Stem Cell Therapy for Erectile Dysfunction: Progress and Future Directions

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Stem Cell Therapy for Erectile Dysfunction: Progress and Future Directions Maarten Albersen, MD, PhD,* Emmanuel Weyne, MD,* and Trinity J. Bivalacqua, MD, PhD† *Laboratory for Experimental Urology, Gene and Stem Cells Applications, Department of Development and Regeneration, University of Leuven, Leuven, Belgium; †The James Buchanan Brady Urological Institute, Department of Urology, Johns Hopkins Medical Institutions, Baltimore, MD, USA DOI: 10.1002/smrj.5

ABSTRACT

Introduction. Erectile dysfunction (ED) is the most common sexual disorder reported by men to their health-care providers and the most investigated male sexual dysfunction. Currently, the treatment of ED focuses on symptomatic relief of ED and therefore tends to provide temporary relief rather than providing a cure or reversing the underlying cause. Recently, stem cell-based therapies have received increasing attention regarding their potential for the recovery of erectile function. Preclinical studies have shown that these cells may reverse pathophysiological changes leading to ED rather than treating the symptom ED. Aim. To review available evidence on the efficacy and mechanisms of action of stem cell application for the treatment of ED. Methods. A nonsystematic review was conducted on the available English literature between 1966 and 2013 on the search engines SciVerse-sciencedirect, SciVerse-scopus, Google Scholar, and PubMed. Results. Several preclinical studies have addressed stem cell-based therapies for the recovery of erectile function following cavernous nerve injury and in Peyronie’s disease, diabetes, aging, and hyperlipidemia. Overall, these studies have shown beneficial effects of stem cell therapy, while evidence on the mechanisms of action of stem cell therapy still varies between studies. While many authors propose engraftment and differentiation of stem cells, a recent paradigm shift toward paracrine mechanisms of action is observed. One clinical study investigated stem cell therapy in diabetic patients, and two more clinical trials are currently recruiting patients. Conclusions. The development of methods to deliver stem cells to the penis has kindled a keen interest in understanding stem cell biology as it related to restoration of normal penile vascular and neuronal homeostasis. The use of stem cells for the treatment of ED represents an exciting new field, which still requires extensive basic research and human trials in diverse ED patient populations in order to define its role in the treatment of ED. Albersen M, Weyne E, and Bivalacqua TJ. Stem cell therapy for erectile dysfunction: Progress and future directions. Sex Med Rev 2013;1:50–64. Key Words. Adipose Tissue-Derived Stem Cells; Aging; Bone Marrow-Derived Stem Cells; Cavernous Nerve Injury; Diabetes; Erectile Dysfunction; Peyronie’s Disease; Regenerative Medicine; Stromal Vascular Fraction

Introduction

E

rectile dysfunction (ED) is defined as the consistent inability to obtain or maintain an erection for satisfactory sexual intercourse [1]. In the past, ED was believed to be primarily due to psychological causes; however, the majority of cases have been attributed to an organic etiology [2]. Although ED patients can have a number of medical conditions, organic ED is usually associ-

Sex Med Rev 2013;1:50–64

ated with vascular risk factors such as diabetes, coronary artery disease, and hypertension [3]. Another major cause of ED occurs after radical prostatectomy (RP) for clinically localized prostate cancer [4]. RP results in significant damage to the neurovascular bundles and autonomic innervation of the penis, thus causing severe end organ damage. The past two decades of sexual medicine research have focused on discovering the molecular © 2013 International Society for Sexual Medicine

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Stem Cell Therapy and Erectile Dysfunction mechanisms involved in the pathogenesis of ED and have provided convincing evidence that ED is predominately a disease of vascular etiology. From these studies, there has been the development of phosphodiesterase type 5 (PDE5) inhibitors that have increased our understanding of the biochemical pathways involved in the erectile process but more importantly provided an effective oral pharmacotherapy for the treatment of ED. Although PDE5 inhibitors are effective in the majority of ED cases, these highly selective medications have been shown to be less effective in certain disease states, such as diabetic ED, post-RP ED, and severe veno-occlusive dysfunction. Approximately 45% of these cases obtain physiologically significant erectile responses for intercourse [5]. Thus, diabetic and post-RP patients represent a hard-treat population of ED patients that would benefit from a more effective treatment modality. Additionally, ED patients report that the most important treatment outcomes and measure of success is the ability of an ED therapy to cure them of their disease [6]. The current ED pharmacotherapies provide symptom relief and do not represent a curative-based approach; thus, the ideal ED therapy would focus on identifying a disease-specific therapy with curative intent. In the present sexual medicine review, we will focus on the current evidence to support the use of stem cells for the treatment of ED, stem cells in erection biology, and discuss stem cell-based strategies in certain ED disease states, such as diabetes, aging, Peyronie’s disease, and cavernous nerve injury. The goal of this communication is to report on the state-of-the-art understanding of stem cells in sexual medicine and highlight the importance of continued research in this important area of research. Methods

An extensive search was conducted on published English language literature between 1966 and 2013 on stem cell therapy for the management of ED. The search engines SciVerse-sciencedirect, SciVerse-scopus, Google Scholar, and PubMed were used, with search terms including “erectile dysfunction,” “stem cells,” “multipotent stromal cells,” “adipose (tissue) derived stem cells,” “bone-marrow derived stem cells,” “animal model,” “diabetes,” “aging,” “Peyronie’s Disease,” and “cavernous nerve injury.” We focused on recent publications (within the last 10 years) as older publications tended to contain

preliminary results that have been more thoroughly or clearly defined in more recent studies. Relevant articles were identified and obtained in full-text form. Abstracts from international meetings were considered for inclusion only if the data had not been published in manuscript form. Information was critically reviewed and synthesized. Reference lists for several of the manuscripts identified via the search were reviewed, and additional relevant citations were obtained from these. The purpose of this study was to identify the most comprehensive and scientifically sound investigations. Thus, not all published or unpublished abstracts in the literature are discussed in the text of this review. In the tables, we have attempted to give an as-completeas-possible overview of the most important preclinical studies on stem cell therapy in the management of ED. Stem Cells and Stem Cell Types

Ever since the invention of the microscope by the Dutchman Antonie van Leeuwenhoek (1632– 1723), man has been interested in cellular biology. The microscope allowed for direct visualization of cells, and cell propagation and differentiation were witnessed for the first time. These technical developments led to the recognition of cells as being the building blocks of life, capable of giving rise to other cells and complete tissues. It was not until early in the 20th century that researchers realized that various types of blood cells all came from one particular type of “stem cell” in the bone marrow, which in turn encouraged physicians to administer bone marrow by mouth to patients with anemia or leukemia. A breakthrough in the discovery of stem cells followed in 1963, when it was discovered by Ernest A. McCulloch and James E. Till that normal mouse blood-forming tissue contains a class of cells that, upon being transplanted into heavily irradiated mice, can proliferate and form macroscopic colonies in the spleen [7]. They noted that within a given colony, differentiation is occurring along three cellular lineages—erythrocytic, granulocytic, and megakaryocytic cells, respectively. Later, it was shown that mice with defective marrow could be restored to health with infusions into the blood stream of marrow taken from other mice. Since that time, bone marrow transplants have been performed safely and efficiently, and in fact, bone marrow transplantation represents the first form of stem cell therapy. In 1998, James Thomson first successfully removed cells from Sex Med Rev 2013;1:50–64

