Minimally Invasive Therapy for Urinary Incontinence and Pelvic Organ Prolapse (Current Clinical Urology) 2014th

10. Stem Cells for the Treatment of Stress Urinary Incontinence

Ervin Kocjancic , Karan Motiani2 and Jaspreet Joneja3

(1)

Department of Urology, Pelvic Health and Reconstructive Urology, College of Medicine, University of Illinois at Chicago, 722 W. Maxwell St. Suite 224, Chicago, IL 60607, USA

(2)

Department of Surgery, University of Cincinnati, College of Medicine, Cincinnati, OH, USA

(3)

Department of Urology, University of Illinois at Chicago, Chicago, IL, USA

Ervin Kocjancic

Email: ervkoc@gmail.com

Abstract

Currently, midurethral tapes represent the gold standard in the treatment of stress urinary incontinence. The tapes, regardless of the rout through which they are deployed, restore the supporting function of ligaments and fascia structure that stabilize urethra during sudden increase in the intra-abdominal pressure. Despite excellent success rates, the tapes are not risk-free. One of the concerns with these procedures is related to the fact that the vast majority of the slings are made of polypropylene and are currently under FDA scrutiny. The stem cells injected in the urethra have the potential to restore the functional, contractile response of the striated sphincter. The potential advantage is enormous as this therapy could treat stress urinary incontinence with a minimal risk. The initial results are promising; however, there are many questions that remain unanswered. Larger and longer clinical trials are necessary to move from a promising cure to the new gold standard and a permanent cure of stress urinary incontinence.

Stress urinary incontinence (SUI) is the most common form of urinary incontinence, which affects approximately 200 million people worldwide [1]. SUI is defined as the involuntary loss of urine associated with physical activities that increase intra-abdominal and bladder pressure. Intrinsic urethral rhabdosphincter deficiency and urethral hypermobility are the most commonly cited etiologies of SUI, which are targets for treatment [12]. Current treatment modalities include pharmacotherapy, injectable bulking agents, pelvic floor muscle therapy, weight loss, and slings [3]. The current standards of care have varying rates of success, failure, and significant side effect profiles that differ from patient to patient. As a result, there has been a push for new therapies to treat SUI. One of the most promising areas of current research into SUI therapy is the use of stem cell-based therapy for urethral rhabdosphincter regeneration as current research shows that age-related apoptosis of the sphincter is a predominant etiology of SUI.

Briefly, stem cells are undifferentiated cells that have the ability to self-renew or differentiate based upon the organism’s needs. Self-renewal occurs to ensure that the stem cell population is maintained while differentiation occurs in order to produce more specialized cells that can be used for necessary biological functions. Furthermore, there are two broad types of stem cells: embryonic and adult. Embryonic stem cells (ESCs) are obtained from the inner cell mass of embryonic blastocysts and have the greatest ability to differentiate into many tissue types. The use of these cells is challenging due to ethical and regulatory issues. Adult stem cells are obtainable from various tissues such as bone marrow stroma, muscle, adipose, and blood but are limited in their abilities to differentiate into various tissue types [4]. The benefit with these cells is derived from the ease with which they are obtained as well as the lack of regulatory issues. Once obtained, the cells are cultivated in a laboratory-based environment where they can be grown, replicated, and selected for the desired cell type. Once the selected cell type is extracted, it must be reimplanted in the desired location where regenerative repair will occur [4].

With regard to SUI therapy, the goal of treatment is to cause regenerative repair of the deficient urethral rhabdosphincter by using stem cells to replace, repair, and enhance the biological function of the damaged tissue [4]. Adult stem cells are of the greatest interest in current research studies due to aforementioned reasons. Additionally, adult stem cells can differentiate into myogenic cells that are transplantable back into the host that they were harvested from. Bone marrow stromal cells have been cited as a common source of multipotent mesenchymal adult stem cells. These cells are able to differentiate into adipogenic, osteogenic, chondrogenic, and, most importantly, myogenic cells [4]. The limitations of using this source are the pain and complications associated with procurement. Furthermore, the yield of myogenic cells from bone marrow has been shown to be lower than other sources [5]. Adipose-derived stem cells have gained much attention as these cells are relatively easy to harvest from fat tissues and can differentiate into adipogenic, chodrogenic, osteogenic, myogenic, and neurogenic cells. When transplanted, these cells can act as bulking agents as well as induce myogenic regeneration [5]. The final source of adult stem cells that is commonly used in SUI therapy is muscle-derived. These cells are taken from muscle biopsies that can be performed with local anesthesia. The cell types that can be obtained from this source include myoblasts, satellite cells, muscle progenitor cells, and muscle-derived stem cells [4]. Periurethral transplantation of these cells allows for formation of new myofibers and innervations due to the release of neurotrophins [4]. The limitations of using these types of cells include poor survival of injected cells, which is currently being investigated in the literature [1].

