCs to increase their implantation
and was found to augment erectile function [125]. Adding low-dose
tional and histological assessment. The most widely used method is
measure the intracavernosal pressure (ICP) upon electrostimulation of
the cavernosal nerves. Typically, 1.5 mA, 20 Hz, and 0.2 ms pulse
width would result in elevation of ICP from 20 cm H2O to 100 cm H2O
in normal rats approaching systemic blood pressure, while in ED rats
ICP is around 30 cm H2O. An elevation of ICP to above 70 cm H2O
would be considered an indication of successful treatment. Alternative-
ly, ICP can be normalized to mean arterial pressure (MAP) in order to
measure erectile function, which is around 0.8 (ICP/MAP) in normal
rats.
This method requires laparotomy, therefore it is usually done at the
time of sacrificing the animal for tissue harvesting, which is 4 weeks
post-SC transplantation. When tissues are harvested, they are examined
using immunohistochemistry (IH) and immunofluorescence (IF) for lo-
cation of SCs, correlation of their function with the function, and/or
presence of SC differentiation. In order to identify SCs, pre-labeling is
typically done [10].

Table 2
Preclinical trials for stem cell treatment of erectile dysfunction.

Trial year First author Animal model Stem cell type Method of Reference
transplantation

2004 Bochinski CN injury rat Allogeneic ESC IC or Intra-MPG [70]

2006 Kim CN injury rat Allogeneic SKMSC IC [95]
2009 Fall CN injury rat Allogeneic BMSC IC [87]
2010 Albersen CN injury rat Autologous ADSC IC [108]
2010 Kendirci CN injury rat Allogeneic BMSC IC [88]
2011 Lin CN injury rat Autologous ADSC Nerve graft [120]
2011 Woo CN injury rat Allogeneic SKMSC IC [97]
2012 Qiu CN injury rat Autologous SVF IC [103]
2012 Kovanecz CN injury rat Mouse SKMSC IC [126]
2012 Kim CN injury rat Allogeneic BMSC CN scaffold [89]
2012 Fandel CN injury rat Autologous ADSC IC [112]
2012 Piao CN injury rat Human ADSC CN scaffold [114]
2013 Jeong CN injury rat Human ADSC CN scaffold [116]
2013 Kim CN injury rat Human ADSC CN scaffold [115]
2013 You CN injury rat Human BMSC IC + periprostatic [90]
2013 You CN injury rat Human ADSC IC + periprostatic [118]
2013 Choi CN injury rat Human testis SC Periprostatic [110]
2013 Ying CN injury rat Autologous ADSC IC [129]
2014 Ying CN injury rat Autologous ADSC Vein graft [122]
2014 Lee CN injury rat Human ADSC IC [117]
2014 Ryu CN injury mouse Allogenic clonal BMSC IC + IP [119]
2012 Qiu Radiation injury rat Allogeneic ADSC Intra-MPG [121]
2007 Bivalacqua Aging rat Allogeneic BMSC IC [91]
2008 Nolazco Aging rat Mouse SKMSC IC [96]
2010 Abdel Aziz Aging rat Allogeneic BMSC IC [81]
2010 Garcia DM rat Autologous ADSC IC [30]
2011 Gou DM rat Allogeneic EPC IC [78]
2011 Qiu DM rat Allogeneic BMSC IC [83]
2012 Qiu DM rat Allogeneic BMSC IC [84]
2012 Sun DM rat Allogeneic BMSC IC [86]
2012 Nishimatsu DM rat Allogeneic ADSC IC [130]
2013 He DM rat Allogeneic BMSC IC [85]
2013 Liu DM rat Human ADSC IC [124]
2014 Ouyang DM rat Human USC IC [111]
2012 Ryu DM mouse Syngeneic SVF IC [104]
2014 Das DM mouse Human SVF IC [131]
2010 Huang Hyperlipidemia rat Autologous ADSC IC [35]
2012 Ma TA injury rat Autologous ADSC SIS graft [53]
2013 Castiglione TA injury rat Human ADSC Intratunical [54]
2014 Gokce TA injury rat Autologous ADSC Intratunical [123]

Please cite this article as: A. Alwaal, et al., Stem cell treatment of erectile dysfunction, Adv. Drug Deliv. Rev. (2014), http://dx.doi.org/10.1016/
j.addr.2014.11.012
6 A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx
6. Clinical trials
There has been only one clinical trial assessing the efficacy of SCs for
ED. Bahk et al. from Korea investigated the effect of IC injection of um-
bilical cord SCs in 7 patients with DM [127]. This demonstrated im-
proved erectile function and was initially promising. Clinical trials on a
larger scale are needed in order to validate these results and clarify
their exact effects in humans. Currently, two phase I–II SC clinical trials
are recruiting patients with ED. In France, IC injection of BMSCs is
being evaluated in post-RP patients (Identifier: NCT01089387),
while in the United States the effects of IC injection of AD-SVF in organic
ED (vasculogenic and neural) are being assessed (Identifier:
NCT01601353).
7. Immunocompatibilty
The least immunogenic SC transplantation would require autolo-
gous SCs. However, even with some of the easier methods of SC extrac-
tion such as in the case of ADSCs, a surgical procedure is still involved
and that by itself may adversely affect the outcome of SC transplanta-
tion. Therefore, more recent SC preclinical studies have increasingly uti-
lized allogeneic and xenogeneic SC transplantations as alternatives.
Allogeneic and xenogeneic SCs do not incite immune response because
they lack T-cell co-stimulatory molecules, and thus, transplantation
with such cells is an attractive alternative to the potentially costly and
inconvenient autotransplantation without the need for immunosup-
pression [128].
8. Future directions
SC therapy represents a very promising treatment option for ED
cases not responsive or partially responsive to PDE5Is. IC injection is
the most commonly used method for SC delivery, in both preclinical
and clinical trials. Several more cumbersome methods have been
attempted but did not prove to be advantageous. The intravenous
route has shown promise and should be further tested in additional pre-
clinical studies before adoption for clinical trials. The issue of allogeneic
versus autologous source also needs to be further clarified. Among the
different SCs used, ADSC represents the most widely and easiest SC to
work with. But, whether ADSC or AD-SVF is a better choice needs to
be answered through additional comparative studies. Timing of SC in-
jection represents another issue despite the demonstration that both
immediate and delayed injections had similar long-term positive treat-
ment outcome [103]. The dosage of SCs requires further testing as the
currently chosen doses of half million to 2 million cells in the rat were
arbitrary. Pharmacological modifications to the SCs have not been
attempted in the treatment of ED, but might be a future target in
order to influence self-renewal capabilities and thereby prolonging
their therapeutic effect. In addition to the two current clinical trials,
more should be conducted so as to adequately cope with increasing de-
mand from patients. All these tasks are difficult but extremely impor-
tant in order to develop better treatment options for this common and
important disease. Table 2 provides a summary of all preclinical trials
using SC for ED treatment.
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