52 spare embryos at fertility clinics and grew them in the laboratory [8]. He thereby established the world’s first human embryonic stem cell line that still exists today. This discovery launched stem cell research into the limelight, and ever since, a plethora of evidence has emerged to illustrate that these embryonic stem cells can differentiate in any cell type in the body, thus potentially having the capability to replace or regenerate any diseased, injured, or purposefully removed cells and tissues. Based on these observations, the classical definition of a stem cell requires that it possess two properties: (i) “self-renewal,” indicating that the cell must be able to undergo numerous divisions without losing its undifferentiated state. Two mechanisms ensure the maintenance of a stem cell line in vivo: “asymmetric division,” in which a father (stem) cell divides into an exact copy of itself and a more differentiated daughter cell, and “stochastic differentiation,” in which one stem cell divides into two exact copies of itself, while another divides into two more differentiated daughter cells; (ii) “potency” or the ability to differentiate into a multitude of specialized cells. Strictu sensu, this requires stem cells to be either totipotent or pluripotent, thus giving rise to any mature cell type, although multipotent progenitor cells are also often referred to as stem cells. Totipotent stem cells are produced from the fusion of an egg and sperm cell, and cells of the early embryo remain totipotent up till the 16-cell stage. Pluripotent stem cells are derived from the inner cell mass of the embryo and can form any of the more than 200 different cell types in the human body. Multipotent stem cells are derived from fetal tissue, cord blood, and adult tissues, and can form all cell types within the embryonic lineage to which these cells belong. These two essential properties have led to stem cell use as a therapeutic agent based on the assumption and observation that exogenously administered stem cells are able to engraft in the diseased host tissue, where they further divide and differentiate to give rise to a healthy new cell population to replace those populations destroyed by disease or injury. However, recent research illustrates that certain widely used stem cell populations, such as mesenchymal multipotent cells, may exert their beneficial effects through a paracrine-dependent fashion rather than by engraftment and differentiation [9]. These new observations are at this time inducing a paradigm shift on the mechanism of action of stem cell therapy, as further detailed later. Sex Med Rev 2013;1:50–64

Albersen et al.

Multipotent Mesenchymal Stromal Cells Multipotent mesenchymal stromal cells, also termed mesenchymal stem cells (MSCs), were first described by Friedenstein and colleagues in 1968 as fibroblast-like cells derived from the bone marrow that generated colonies when plated at low densities [10]. The International Society for Cellular Therapy has provided the following minimum criteria for defining multipotent human MSCs [11]: 1. Plastic-adherent when maintained in standard culture conditions. 2. MSC must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14, CD11b, CD79-a, CD19, and HLA-DR surface molecules. 3. MSC must differentiate into osteoblasts, adipocytes, and chondroblasts ex vivo.

Bone Marrow-Derived MSC While it is estimated that MSCs represent only 1 in 10,000–1,000,000 of the total nucleated cells within bone marrow aspirates, they are easily cultured and expanded ex vivo under specific conditions. They can easily be maintained for as many as 40 population doublings, resulting in cellular yields sufficient for most clinical cell therapy strategies in a time frame of 8–10 weeks. As they are well characterized and have been extensively studied, bone marrow-derived MSCs (BM-MSCs) remain the principal source of MSCs for most preclinical and clinical studies. However, isolation of MSCs has been reported from several tissues, including adipose tissue, liver, muscle, amniotic fluid, placenta, umbilical cord blood, and dental pulp. It is believed that MSCs represent a population of cells responsible for local growth and regeneration of the tissue in which they reside, and it is conceivable that there may be a distinct population of MSC-like cell population endogenous to every tissue in the human body [9,12]. Adipose Tissue-Derived MSC Of the various tissues in which MSCs have been isolated, the fat tissue has been of particular interest for ED researchers investigating cellular therapy. Adipose tissue-derived stem cells (ADSCs) are a distinct population of MSCs residing at the perivascular niche in adipose tissue [13,14]. To obtain these cells, resected fat or lipoaspirate is minced and incubated with collagenase in order to dissociate the extracellular matrix. By centrifugation, floating mature adipocytes are then separated from the pelleted stromal vascular fraction (SVF)

Stem Cell Therapy and Erectile Dysfunction [15]. Because the SVF comprises a heterogeneous cell population, the final isolation step consists of plating these cells in order to select the adherent population by successive washings. They share many characteristics with BM-MSCs pertaining to morphology, phenotype, ex vivo differentiation ability, and they have a highly similar gene expression profile. However, the frequency of these cells is 100- to 500-fold higher in adipose tissue compared with the frequency of MSCs in bone marrow [9]. Furthermore, while bone marrow is obtainable in the gram range by a painful marrow aspiration, hundreds of grams of adipose tissue can be obtained with minimally invasive procedures under local anesthesia or via liposuction. The possibility of harvesting tissue in the hundreds of grams range allows for direct reinjection of cells such as adipose-derived SVF in the same surgical procedures during which they were harvested. Although SVF cells are not ADSCs per se, human clinical trials have recently been initiated employing injection of uncultured SVF during breast reconstruction surgery and to treat myocardial infarction, traumatic calvaria defects, lipodystrophy, and type I and II diabetes [16]. As there is no need for expanding cells in culture to obtain sufficient numbers for cellular therapy, the risks of contamination or dedifferentiation of cells are minimized [17]. Both BM-MSC and ADSC are establishing an impressive track record of high efficacy in various preclinical disease models, and their success in clinical trials for various cardiovascular, inflammatory, fibrotic, and degenerative diseases guarantees further expansion of the applicability of these cells for other vascular disease such as ED. They further have a proven beneficial safety profile in human use and do not bear the risk of teratoma formation, as embryonic or induced pluripotent stem cells do. Furthermore, by transplanting these cells in an autologous or allogeneic fashion, ethical issues concerning the use of embryonic material are avoided. As illustrated later, most researchers have chosen either of these two stem cell sources to investigate their application either in the preclinical setting or in upcoming clinical trials, resulting in astonishing results on erectile improvement. The Use of Stem Cells for ED

Cavernous Nerve Injury Injury to the neurovascular bundle that innervates the penis is a frequent complication of RP and

