**4. Preclinical and clinical studies**

Several animal studies have been addressing the problem of stress urinary incontinence (SUI) and different strategies have been tested to restore continence, either by applying pharmaco‐ logical therapies, bulking agents, sling surgical procedures or cell-based therapies [94]. Until now, the first three strategies mentioned above are commonly applied in clinics. However, the outcomes are associated with adverse events and limited effectiveness in middle and long terms [95-97]. Therefore, cell-based therapies are aiming to bring new solutions to the treat‐ ment of SUI. Numerous preclinical studies have been implementing stem/progenitor cell injections for restoration of muscle contraction in SUI. Animal models that mimic SUI are crucial for the understanding of effects and benefits of the different therapies options.

#### **4.1. Animal model**

Another type of cells is isolated from muscle biopsies through a series of preplating stages. These cells are also recognized to have a myogenic profile and are capable to fuse and form skeletal muscles fibers. They are known as muscle-derived stem cells (MDSC) with character‐ istics of non-committed progenitor cells [75, 76] and are most probably originating from blood vessel walls [77]. Similarly, other cells types isolated from the muscle compartment such as mesoangioblasts and pericytes are involved in the muscle regeneration but are of nonmyogenic origin. These are vessel-associated progenitors, not expressing myogenic markers such as Myf5 and MyoD even though they can differentiate to myotubes and fuse to form fibers [78-80]. More cell types with non-myogenic profile are found in the skeletal muscle and have recently been demonstrated to form fibers. Hence, skeletal myogenic precursors or muscle stem cells sorted by FACS are capable to reconstitute fibers in rodent models [72, 81]. The first type of cells is characterized by expression of β1-integrin (adhesion protein) and CXCR4 (SDF-1 receptor), the second type by α7-integrin (adhesion protein) and CD34 markers. Side popula‐ tions are also isolated from muscle tissues and are expressing specific surface markers [82]. They are distinct from satellite cells and have been used successfully in muscle regeneration in rodent models [83-87]. Surprisingly, more types of cells of the skeletal muscle tissue can contribute to muscle regeneration. In fact, recently, a new type of myogenic cells, localized in the area of the interstitium between muscle fibers, has been characterized and is known as PW1-interstitial cells (PICs). They are characterized as positive for cell stress mediator PW1 but negative for Pax7; though they possess myogenic profile *in vitro* and lead to muscle regeneration *in vivo*, which includes the generation of satellite cells [88]. Hence, various types of cells isolated from skeletal tissue either mechanically or by flow cytometry are capable to regenerate muscle. In addition to the muscle there are more sources of stem/precursor cells

Mesenchymal stem cells (MSC) are procured from bone marrow biopsies and are multipotent stem cells that give rise also to skeletal muscle fibers and participate to restore the satellite cell niche [89]. These cells are well characterized and involved in many different applications due to their multipotency as it is the case for adipose-derived stem cells (ADSC). The latter are easily harvested by liposuction, cultured *in vitro* and injected to restore muscle in the case of SUI [90, 91]. Embryonic stem cells, induced pluripotent stem cells and umbilical cord blood have been demonstrated to be good alternatives for skeletal muscle regeneration [74, 92, 93]. However, precaution should be taken when these types of cells are considered for further development in clinics, as different types of viruses are used during the process of myogenic induction. In addition, there are still potential tumorigenicity issues with this sort of cells that

Hence, the sources of stem/progenitor cells for skeletal muscle regeneration are large. Though, several important factors need to be considered when choosing the optimal source for treating patients. Autologous cell therapy avoids immunogenic reaction and therefore complications after the implantation procedure. Therefore, autologous satellite/muscle precursor cells are advantageous for muscle regeneration. They are committed to muscle restoration and

isolated from other compartments.

686 Regenerative Medicine and Tissue Engineering

**3.3. Sources outside of the skeletal muscle compartment**

need to be solved before further clinical application.

To stimulate SUI in animals, various methods were applied. The goal is to injure one or several aspects of the urinary continence mechanisms to provoke incontinence as found in patients. The methods comprise the compression of the muscular and neurological system involved in continence by vaginal distension [98, 99], crush of the pudental nerve [100], damaging of anatomic supports such as fascia and pubourethral ligament [101-104] or destruction of intrinsic urethra by periurethral cauterization, urethral sphincterectomy, pudendal nerve transection and botulinum toxin periurethral injection [105-112]. One has to note that vaginal distension or pudendal nerve injury are relatively limited models due to the fact that the injury is naturally recovered after 2 weeks and thereby does not mimic an irreversible SUI. Eberli et al. have been describing a large animal model for SUI that was followed for 6 months. In this study, the sphincter muscle of dogs has been irreversi‐ bly damaged by surgically removing part of it. During the follow-up, the dogs were permanently affected by this procedure with long term decrease in sphincter pressures [107].

