ADR-12708; No of Pages 8 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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Stem cell treatment of erectile dysfunction☆,☆☆ Amjad Alwaal a,b,⁎, Uwais B. Zaid a, Ching-Shwun Lin a, Tom F. Lue a a b

Department of Urology, University of California, San Francisco, CA, USA King Abdul Aziz University, Jeddah, Saudi Arabia

a r t i c l e

i n f o

Article history: Accepted 8 November 2014 Available online xxxx Keywords: Erectile dysfunction Stem cell therapy

a b s t r a c t Erectile Dysfunction (ED) is a common disease that typically affects older men. While oral type-5 phosphodieserase inhibitors (PDE5Is) represent a successful first-line therapy, many patients do not respond to this treatment leading 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 database search of all relevant articles, we present a summary of the scientific principles behind SCs and their 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 . . . . . . . . . . . . . . . . . . 1.1. ED . . . . . . . . . . . . . . . . . . . 1.2. Mechanism of erection . . . . . . . . . . 1.3. Aging and ED . . . . . . . . . . . . . . 1.4. Metabolic syndrome . . . . . . . . . . . 1.5. Prostate cancer therapy-related ED . . . . 1.6. Peyronie's disease (PD) . . . . . . . . . 2. Types of stem cells used in ED . . . . . . . . . . 2.1. ESC . . . . . . . . . . . . . . . . . . 2.2. Endothelial progenitor stem cells (EPSCs) . 2.3. Bone marrow-derived stem cell (BMSC) . . 2.4. Skeletal muscle-derived stem cell (SKMSC) 2.5. Neural crest SCs . . . . . . . . . . . . . 2.6. Adipose tissue-derived stem cell (ADSC) . . 2.7. Testis SC . . . . . . . . . . . . . . . . 2.8. Human urine SC (USC) . . . . . . . . . . 3. Methods of SC delivery for ED treatment . . . . . 4. Methods to enhance the therapeutic effects of SCs 5. Assessment of outcome . . . . . . . . . . . . . 6. Clinical trials . . . . . . . . . . . . . . . . . 7. Immunocompatibilty . . . . . . . . . . . . . . 8. Future directions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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☆ 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: [email protected] (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

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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 wellbeing and relationship with his partner [1]. ED is defined as the inability to achieve or maintain a penile erection satisfactory for sexual intercourse [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 remain 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 certain 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 mechanism. 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 activated, inducing contraction of the cavernosal smooth muscles with resultant diminished blood in-flow and reduced size of the lacunar spaces, which in turn allows more venous out-flow through the subtunical venules [12]. 1.3. Aging and ED Different diseases and conditions cause ED through different mechanisms. 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 collagen 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].

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 endothelial cells. Hyperglycemia is also associated with smooth muscle dysfunction 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 DMassociated ED due to SCs' regenerative ability and their potential to restore 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 highdensity 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 posttreatment 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 injury to the cavernosal nerve fibers that run along the posterolateral aspect of the prostate. Nerve-sparing RP has been introduced in order to preserve the cavernosal nerves. However, despite the continued refinement 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 degeneration [23,45]. PDE5Is are being prescribed routinely in many centers post-RP as part of the “penile rehabilitation protocol”. The exact mechanism 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 excising the plaque followed by patch grafting the tunica albuginea (TA)

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

A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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.

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The SC niche is a microenvironment that supports SCs in their quiescent state and promote SC self-renewal or tissue regeneration upon tissue 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 interaction between SCs and their neighboring cells, interaction with surrounding 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 potential. 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 production of growth factors leading some researchers to replicate this niche environment in vitro [69]. 2.1. ESC

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 specialized 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]. Totipotent 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 blastocyst [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 debatable [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 between them have not been fully elucidated [65].

ESCs are pluripotent cells that had been studied only once in ED research. 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 celllike characteristics and display several endothelial markers such as CD31, CD34, VEGFR2, Tie-2, and eNOS. In addition, they form vessellike structures on matrigel [71]. This discovery has generated promise for using EPSCs in angiogenic therapy. However, there have been debates 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 behavior 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 – 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 – Neuronal and endothelial dysfunction – Reduction in cavernosal NO levels – Injury to the CN fibers followed by smooth muscle degeneration – Veno-occlusive dysfunction – Intracorporal fibrosis