53 radical pelvic surgery in general, leading to severe and difficult-to-treat ED. Despite the advent of “nerve-sparing” operative techniques, ED remains to be a highly frequent complication of pelvic surgery [4]. Therefore, it is and has been one of the most researched subtypes of ED. Following cavernous nerve injury, neuropraxia (due to compression of the nerve or temporal ischemia) or axonotmesis (as a result of more profound damage, such as stretching, crushing, or contusion) occurs, even when an attempt is made to spare the nerve. In case of neuropraxia, the nerve recovers spontaneously. If axonotmesis occurs, the nerve sheets remains intact, while the axon is lost in a process termed “Wallerian degeneration,” and regeneration of an axon is required to reinnervate the target organ, which in this case are the bilateral corpora cavernosa and more specifically the endothelial and smooth muscle cells located in these corporal bodies and their supplying arteries [18]. As a result of temporal denervation, apoptosis occurs in the endothelial and smooth muscle cells of the penis resulting in loss of these cell types and replacement of smooth muscle tissue with collagen, leading to corporal fibrosis and eventually venous leak during the rigid phase of erection [19]. Stem cell studies in the rat model of cavernous nerve injury have targeted and investigated the effects of stem cells on various steps in this process, including nerve regeneration, but also smooth muscle preservation and apoptosis prevention, in an attempt to keep the corpus cavernosum in a healthy state while nerve regeneration occurs. Other studies have investigated a more profound injury termed “neuronotmesis,” in which not only the axon but also the encapsulating connective tissue loses its continuity. The last (extreme) degree of neuronotmesis is nerve transaction or removal. Even in transection studies, beneficial effects of stem cells application have been reported. Stem cell studies geared toward preservation of ED in cavernous nerve injury are summarized in Table 1. The first attempt in restoring erectile function following cavernous nerve injury (CNI) using stem cells was made in 2004 by Bochinski and colleagues from the UCSF laboratory, who injected green fluoresecent protein (GFP)-labeled neural embryonic stem cells either into the corpora cavernosa or adjacent to the major pelvic ganglion (MPG) of rats following a crush injury of the cavernous nerve [20]. The authors observed partial preservation of erectile function following cavernous nerve injury in rats treated with stem cells. In Sex Med Rev 2013;1:50–64

Cavernous nerve injury crush (rat)

Cavernous nerve crush injury (rat) Cavernous nerve resection (rat)

Cavernous nerve crush injury (rat)

Cavernous nerve crush injury (rat)

Cavernous nerve crush injury (rat)

Cavernous nerve crush injury (rat) Cavernous nerve crush injury (rat)

Cavernous nerve crush injury (rat)

Pelvic irradiation

Cavernous nerve crush injury (rat) Cavernous nerve crush injury (rat)

Bochinski et al. (2004) [20]

Kim et al. (2006) [21]

Kendirci et al. (2010) [23]

Albersen et al. (2010) [24]

Fandel et al. (2011) [25]

Woo et al. (2011) [26]

Sex Med Rev 2013;1:50–64

Qiu et al. (2012) [17]

Qiu et al. (2012) [28]

Kim et al. (2012) [29]

Cavernous nerve crush injury (rat)

You et al. (2013) [34]

Unlabeled xenogeneic BM-MSC; intracavernous injection or combined intracavernous and periprostatic (fibrin glue-based) implantation Unlabeled xenogeneic ADSC; intracavernous injection or periprostatic implantation, or combined

Xenogenic (human) ADSC embedded in a NGF-hydrogel

Unlabeled allogeneic ADSC; intracavernous injection

PKH-26-labeled allogeneic BM-MSC embedded in Matrixen; local application on crush site Xenogenic (human) ADSC embedded in a BDNF/PLGA porous membrane system

EdU-labeled autologous ADSC; IV injection

Autologous adipose tissue-derived stromal vascular fraction, immediate or delayed intracavernous injection

EdU-labeled autologous or allogeneic ADSC; intracavernous injection

PKH-26-labeled allogeneic MDSC

EdU-labeled autologous ADSC; intracavernous or perineural injection

Allogeneic BM-MSC from transgenic GFP-positive rats (either or not selected for P75 NGF receptor expression); intracavernous injection EdU-labeled autologous ADSC or ADSC-lysate intracavernous injection

Allogeneic bone marrow mononuclear cells labeled with PKH-26 dye; intracavernous injection

MDSC transfected with LacZ; intracavernous injection

Neuronal embryonic stem cells labeled with GFP; intracavernous injection or direct injection into MPG

Type of stem cell + mode of administration

4 weeks

4 weeks

4 weeks

3 months

4 weeks

4 weeks

6 weeks

3 months

2 or 7 days

4 weeks

1, 3, 7, and 28 days

4 weeks

4 weeks

3 and 5 weeks

2 and 4 weeks

3 months

Time point of evaluation

Improved ICP to CNS in three treatment groups, no additional effect of combination therapy

Improved ICP to CNS in both groups

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS in both groups

N/A

Improved ICP to CNS

Improved ICP to CNS in the intracavernous group; no effect of perineural injection

Improved ICP to CNS in both groups

Improved ICP to CNS (P75-group)

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS in both groups

Functional outcome

Improved smooth muscle content (trichrome)

Preservation of nNOS and smooth muscle content in the penis (IF); prevention of cavernous fibrosis; no significant cell incorporation at 4 weeks Increased smooth/muscle to collagen ratio (trichrome) in the erectile tissue and time-dependent increase in nNOS expression (IF) in the dorsal nerve after intracavernous injection Intracavernously injected cells are recruited to the MPG in injured animals, not in sham animals in early days after injury, but are not permanently engrafted Increased cGMP levels in penile tissue; cavernosal dye retention after 4 weeks Both autologous and allogeneic ADSC exit the corpus cavernosum within days after injection and cavernous nerve injury and migrate preferentially toward the bone marrow (IF) Improved nNOS and neurofilament staining in the dorsal penile nerve (IF); improved smooth muscle/collagen ratio in the erectile tissue (IF + trichrome) Improved nNOS expression in dorsal penile nerve and MPG (IF), improved smooth muscle content (IF), EdU-positive cells migrated into the MPG Improved nNOS and eNOS protein levels (WB); label retention in the MPG (IF) Increased smooth muscle/collagen ratio (trichrome), nNOS content (IF), phospho-eNOS protein expression (WB), and cGMP level (ELISA) Increased NADPH diaphorase staining of penile nerves (HC), increased nNOS levels (WB) Increased smooth muscle content, increased eNOS protein expression (WB) Improved smooth muscle content (trichrome), improved nNOS expression (IF)

Improved neurofilament staining in dorsal and cavernous nerves, improved; improved NADPH (NOS) diaphorase staining in dorsal nerves; no evidence of incorporation of cells Increased cavernosal level of PGP 9.5 (IF); persistent LacZ expression (staining for LacZ) Improved nNOS and eNOS (PCR, WB), decreased apoptosis (TUNEL), persistence of PKH-26 dye in the corpus cavernosum at 5 weeks Rare long-term engraftment of GFP-positive cells, displaying mesenchymal morphology (no differentiation)