#### **4.2. Preclinical studies**

Rats are the preferred animal models for studying safety and efficacy of several cell types for treatment of SUI (table.1).


Abbreviations: ADSC, adipose-derived stem cells; BMSC, bone-marrow mesenchymal stem cells; MDC, muscle-derived cells; MDSC, muscle-derived stem cells; MPC, muscle precursor cells; MSC, mesenchymal stem cells; UCBSC, umbilical cord blood stem cells;

**Table 1.** Animal studies for treating stress urinary incontinence based on cell therapy.

Muscle derived cells were the first cells to be used for urethral regeneration and to demonstrate that cell therapy might represent an option for the treatment of SUI. Hence, MDSC, myoblasts, MPCs or muscle fibers injected around the injured area were surviving, participating in fiber formation and re-establishing muscle contractility [113-118]. In rat models, it was shown that the injected MDSC – isolated by preplating procedures or FACS-sorted - were participating actively in muscle regeneration for up to 3 months [119, 120]. Interestingly, in a dog study, a rare large animal model for SUI, transplanted MPCs were efficiently restoring the sphincter pressure to 80% of normal values during a half year follow-up period [121]. Concerning the speed of regeneration, Cannon et al. noticed 87% recovery after only 2 weeks post-injection and Chermansky et al. a full recovery after 4 weeks with myoblast and 6 weeks with MDSC [113, 116, 122]. Hence, muscle derived cells are able to incorporate the urethral structure and help recovering continence by reconstructing new fibers and connections with the surrounding cells – nerves, Schwan cells, vessels etc. [119, 121]. However, they are not the only kind of cells facilitating this cell therapy. Bone marrow derived mesenchymal stem cells, adipose-derived stem cells, umbilical cord blood stem cells (UCBSC) have been proven to also restore continence in animal SUI models [123-135]. In fact, different studied showed that these cells are contri‐ buting to the formation of fibers and contractile muscles which permit to control urinary leakage. When compared to common procedures for treatment of SUI such as the injection of collagen bulking agent, ADSC cell therapy provided better results [132]. Moreover, the association of cells with biomaterials seems to enable further improvements as observed with BMSC and sling systems or MDSC with fibrin glue [135, 136]. To improve this cell therapy model, Zhao et al. took account of the fact that muscle regeneration is an interaction process involving paracrine factors produced by surroundings cells and combined with ADSC the nerve growth factors. This method stimulated muscle regeneration and demonstrated that combining different cell types could be beneficial for muscle restoration in SUI.

#### **4.3. Clinical trials**

**Cell type**

MDSC

MDC fibroblasts

MDSC\_FACS sorted

Myoblasts / ADSC

blood stem cells;

**Animal model**

688 Regenerative Medicine and Tissue Engineering

SCID mice /

Rats

**SUI model Injection**

MPC Mice Noxetin Urethral 2 to 4 Yiou et al. 2002 MDSC Rats Sciatic nerve section Urethral 4 Lee et al. 2002 MDPC Rats Sciatic nerve section Urethral 2 Cannon et al. 2003 MPC Rats Electrocoagulation Urethral 0.7 to 4 Yiou et al. 2003 MDSC Rats Pudendal nerve section Urethral 12 Lee et al. 2004

MDC Rats Electrocauterization Urethral 2 to 6 Chermansky

MDSC Rats Sciatic nerve section Urethral sling 2 Cannon et al. 2005

Myoblasts Rats Cryoinjury / noxecin Urethral 1 to 6 Praud et al. 2007

ADSC Rats Vaginal dilatation Urethral 4 Lin et al. 2010 BMSC Rats Sciatic nerve section Urethral sling 4 to 12 Zou et al. 2010

MDSC Rats Pudendal nerve section Urethral 1 to 4 Xu et al. 2010

UCBSC Rats Electrocauterization Urethral 2 to 4 Lim et al. 2010 BMSC Rats Pudendal nerve section Urethral 4 to 8 Corcos et al. 2011 BMSC Rats Pudendal nerve section Urethral 4 Kim et al. 2011

ADSC Rats Pudendal nerve section Periurethral 3 to 4 Wu et al. 2011

ADSC Rats Pudendal nerve section Periurethral 8 Zhao et al. 2011 MPC Dogs Urethral sphincterecomy Periurethral 24 Eberli et al. 2012

Abbreviations: ADSC, adipose-derived stem cells; BMSC, bone-marrow mesenchymal stem cells; MDC, muscle-derived cells; MDSC, muscle-derived stem cells; MPC, muscle precursor cells; MSC, mesenchymal stem cells; UCBSC, umbilical cord