DM

Hyperlipidemia Prostate cancer treatment (RP and RT) PD

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

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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 costimulatory 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 different 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 extensively studied because of their prolonged proliferation, low immunogenicity, 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 function 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 therapeutic 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 abundant tissue sources [10]. There are two populations of SCs derived from adipose tissue that have been used in ED and non-ED research. The adipose derived-stromal vascular fraction (AD-SVF) cells, which are derived from the pellet fraction of collagenase-digested adipose tissue homogenate [100], and ADSCs, which are derived from SVF and are adherent 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 endothelial 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 preclinical 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

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 differentiate 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 delivery 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 located 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 demonstrated regenerative effects, however this route has not been extensively 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 direct 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 intraperitoneal injection of SCs was less effective than IC injection in restoring 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 recovery of erectile function, the results did not reach a statistical significance [120]. Further technical refinement is needed, but given the good response 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 improvement 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].

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

A. Alwaal et al. / Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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].

4. Methods to enhance the therapeutic effects of SCs In order to boost the therapeutic potential of SC therapy, several preclinical trials attempted to improve SC performance by genetically altering 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 injury 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

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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 functional 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. Alternatively, 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 location 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 transplantation

Reference

2004 2006 2009 2010 2010 2011 2011 2012 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2014 2014 2014 2012 2007 2008 2010 2010 2011 2011 2012 2012 2012 2013 2013 2014 2012 2014 2010 2012 2013 2014

Bochinski Kim Fall Albersen Kendirci Lin Woo Qiu Kovanecz Kim Fandel Piao Jeong Kim You You Choi Ying Ying Lee Ryu Qiu Bivalacqua Nolazco Abdel Aziz Garcia Gou Qiu Qiu Sun Nishimatsu He Liu Ouyang Ryu Das Huang Ma Castiglione Gokce

CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury rat CN injury mouse Radiation injury rat Aging rat Aging rat Aging rat DM rat DM rat DM rat DM rat DM rat DM rat DM rat DM rat DM rat DM mouse DM mouse Hyperlipidemia rat TA injury rat TA injury rat TA injury rat

Allogeneic ESC Allogeneic SKMSC Allogeneic BMSC Autologous ADSC Allogeneic BMSC Autologous ADSC Allogeneic SKMSC Autologous SVF Mouse SKMSC Allogeneic BMSC Autologous ADSC Human ADSC Human ADSC Human ADSC Human BMSC Human ADSC Human testis SC Autologous ADSC Autologous ADSC Human ADSC Allogenic clonal BMSC Allogeneic ADSC Allogeneic BMSC Mouse SKMSC Allogeneic BMSC Autologous ADSC Allogeneic EPC Allogeneic BMSC Allogeneic BMSC Allogeneic BMSC Allogeneic ADSC Allogeneic BMSC Human ADSC Human USC Syngeneic SVF Human SVF Autologous ADSC Autologous ADSC Human ADSC Autologous ADSC

IC or Intra-MPG IC IC IC IC Nerve graft IC IC IC CN scaffold IC CN scaffold CN scaffold CN scaffold IC + periprostatic IC + periprostatic Periprostatic IC Vein graft IC IC + IP Intra-MPG IC IC IC IC IC IC IC IC IC IC IC IC IC IC IC SIS graft Intratunical Intratunical

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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 umbilical cord SCs in 7 patients with DM [127]. This demonstrated improved 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 autologous SCs. However, even with some of the easier methods of SC extraction such as in the case of ADSCs, a surgical procedure is still involved and that by itself may adversely affect the outcome of SC transplantation. Therefore, more recent SC preclinical studies have increasingly utilized 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 immunosuppression [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 preclinical 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 injection represents another issue despite the demonstration that both immediate and delayed injections had similar long-term positive treatment 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 demand from patients. All these tasks are difficult but extremely important 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. References [1] J.J. Sánchez-Cruz, A. Cabrera-León, A. Martı́n-Morales, A. Fernández, R. Burgos, J. Rejas, Male erectile dysfunction and health-related quality of life, Eur. Urol. 44 (2003) 245–253. [2] N.C.C. Impotence. NIH consensus development panel on impotence, JAMA 270 (1993) 83–90. [3] E.O. Laumann, S. West, D. Glasser, C. Carson, R. Rosen, J.-h. Kang, Prevalence and correlates of erectile dysfunction by race and ethnicity among men aged 40 or older in the United States: from the male attitudes regarding sexual health survey, J. Sex. Med. 4 (2007) 57–65. [4] R. Shamloul, H. Ghanem, Erectile dysfunction, The Lancet 381 (2013) 153–165.

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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