Molecular and histological results

ADSC = adipose-derived stem cell; BDNF = brain-derived neurotrophic factor; BM-MSC = bone marrow-derived mesenchymal stem cell; cGMP = cyclic guanosine monophosphate; CNS = cavernous nerve electrostimulation; EdU = 5-ethynyl-2′-deoxyuridine; ELISA = enzyme-linked immunosorbent assay; eNOS = endothelial nitric oxide synthase; GFP = green fluorescent protein; HC = histochemistry; ICP = intracavernous pressure; IF = immunofluorescence; IV = intravenous; LacZ = beta-galactosidase; MDSC = musclederived stem cell; MPG = major pelvic ganglion; N/A = not available; NADPH = nicotinamide adenine dinucleotide phosphate; NGF = nerve growth factor; nNOS = neuronal nitric oxide synthase; PCR = polymerase chain reaction; PKH-26 = cell membrane marker; PGP 9.5 = protein gene product 9.5 marker; PLGA = poly lactic-co-glycolic acid; TUNEL = terminal deoxynucleotydyl transferase-mediated dUTP nick end labeling; WB = Western blot.

You et al. (2013) [33]

Kim et al. (2013) [32]

Cavernous nerve crush injury (rat) Cavernous nerve crush injury (rat) Cavernous nerve crush injury (rat)

Ying et al. (2012) [31]

Piao et al. (2012) [30]

Lin et al. (2011) [27]

Fall et al. (2009) [22]

Pathophysiology

Preclinical stem cell studies in cavernous nerve injury-induced erectile dysfunction

Author (year)

Table 1

54 Albersen et al.

Stem Cell Therapy and Erectile Dysfunction treated groups, neurofilament and neuronal nitric oxide synthase (nNOS) staining showed increased neuroregeneration or nerve preservation compared with injured controls. Interestingly, in both treated groups, there was no direct evidence of engrafted stem cells after harvesting the tissue. It took until 2010 till Kendirci et al. [23] and Albersen et al. [24] published the first reports of MSC therapy in cavernous nerve crush injuryinduced ED. While both studies used different MSC populations (Kendirici allogeneic BM-MSC and Albersen autologous ADSC), the outcomes were very similar. Both manuscripts reported an increased erectile response to cavernous nerve electrostimulation indicative of either inhibition of nerve damage (apoptosis after nerve crush) or enhanced nerve regeneration. In both studies, a small number of detectable labeled cells were found in the corpora cavernosa 4 weeks after intracavernous injection of MSCs. A shared conclusion was that MSC may be exerting their beneficial effects in the nerve crush model in a paracrine fashion [23,24,35]. This was supported by Kendirci et al. by the finding that MSC are capable of secreting a broad variety of neurotrophic factors, and by Albersen et al. by showing partial recovery of erectile function after injection of ADSC-derived cell lysate. In vitro, this opinion was further strengthened by Zhang and coworkers, who showed increased neurite sprouting in MPG cultures when exposed to ADSCconditioned medium [36]. While these studies showed preservation of nNOS expression and neural responses to electrostimulation, they failed in illustrating whether these observations were a result of neuroprotection or (enhancement of) nerve regeneration. Furthermore, they reported on a lack of cellular engraftment in the corporal tissues but did not investigate the fate of injected cells. Fandel et al. and Lin et al. filled this gap by tracking labeled cells throughout the rat’s body after ADSC injection [25,27,37]. A rapid and time-dependent disappearance of intracavernously injected ADSC from the penis was noted. Also noteworthy is that the number of ADSC in the MPG of cavernous nerve injured rats increased incrementally from 1 to 7 days following injection. In the same time frame, it was observed that the number of ADSC appearing in the bone marrow decreased. Such an inverse relationship suggests that intracavernously injected ADSC exit the penis and travel preferentially to bone marrow; however, when an injury is present, a portion of the injected ADSC is

55 recruited from the penis or systemic circulation to the injury site. Of interest, at 28 days following injection, examination of the MPG revealed that ADSC had exited the ganglia at the time that recovery of erectile function was established. Again, these observations contradicted the theory that stem cells would engraft and differentiate into host cell types (in this case, neurons or glial cells) to enhance erectile function. Interestingly, Qiu et al. confirmed ADSC homing to the major pelvic ganglia in a rat model for pelvic irradiationinduced ED after intravenous injection of these cells [28]. It was proposed that ganglia cells provide clues to their surroundings after injury, including the secretion of chemotactic molecules that draw exogenously administered stem cells from the circulation to initiate repair pathways in analogy with the animal’s own injury-healing mechanisms based on attraction of endogenous stem cells that are dislodged from their niche upon activation of chemokine-mediated signaling pathways after tissue-injury [9,38,39] (Albersen et al., unpublished data). Based on the earlier mentioned observations, it is becoming increasingly clear that—in the case of an acute injury such as cavernous nerve crushing—stem cells migrate to injured tissue and act as a local “factory” of molecules, promoting neuroprotection, modulation of inflammatory response, reduction of scar, and facilitation of axonal regeneration and plastic rewiring of neuronal connections. Indeed, the multiple time-point approach that was used for investigating cell trafficking was further employed by Fandel and coworkers to illustrate that nNOS expression in the penile nerves increased in a timedependent fashion indicative of nerve regeneration rather than preservation after injury [25]. These observations were only made in the group where stem cells had effectively migrated to the MPG confirming the essential process of stem cell recruitment and tissue repair. Besides intracavernous or intravenous injection, various authors have made attempts to directly influence nerve regeneration by implanting the stem cells in close proximity to the cavernous nerve following injury or next to the neuronal cell bodies in the MPG [20,30,32–34,40]. Because of their anatomical characteristics and their small size, direct injection of a cell suspension into the cavernous nerve itself or the MPG is extremely difficult, as the cells would immediately disperse throughout the peritoneal cavity; in rodents, unlike in humans, the testicles, prostate (and thus the cavernous nerves and ganglia, which are Sex Med Rev 2013;1:50–64

56 located at the lateral prostate surface), and bladder are not anatomically separated from the abdomen by a peritoneal coverage. In order to keep transplanted stem cells in close proximity to the cavernous nerve, various authors have attempted to implant cells immobilized in a carrier that can easily be kept in place. Piao et al. used a poly lactic-co-glycolic acid membrane containing brain-derived growth factor (BDNF) either alone or transplanted with ADSC and compared the effects of this tissue-engineered sheet with periprostatic implantation of cell suspension of ADSC [30]. They found beneficial effects of the combined ADSC and BDNF-loaded membranes compared with either ADSC or the BDNF-loaded membrane alone. These effects were the result of increased nitrinergic nerve regeneration as evidenced by enhanced nNOS staining in the penile nerves and elevated levels of cyclic guanosine monophosphate (cGMP) in the penile tissue. Secondarily (as cells were not implanted directly in the corporal tissue), the corpora cavernosa histological architecture was preserved in terms of smooth muscle/collagen ratios. The authors suggested that the membrane contributed to ADSCs engraftment into the cavernous nerve. Similar results were obtained by the same group with the use of ADSC embedded in a nerve growth factorloaded hydrogel [32]. Others have used a collagencontaining gel to retain MSC in close proximity to the MPG and again observed improved erectile recovery and increased penile nNOS expression after cavernous nerve crush injury [29]. In a cavernous nerve resection model, Lin and colleagues showed that bridging the nerve gap with adipose matrix loaded with ADSC resulted in increased cavernous nerve sprouting compared with unseeded matrix; however, because of the model chosen, there was no significant effect on nerve electrical stimulation-induced intracavernous pressure (ICP) increases [40].