Why Stem Cells

In the last decade the use of human stem cells has been presented as a potential breakthrough in the treatment of several different types of lower urinary tract diseases. The regenerative medicine held great expectations in the treatment of functional disorders of lower urinary tract system such as urinary incontinence, neurogenic bladder, and urethral trauma. While these approaches hold much promise, the practical results and treatments available to patients have been slow to materialize. We did not see the rapid translation of research done in the labs into the clinical treatments.

The initial concept of cell-based therapy for SUI was based on the hypothesis that injection of skeletal myoblasts into a dysfunctional urethra could “recharge” the sphincter muscle [6]. The further evolution was the injection of stem cells derived from skeletal muscle cells. In 2002 Yiou et al. published their results on skeletal-derived stem cells injection in a murine model of urethral sphincter lesion [7]. These initial results were followed by other preclinical and clinical studies (Table 10.1). The next step was represented by the utilization of nonskeletal stem cells such as those derived from adipose tissue or stem cells derived from bone marrow [8].

Table 10.1

Clinical trials for stem cell use in stress urinary incontinence

Study

Patients

Stem cell type

Administration method

Follow-up

Se`be P, Doucet C et al.

12 females

Autologous mesenchyme-derived stem cells (MDSC)

Intrasphincteric injection

1 year: 3 patients dry, 7 patients improved on 24 h pad test but not on voiding diary, 2 patients slightly worse

Lee CN, Jang JB et al.

39 females

Allogenic human umbilical cord blood stem cells

Urethral submucosal injection

1 year: the effectiveness was similar to conventional bulking agents

Herschorn S, Carr L et al.

29 females

Autologous MDSCs

Intrasphincteric injections; two doses given

1 year: showing 50 % success rate with no leakage

Mitterberger A, Pinggera GM et al.

20 females

Skeletal muscle-derived autologous fibroblasts and myoblasts

Transurethral U/S-guided injections of fibroblasts into urethral submucosa and myoblasts into sphincter

1 year: 18/20 patients were cured while the remaining 2 were improved. 2 years: 16/20 were still cured, 2 others were improved, 2 lost to follow up

Carr LK, Steele D et al.

8 females

Autologous MDSC

Transurethral injection

1 year: 1/8 cured, 5/8 improved, no serious adverse reactions reported

Strasser H, Marksteiner R et al.

42 females

Autologous myoblasts and fibroblasts from skeletal muscle

TUUS-guided injection into rhabdosphincter

1 year: 38/42 completely continent

Blaganje M, Lukanović A et al.

38 females

Autologous myoblast from skeletal muscle

Ultrasound-guided injection into the external urethral sphincter followed by electrical stimulation (ES)

6 week: 78.4 % had negative urinary stress test, 13.5 % considered themselves cured

Sources of Stem Cells

There are two groups of stem cells that have been investigated for a clinical use. They are the pluripotent ESCs and the multipotent adult stem cells. Due to ethical concerns and the possible teratogenicity, the use of ESC has been limited in most of Western Europe and North America.

The adult, somatic, stem cells can be obtained from any vascularized tissue as it is currently thought that they constitute the pericytes of the vessels within the tissue [9]. The common donor sites for adult stem cells are the bone marrow, the skeletal muscle, and the adipose tissue. Each of the above sources has advantages and limitations as summarized in the schematic below.

Bone Marrow

Bone marrow has three types of Stem cells [10]: hemopoietic, endothelial, and mesenchymal (potentially useful for SUI regenerative therapy). The advantages are that they are easy to isolate, easy to cultivate, multipotent, and leak of MHC class I cell surface marker (invisible to the host immune system and as such can potentially be used as “Universal” cells from one donor to a different recipient). Disadvantages are that a bone marrow biopsy is required to harvest the cells, and they represent only a small % of cells in the bone marrow.

Muscle-Derived Stem Cells

Muscle-derived stem cells have the advantages of being easy to harvest (simple muscular biopsy and they have good integration with host muscle tissue. Disadvantage include that it is difficult to obtain a sufficient number of stem cells and keep the cells alive after the transplant. The process requires a specific plating technique and cell expansion technique. It is also a time-consuming process (up to 6 weeks are required from the harvesting to the reimplantation of the stem cells).Also, they express the surface antigens (only autologous transplant is possible).

Adipose-Derived Stem Cells

Advantages are that they are easy to harvest (simple liposuction) and large numbers of multipotent cells are present in the adipose tissue. Disadvantages are that they are difficult to manage, have less favorable homing and implantation, and they express the surface antigens (only autologous transplant is possible).

Are Stem Cells Safe?

Many of the earliest stem cell studies were conducted on cells isolated from tumors rather than from embryos. Of particular interest was research on embryonic carcinoma cells (EC), a type of stem cell derived from teratocarcinoma. The EC research laid the foundation for the later discovery of and subsequent work on ESC. Both ESCs isolated from the mouse (mESCs) and then later from humans (hESCs) shared not only pluripotency with their EC cousins, but also robust tumorigenicity as each readily form teratoma. Surprisingly, decades after the discovery of mESCs, the question of what drives ESCs to form tumors remains largely an open one. This gap in the field is particularly serious as stem cell tumorigenicity represents the key obstacle to the safe use of stem cell-based regenerative medicine therapies [11].