Myofibers Pigs Urethral injury Myofiber

Nerve transection / sphincter injury

BMSC Rats urethrolysis / cardiotoxin Urethral 13

BMSC Rabbits Cryoinjury Urethral 1 to 2

ADSC Rats Pelvic nerve section Periurethral 2 to 4

section

MSC Rats Vaginal dilatation intravenously

**Table 1.** Animal studies for treating stress urinary incontinence based on cell therapy.

BMSC Rats Pudendal, other nerves

**Target organ**

Rats Cryoinjury bladder Bladder 1 to 4 Huard et al. <sup>2002</sup>

Rats Sciatic nerve section Urethral 4 Kwon et al. 2006

Rats Vaginal dilatation Urethral 4 Fu et al. 2010

Urethra and baldder neck

implantation

**Time point Weeks**

**Reference Year**

et al.

4 Lecoeur et al. 2007

Kinebuchi et al.

Imamura et al.

Watanabe et al.

1 to 8 Du et al. 2012

Cruz et al. 2012

0.6 to 1.4

Urethral 4 to 12 Hoshi et al. 2008

2004

2010

2011

2011

Several clinical trials applying cell therapy in SUI have been conducted in the last decade (table.2).

Safety and efficacy of this strategy have been proven with several types of cells in women and men. As the procedures differ from one trial to another straight comparisons between them are difficult. Additionally, the recruited patients suffered from different levels of SUI - from moderate to severe- and some studies even included complementary therapies such as electrical stimulation or exercises to improve the results. Nevertheless, the results were highly promising and have demonstrated that a solution for patients suffering of SUI is within reach. Surprisingly, the first cells to be used in a clinical trial for SUI was not muscle derived cells but chondrocytes isolated from auricular cartilage that were expanded in culture before injection in female patients. Out of 32 treated patients 26 had an improved situation and 50% were continent after one year [137]. This was the only clinical study using chondrocytes for voiding dysfunction. Muscle derived cells (myoblasts and MDSC) are the most frequently used cells in muscle regeneration for SUI in both genders. Myoblasts have been used in many trials and were injected in or around the external urethral sphincter. The efficiency was stated to be


Abbreviations: MDC, muscle-derived cells; MDSC, muscle-derived stem cells; UCBSC, umbilical cord blood stem cells.

**Table 2.** Clinical trials for treating stress urinary incontinence based on cell therapy.

between 50% and 88% in a follow-up of 12 months [138-141]. Even if the designs between studies differ, the combination of cell therapy with electrical stimulation or/and pelvic floor exercises may explain the variation between the values. In fact, a cell therapy with the application of myoblasts alone seems to provide a 50% improvement [139, 141], improving to 78.4% if electrical stimulation is added [138] and reaching 88% with pelvic floor exercises [140]. This approximate comparison can encourage future clinical studies to combine other therapies and exercises with cell therapies in order to optimize the outcome. Myoblasts have also been combined with fibroblasts mixed in a collagen solution. The results were impressive: 79% of treated women and 65% of the men reached continence [142, 143]. As a Lancet publication of this group was retracted, these results should be handled with precaution and should be confirmed by other groups [144]. Other muscle-derived cells have been injected in patients with SUI. Since 2008, MDSC have been applied in several clinical trials [145, 146] with improvement rates of 53% after 1-year follow-up with 10 million cells injected, 63% with 20 million and 67% with 50 million. The efficiency of the cell therapies seems to be dose-depend‐ ent. This was confirmed by Kaufman et al. in a 6-month dose escalating study, where im‐ provements increased with the dose of injected cells. The best results were obtained with 200 million MDSC injected [146]. Interestingly, no serious adverse effects were observed even when numbers of UCBSC as high as 400 million were applied [147]. In this latter case, 72% of 39 patients were more than 50% satisfied 12 months post-injection. This represents another type of cells that is suitable for SUI treatment. Although the cell therapy with UCBSC is allogenic, no immunosuppressive effects were observed during this cure. As a source of multipotent stem cells, ADSC were trusted in recovering the contractility of the sphincter muscle in patients [148]. Certainly, the encouraging preclinical studies enabled transplantation of ADSC in patients suffering from SUI. However, only 3 patients were treated so far. Peri‐ urethral injection of ADSC seems to be safe and showed improvement of the sphincter contraction after 6 months follow-up. The use of total nucleated cells associates with lysates seems to be another good option for treating SUI. This type of cells significantly helped all treated patients in the study: 100% noticed improvement in their situation and 88% reached complete continence after 6 months. Hence, these clinical trials show that different sources of cells were able to improve the continence level of patients suffering from SUI.