Aging Regarding stem cell therapy for causes other than cavernous nerve injury-induced ED, other mechanisms of action of stem cell therapy, including engraftment and differentiation, have been proposed. In ED in the context of aging, vascular disease, or diabetes, there is no specific single injury site with concomitant inflammation and chemokine secretion resulting in stem cell recruitment toward involved tissues. In spite of this fact, various kinds of stem cells have proven helpful in the recovery of erectile function in models for Sex Med Rev 2013;1:50–64

Albersen et al. these disease states. Also, whereas stem cell incorporation was not observed in animal models of cavernous nerve injury, it has been observed in models for chronic disease. Until today, however, the extent to which this incorporation of cells contributes to cure of ED remains controversial. Bivalacqua and colleagues postulated that aging contributes to the development of ED through the occurrence of endothelial dysfunction. They engineered BM-MSC to express endothelial nitric oxide synthase (eNOS) in order to enhance eNOS expression in the penile endothelium and increase bioavailable NO, which diffuses from the endothelium into the underlying smooth muscle cell to relax smooth muscle cell and eventually increase penile blood flow [41]. When BM-MSCs were injected into rats with aging-induced ED (aged 25 months), the eNOS gene-modified BM-MSC was associated with a more rapid improvement in erectile function compared with wild-type MSC, although both modified and unmodified MSC were able to increase erectile function 21 days after injection. Improved erectile function was associated with increased eNOS protein, NOS activity, and cGMP levels in the corpora cavernosa. In addition, unmodified BM-MSCs and injected cells were traced back to the corpus cavernosum where immunofluorescent staining revealed that transplanted cells expressed markers specific for endothelial and smooth muscle cells. Abdel Aziz and coworkers were able to show that the beneficial effects on erectile function of intracavernously injected BM-MSC last up to 3–4 months after injection [42]. They further showed persistence of stem cells in the erectile tissue. However, the morphology of transplanted cells and colocalization with smooth muscle or endothelial markers was not shown. Nolazco et al. have tested the application of muscle-derived stem cells (MDSCs) in aged rats [43]. These cells are harvested from a skeletal muscle biopsy and expanded in vitro to obtain sufficient cell numbers for treatment. Following intracavernous injection of these cells, the authors found activation of an a-smooth muscle actin promotor after injection, suggesting conversion of these cells into smooth muscle-like cells. The authors confirmed this hypothesis with immunofluorescence imaging and observed a complete colocalization of smooth muscle actin and 4′,6-diamidino-2-phenylindole (DAPI) (a nuclear marker, with which injected cells were labeled), leading them to conclude that stem cells could replace corporal smooth muscle cells that are lost or functionally damaged in the

Stem Cell Therapy and Erectile Dysfunction penis during the aging process and, by doing so, restore the normal compliance of the tissue.

Diabetes and Metabolic Syndrome As with aging, the effects of diabetes on erectile function are the result of damage to four essential physiological components of erection, namely vascular/endothelial impairment, reduced nitrinergic innervation, smooth muscle changes, and reduction of corporal compliance. Sexual medicine researchers have examined the effects of intracavernosal stem cell administration on these pathophysiological disturbances. Garcia and colleagues used intracavernous injection of autologous ADSC to treat diabetes type II-induced ED [44]. Erectile responses to cavernous nerve stimulation measured 21 days after injection were significantly better in the ADSCtreated group compared with controls. Upon histological examination of the penile tissue 3 weeks after injection, only a small number of 5-bromo2-deoxyuridine (BrdU)-labeled ADSC was detected within the corporal tissue of the treatment group. Whereas there was no significant incorporation of cells, a significant increase in nNOS in the penile dorsal nerve and in the number of endothelial cells in the corpora cavernosa was observed in the treatment group. In agreement with these findings, Lue’s lab further studied the effects of intracavernous application of autologous ADSCs in an animal model for metabolic syndrome (hyperlipidemia) and again found improvement in erectile function resulting from enhanced endothelial and neural function [45]. This study also was not able to locate a significant number of stem cells that engrafted in the penis in spite of the observed functional and structural improvements. In type I diabetes, BM-MSC therapy has been extensively studied by Qiu and coworkers, who have shown that intracavernous injection of uncommitted BM-MSCs resulted in an increased ICP upon cavernous nerve electrostimulation compared with untreated diabetic controls [46]. The authors claimed that this observed change was the result of increased content of endothelium and smooth muscle in the corpus cavernosum and also noted increased expression of neuronal markers nNOS and neurofilament in dorsal penile nerves using immunofluorescence imaging. They observed colocalization of ample cells labeled with calmodulin, endothelial markers CD31, Von Willenbrand Factor (VWF), or smooth muscle markers alpha actin and calponin. In a later study,

57 the same group was able to partially replicate neurotrophic effects of BM-MSC injection alone with injection of BM-MSC-conditioned medium [47]. In addition, they showed that ex vivo gene modifying the stem cell with vascular endothelial growth factor (VEGF) gene resulted in even better erectile responses in type I diabetic rats [48]. Taken together, these data suggest that MSC therapy in diabetic animal models of ED may provide beneficial erectile effects via paracrine mechanisms. Ryu and coworkers confirmed this finding by providing a negative control experiment, in which they showed that blockage of the VEGF pathway resulted in a blunting of the positive effects of SVF injection in diabetic animals [49]. They further provided data on the blood glucose levels of these animals after SVF therapy and illustrated that the effects on erectile function were not mediated by an improvement of the general diabetic condition of these mice. A summary of stem cell studies in aging, diabetes, and hyperlipidemia-induced ED are listed in Table 2.

Peyronie’s Disease and Corpus Cavernosum Injury Only recently have sexual medicine researchers used stem cell-based therapies to reverse or prevent the pathological characteristics of Peyronie’s disease. Castiglione et al. hypothesized that MSCs have the potential to target inflammatory and fibrotic tissue changes in an early phase because of the beneficial effects of MSC therapy in preclinical animal models of kidney, liver, and pulmonary fibrosis [51]. The exact mechanisms of antifibrotic effects of MSCs remain to be elucidated. Most preclinical studies suggest that MSC work through immunomodulation, thereby limiting the host response to injury and preventing the onset of fibrosis. Another proposed mechanism is the induction of phenotypical changes in resident fibroblast illustrated by reduced collagen and increased hyaluronic acid production in fibroblasts cocultured with MSC [51]. Furthermore, the direct interaction of MSC with the extracellular matrix has been proposed based on their ability to secrete high numbers of matrix metalloproteinases and other matrix-modulating enzymes [52]. With these observations in mind, the authors injected xenogeneic (human) ADSC in the tunica albuginea of rats with experimentally induced Peyronie’s disease. The ADSC therapy was administered during the inflammatory phase of the Peyronie’slike disease state in the rat. Stem cell injection resulted in a nearly complete prevention of Sex Med Rev 2013;1:50–64

Sex Med Rev 2013;1:50–64

Aging (rat)

Aging (rat)

Diabetes type II (rat)

Hyperlipidemia (rat)

Diabetes type I (rat)

Diabetes type I (rat)

Diabetes type I (rat)

Diabetes type I (rat)

Diabetes type 1 (mouse)

Bivalacqua et al. (2007) [41]

Nolazco et al. (2008) [43]

Garcia et al. (2010) [44]

Huang et al. (2010) [45]

Qiu et al. (2011) [46]

Qiu et al. (2012) [48]

Sun et al. (2012) [47]

Nishimatsu et al. (2012) [50]

Ryu et al. (2012) [49]

Allogeneic adipose tissue-derived stromal vascular fraction from GFP+ mice

ADSC (auto- or allogeneic not reported), either or not with blunted expression of andromedullin

Allogeneic BM-MSC, CM-DiI-labeled, or BM-MSC-conditioned medium

Allogeneic BM-MSC, CM-DiI-labeled, or BM-MSC transfected with VEGF; intracavernous injection

Allogeneic BM-MSC, CM-DiI labeled; intracavernous injection

EdU-labeled autologous ADSC; intracavernous injection

Autologous ADSC; BrdU-labeled, intracavernous injection

Xenogeneic (mouse) MDSC labeled with DAPI; intracavernous injection

Allogeneic BM-MSC; or BM-MSC transfected with eNOS or LacZ; intracavernous injection

Type of stem cell + mode of administration

2 weeks

4 weeks

4 weeks

4 weeks

4 weeks

4 weeks

3 weeks

2 and 4 weeks

7 and 21 days

Time point of (functional) evaluation

Improved ICP to CNS in ADSC; this result was absent after genetic knock-down of andromedullin Improved ICP to CNS; this result was absent after addition of a VEGF-blocker

Improved ICP to CNS in both groups

Improved ICP to CNS in both groups

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Improved ICP to CNS

Functional outcome

Increased eNOS phosphorylation, and cGMP expression; increased cavernous VEGF-A expression and increased presence of CD34(+)CD31(-)cells

Cells expressing LacZ were found in the erectile tissue up to 21 days. Colocalization of LacZ with endothelial and smooth muscle markers (IF). Increased eNOS expression (WB) and activity, increased cGMP levels (ELISA). Colocalization of DAPI and a-smooth muscle actin and smoothelin; increased a-smooth muscle actin (WB) Increased nNOS levels in dorsal penile nerve (IHC) and endothelial cells in the corpus cavernosum (IHC) Increased nNOS levels in dorsal penile nerve (IHC) and endothelial cells in the corpus cavernosum (IHC); no significant cell incorporation at 4 weeks Increased smooth muscle and endothelial markers (IF); few CM-DiI-positive cells at 4 weeks colocalized with smooth muscle and endothelial markers Increased smooth muscle and endothelial markers (IF); more pronounced in transfected cell group; enhanced penile VEGF expression (ELISA) In vitro: secretion of neurotrophic molecules by BM-MSC; in vivo: improved nNOS and neurofilament staining in the dorsal penile nerve (IF); time-dependent decrease in the number of BM-MSCs in the corpus cavernosum following injection Increased VE-cadherin and eNOS (WB)

Molecular and histological results

ADSC = adipose-derived stem cell; BM-MSC = bone marrow-derived mesenchymal stem cell; BrdU = 5-bromo-2-deoxyuridine; cGMP = cyclic guanosine monophosphate; CM-DiI = membrane/lipophilic marker; CNS = cavernous nerve electrostimulation; DAPI = 4′,6-diamidino-2-phenylindole (chromatin marker); EdU = 5-ethynyl-2′-deoxyuridine; ELISA = enzyme-linked immunosorbent assay; eNOS = endothelial nitric oxide synthase; GFP = green fluorescent protein; ICP = intracavernous pressure; IF = immunofluorescence; IHC = immunohistochemistry; LacZ = beta-galactosidase; MDSC = muscle-derived stem cell; nNOS = neuronal nitric oxide synthase; VE = vascular-endothelial; VEGF = vascular endothelial growth factor; WB = Western blot.

Pathophysiology

Preclinical stem cell studies in aging, diabetes, and hyperlipidemia-induced erectile dysfunction

Author (year)

Table 2

58 Albersen et al.

Stem Cell Therapy and Erectile Dysfunction elastosis and fibrosis of the tunica albuginea and interestingly rescued erectile function in these rats, as illustrated by a complete restoration of the ICP increase during cavernous nerve electrostimulation 5 weeks after stem cell injection. After 5 weeks, only few labeled ADSC were found in the penises of treated rats. While this study provided a proof of principle for stem cell efficacy in Peyronie’s disease, most patients come to see their health-care provider in later disease stages; thus, these results cannot be directly translated to clinical application. Ferretti et al., however, targeted the disease in the chronic phase and injected autologous ADSC in rats with established Peyronie’s plaques and curvature [53]. They showed that injection of ADSC in the plaque after mechanical penile remodeling resulted in decreased penile curvature at 2 months after injection. In this study, no difference was observed in erectile function. The authors investigated the penile tissue after stem cell injection and proposed that neoangiogenesis is a potential mechanism that can explain this phenomenon. These two studies provide a foundation for stem cell therapy as a novel application within the field of Peyronie’s disease research and, although only providing a proof of concept, may provide future hope for men dealing with this difficult-totreat disease. However, more work is needed both in the translational phase as well as in elucidating how these effects are established. Only one manuscript has described the effects of stem cells in healing of cavernosal injury [54]. A study of An et al. provided some interesting detail on how MDSCs can aid in wound healing, in particular in the healing process of the corpus cavernosum after injury. The authors developed a model for penile injury in which a section of the corpus cavernosum was removed in rabbits after which either MDSC or MDSC transfected with VEGF were injected into the wound area. This treatment resulted in significantly smaller scars in magnetic resonance imaging studies and better erectile function in vivo. Thirty days after the injury and cell injection, the authors observed that the number of GFP-labeled MDSC in the corpus cavernosum of MDSC-VEGF groups was significantly higher than that in the MDSC group at different time points after cells transplantation. They postulate that VEGF transfection may have a beneficial effect on the survival of transplanted MDSC. Furthermore, immunohistochemical staining showed that the numbers of smooth muscle and endothelial cells increased in the

59 injured sites of MDSC-VEGF-treated rabbits. While traumatic penile injury is an extremely rare condition, these results may be translated to men undergoing partial penile resection or penile reconstructive surgery, for example in the case of severe Peyronie’s disease.

Translational Studies in the Use of Stem Cells for ED The Use of Mononuclear Cell Fractions of the Bone Marrow and Adipose Tissue Cell culture expansion of stem cells is often required to reach sufficient cell numbers for therapeutic purposes, which are typically in the range of 1–20 million per kilogram of body weight. This holds true for bone marrow as well as adipose tissue-derived MSC. However, current major restrictions regarding the use of cultured cells in human subjects include contamination, unexpected cell differentiation, and possible tumorigenesis [15,17]. Unlike other stem cells that are present at low concentrations in their native tissues, ADSC have been shown to be abundantly present in adipose tissue. Most importantly, compared with other stem cell sources such as the bone marrow, large amounts of adipose tissue can be harvested with minimal or no side effects from human subjects. This provides a new perspective to use adipose tissue-derived cells in the clinical setting. Adipose tissue is composed of two main cell populations: mature adipocytes and the SVF. SVF is a heterogeneous fraction containing preadipocytes, mature endothelial cells, vascular smooth muscle cells, macrophages, fibroblasts, and a large population of stem/progenitor cells [15]. It has been proposed that the use of SVF, rather than isolated and cultured stem cell populations, may be beneficial because of the ease of harvest and the reduced risk of these cellular products. Qiu et al. elegantly demonstrated that in the cavernous nerve injury model, the use of autologous freshly isolated SVF is equally effective in restoring erectile function as the use of autologous cultured ADSC [17]. This benefit was not only evidenced in terms of function but also rigorous improvements in neural and smooth muscle integrity, and a reduction of corpus cavernosum fibrosis were observed with both therapeutic approaches. With the observation of the beneficial effects of SVF replicating those of ADSC, a novel approach that is directly applicable in the clinical setting has been identified, and initial clinical studies have been initiated with the use of SVF for the treatment of ED. Liposuction and injection of SVF during the same procedure as RP may be a reasonSex Med Rev 2013;1:50–64

60 able treatment option for preoperatively motivated patients. Ryu and coworkers tested the applicability of SVF in the setting of diabetes and confirmed an improvement in endothelial structure and function resulting in improved erectile function after intracavernous injection of SVF in diabetic mice [49]. From other medical fields, such as myocardial infarction models, we have learned that the use of the mononuclear fraction of cells found in bone marrow can replace cultured BM-MSC. Just like SVF, the bone marrow mononuclear fraction can be harvested and prepared for autologous injection in several hours and thus could be an attractive therapeutic alternative to BM-MSC. Fall and colleagues have used this cell suspension in a cavernous nerve resection rat model and observed decreased apoptosis and improvements in the expression of nNOS in the penile tissue [22]. A recovery of erectile function was not observed in a 3-week time frame, but this is likely due to the choice of nerve resection rather than a less severe model of cavernous nerve injury.

Allogeneic and Xenogeneic Transplantation As an alternative to freshly isolated cell products, standardized MSC cell lines produced in a GMP facility can be extensively characterized and tested for their safety and efficacy. This provides the advantage of working with a highly standardized product of which its in vivo behavior can be quite accurately predicted based on previous studies. A possible disadvantage however is that these cells are always allogeneic. Lin and colleagues have studied migratory behavior of autologous and allogeneic ADSC in the cavernous nerve injury rat model and found no differences [27]. Kim et al. from Korea aimed at testing efficacy and safety of allogeneic (transplantation of Sprague-Dawley cells into Sprague-Dawley rats) and xenogeneic (transplantation of Wistar cells into SpragueDawley rats) ADSC transplantation for the treatment of cavernous nerve injury-induced ED [55]. They concluded that erectile function in rats treated with allogeneic and even xenogeneic stem cells was well preserved and did not observe any difference in immunogenicity of these MSC preparations. Castiglione et al. used human (xenogeneic) ADSC in rats with experimental Peyronie’s disease and reported no adverse events [51]. In addition, all studies on BM-MSC mentioned in this review have shown excellent results of allogeneic stem cell transplantation. The effectiveness and lack of a major immune response to allogeneic MSC transplantation is likely explained Sex Med Rev 2013;1:50–64

Albersen et al. by the expression of human leukocyte antigen (major histocompatibility complex [MHC]) class I but no MHC class II molecules on their cell surface. Therefore, MSC could be considered as being hypoimmunogenic and even have been used for immunosuppression purposes. Both the use of freshly isolated cell fractions and highly standardized allogeneic stem cell products make the step to clinical translation of cellular therapy for ED feasible, which is illustrated by the recent initiation of clinical trials with the use of these types of cellular products (see section “Future Perspectives and Considerations for the Use of Stem Cells in Humans”). Limitations in Stem Cell Research in ED

Route of Administration In most stem cell studies targeting ED, intracavernous injection was chosen as a route of administration of these cells. The choice for this route of administration was based on extensive human experience with intracavernous injection of vasoactive substances and because many preceding studies investigating penile growth factor therapy and gene-therapy targeting the endothelium or smooth muscle [56] had also used intracavernous injection with excellent results. However, the choice for intracavernous injection of stem cells alone or fractions of stem cell lysate are not supported by solid experimental evidence [57]. Only one study compared intracavernous injection of ADSC to injection in the dorsal penile perineural space, and two other studies investigated intracavernous and periprostatic implantation of stem cells [20,25,34]. In the first, the intracavernous injection was the only effective strategy; in the latter two, there were no major functional or structural differences. These studies were all conducted in the cavernous nerve injury model. Another study showed improved erectile function after pelvic irradiation when ADSC were injected intravenously but did not compare these results with intracavernously injected cells [28]. As the corpus cavernosum is comprised of sinusoids, which are essentially bundled venules, it is hypothesized that intracavernous injection is essentially like intravenous injection. The placement of a constrictive band at the base of the penis does not seem to limit stem cell disappearance from the penis. The highly perfused nature of the penile sinusoids allows for direct access of intracavernously injected stem cells to the systemic circulation from where they migrate toward

Stem Cell Therapy and Erectile Dysfunction injury sites and their natural niche. As stem cell recruitment to the MPG seems essential in the initiation of neuroregeneration (at least in the cavernous nerve injury model), it remains an interesting question whether intravenous injection would perform just as well as intracavernous injection [25]. Another interesting aspect of the rapid systemic uptake of intracavernously injected stem cells is that their benefits may be systemic rather than localized in diseases without a single pathological focus on the penis or the major pelvic ganglia. For example, the first study that controlled for blood glucose levels in stem cell therapy for diabetesinduced ED only just appeared in the literature, while many before have reported on beneficial effects of stem cell therapy on diabetes-related ED [49]. It is plausible that the mechanisms of action of stem cell therapy in chronic conditions such as aging, hyperlipidemia, and diabetes types 1 and 2 may be systemic rather than focused in tissues related to erectile function. Supportive of this hypothesis is a human trial employing umbilical cord stem cells for diabetes-related ED in seven patients; after intracavernosal transplant of these cells, blood glucose levels decreased, antidiabetic medication dosages were reduced, and glycosylated hemoglobin levels improved for up to 4 months [58].

Tracking of Stem Cells after Injection For the study of fate of stem cells after injection, there is a need to label cells to allow for their identification in the host tissue. Various methods have been used to label stem cells before injection, each with their advantages and disadvantages [37]. Lipophilic fluorescent dyes such as CM-DiI and PKH-26, which are dissolved in the cell membrane or cytoplasm, and are easy to use as simple addition of dyes to the cell culture dish is sufficient for labeling. One of the main disadvantages of these labels is that surrounding cells easily absorb and express the fluorescent dye, or labeled membrane fragments may be phagocytosed by macrophages upon disintegration of the cell membrane during apoptosis or cell death in general [35]. This may result in a false-positive idea of cell retention in the host tissue. Furthermore, in longterm studies, the fluorochromes may fade [37]. Other labels, such as 5-ethynyl-2′-deoxyuridine (EdU) and BrdU, are analogous of nucleotides (thymidine) and are taken up in the cell’s DNA [24,37,44,57]. The advantage of these labels is that the fluorescent azide is added upon detection, and

61 fading is not an issue. Furthermore, colocalization of the cell label with nuclear markers such as DAPI makes it easy to discern phagocytosed cell fragments from live cells with intact nuclei [24]. It has been proposed that these labels may influence cell behavior and division because of their nature of intercalation in the DNA, but a recent study did not confirm these toxic effects (of EdU) on ADSC [59]. However, upon division of stem cells into daughter cells, the number of intercalated molecules is reduced by half per division and labeling intensity, thus may be divided in half upon each cell division. Furthermore, after transplantation, BrdU-labeled cells are detected with an anti-BrdU antibody. However, in order to expose the incorporated BrdU, which is buried in the densely packed chromosomes, tissue samples must be pretreated in DNA denaturing conditions with strong acids and high temperature. This harsh treatment invariably degrades the tissue structure, resulting in distorted histological images [57]. As EdU is chemically detected, this is not the case for EdU labeling of cells. In rare instances, DAPI was used to label cells before injection. However, it is a poor label for living cells as it does not penetrate intact cell membrane well. Furthermore, the noncovalent binding to DNA results in possible dissociation of DAPI from the DNA of labeled cells after transplantation and can be adsorbed by host cells resulting in false-positive detection. Genetic labeling, such as by incorporation of the GFP gene or the LacZ gene (which encodes for b-galactosidase) into the stem cells’ DNA results in stable gene label expression [41,42]. However, with subsequent cell division, labeling efficiency may reduce over time and specific issues may be high tissue autofluorescence in the green channel with GFP-labeled cells, and the need for fresh frozen, nonfixed tissue for the detection of LacZ (the enzyme converts a colorless substrate into a blue end product and is inactive after fixation of tissue), making efficient colocalization studies difficult. Another highly reliable method of graft cell identification is the detection of Y-chromosome by fluorescence in situ hybridization. This technique is commonly employed in studies investigating male cell transplantation into female hosts, although this is—due to obvious reasons—not possible in ED studies. While these labels help us to study cell behavior after injection or transplantation, they obviously have their drawbacks that need to be taken into account when interpreting stem cell studies in ED. Sex Med Rev 2013;1:50–64

62 Lack of recognition of these limitations (e.g., the staining of nuclear labels in cytoplasm of cells being accepted as a proof of cell retention) has led to erroneous interpretation of cell engraftment in the past. Furthermore, lack of quality and absence of high magnification images of engrafted or differentiated cells are encountered frequently in stem cell literature [57]. Careful review and interpretation of stem cell studies is therefore warranted and essential before translating these study results into the human situation. Future Perspectives and Considerations for the Use of Stem Cells in Humans

Research into stem cell therapy for ED is exciting, and therapeutic success of these pioneering studies suggests this approach may offer an effective, long-lasting treatment option or potentially a possible curative treatment regimen for severe ED. The observation that distinct stem cell populations have exhibited milieu-dependent differentiation and functional recovery in models of ED implies that a variety of cell-based approaches may prove efficacious for both vasculogenic- and neurogenic-mediated penile vascular dysfunction. However, as we encountered in our early experience with defining the molecular basis of erection and development of effective pharmacotherapies for ED, it will take extensive preclinical evidence before we ultimately learn the true therapeutic efficacy. We have recently learned that human umbilical cord blood stem cells can have beneficial effects on erectile function when administered into the penis of men with severe type 2 diabetes [58]. However, this effect was short-lived and not durable. Therefore, more research in both preclinical and early phases 1 and 2 clinical trials must be performed before we can define the role of stem cell-based therapies for ED. At the current time, there are two human clinical trials that are enrolling patients in both France and the United States utilizing intracavernous bone marrow-derived mononuclear fraction injected into the corpora cavernosa for the treatment of post-RP ED (NCT01089387; http://www. clinicaltrials.gov) and SVF for the treatment of ED (NCT01601353; http://www.clinicaltrials. gov). The primary goal of these early phases 1 and 2 clinical trials is safety with secondary end points of efficacy. We will eagerly await the results of these human clinical trials that will further our understanding of the use of stem cell-based therapies for the treatment of ED. Sex Med Rev 2013;1:50–64

Albersen et al. Conclusions

The development of methods to deliver stem cells to the penis has kindled a keen interest in understanding stem cell biology as it related to restoration of normal penile vascular and neuronal homeostasis. The application of stem cell-based therapies for the treatment of ED represents an exciting new field, which still requires extensive basic research and human trials in diverse ED patient populations in order to define its role in the treatment of human male ED. Corresponding Author: Maarten Albersen, MD, PhD, Department of Urology, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium. Tel: +32-16-3469-30; Fax: +32-16-34-69-31; E-mail: maarten. [email protected] Conflict of Interest: The authors report no conflicts of interest. Statement of Authorship

Category 1 (a) Conception and Design Maarten Albersen; Trinity J. Bivalacqua (b) Acquisition of Data Maarten Albersen; Emmanuel Weyne (c) Analysis and Interpretation of Data Maarten Albersen; Trinity J. Bivalacqua; Emmanuel Weyne

Category 2 (a) Drafting the Article Maarten Albersen; Trinity J. Bivalacqua (b) Revising It for Intellectual Content Trinity J. Bivalacqua; Maarten Albersen; Emmanuel Weyne

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