The molecular basis of the tumorigenicity of pluripotent cells lies in their cancer-resembling properties, namely their ability to self-renew and proliferate, lack of contact inhibition, and telomerase activity which are promoted by several molecular processes and have been described in detail by Blum and Benvenisty [1213]. On closer examination of the karyotype of ESCs, polyploidy has been observed in mESCs despite an intact spindle assembly checkpoint (SAC) [14]; Mantel and co-workers demonstrated that mESCs and hESCs have an intact SAC, but that mitotic failure-induced polyploidy does not lead to apoptosis of these cells. Mitotic errors often occur in rapidly proliferating cells and the SAC, typically leading to apoptosis of these cells and the necessity for genome maintenance. Uncoupling of this mechanism will lead to karyotypic abnormalities in ESC culture. In addition, when inducing double-strand breaks in the DNA of mouse ESCs and mouse embryoid bodies (EBs) by the DNA-damaging agent etoposide, ESCs were resistant to apoptosis whereas the EBs underwent caspase-3-dependent apoptosis. This suggests that polyploidy tolerance in ESCs will give way to apoptosis upon lineage-specific differentiation. This hypothesis has been confirmed for mouse as well as human mononuclear polyploid/aneuploid cells, indicating that checkpoint–apoptosis uncoupling is an intrinsic behavior of mESCs and hESCs [15].

The examination of gene expression in several human tumors has shown that the pluripotency markers used in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) are directly oncogenic (e.g., c-Myc and Klf4) or are involved in tumorigenesis (e.g., Sox2Nanog, and Oct3/4). Targets of NanogSox2, and Oct4 that encode for transcription regulators are overexpressed and prove especially active in high-grade breast tumors [1116]. Upregulation of Nanog and Oct4 is also associated with poor outcomes in patients with oral cancer [1718]. The pluripotent genes Oct3/4Sox2Nanog, c-Myc, and Klf4were also present in prostate tumor cell lines, as well as in primary prostate tumor tissue. Furthermore, injection of these tumor cells containing the pluripotent genes created strong tumorigenicity in immunodeficient mice [19].

The biological link between pluripotency and tumorigenicity can perhaps best be described by c-Myc. Although not required for the induction of pluripotency in iPSCs, c-Myc augments the reprogramming ability of Oct4Sox2, and Klf4, inhibits differentiation, and promotes proliferation. Myc proteins have been shown to influence several thousand genes. The majority of these are upregulated and involved in cell growth; the few genes that are downregulated are involved in cell cycle arrest, cell adhesion, and cell–cell communication. The influence of Myc on these nuclear processes is oncogenic [2021].

However, not all members of the Myc family possess the same properties. L-Myc, for instance, has a decreased transformation activity and only few human cancers are associated with an aberrant expression of L-Myc. A recent study reported the creation of iPSCs with L-Myc instead of c-Myc and chimeric mice derived from these L-Myc iPSCs showed reduced tumorigenicity [19].

Recently, 21 genes were found to be highly expressed in hESCs as well as in teratomas [13]. These genes were then credited as known oncogenes, if they were involved in cell cycle progression, inhibition of apoptosis, signal transduction, transcription, or translation. Of these 21 genes, Survivin (BIRC5) was found to be the strongest candidate gene. Survivin is an anti-apoptotic gene that is also involved in mitotic regulation and is expressed in the majority of cancers [22]. It was highly expressed in hESCs and teratomas and downregulated in mature EBs [23].

From these insights into the gene expression of hESCs and iPSCs, it can be concluded that tumor formation is not dependent on the presence of EC-like cells, but is more an intrinsic property of pluripotent cells. Furthermore, this tumorigenicity of pluripotent cells is reduced upon differentiation. The dilemma now facing investigators is that in reducing the tumorigenicity of stem cells, the very essence of stem cells that makes them useful also has to be reduced, namely self-renewal and pluripotency. The “best” ESCs are often considered to be those that grow fairly rapidly, form tightly packed colonies, have a low rate of spontaneous differentiation, and are readily passageable. The simplest way to slow or even eliminate the tumorigenicity of normal stem cells prior to transplantation may be to take advantage of their natural “brakes” or pluripotency by partially differentiating them into progenitors [24].

Introduction of a Stem Cell-Specific Suicide Gene

The stable genetic introduction of a suicide gene such as thymidine kinase (tk) into stem cells has been reported to be effective in combination with Ganciclovir (Gan) treatment [25].

Directed Killing of Residual Stem Cells Based on a Nongenetic Method

The most promising approach to this end is to use killer antibodies directed against antigens present on the surface of hESC such as SSEA-4 or a member of the TRA family [26].

Use Stem Cells Themselves for Transplant, but First Eliminate Tumor Forming Potential Without Genetic Modification

In theory, the simplest approach to regenerative medicine and the one expected to lead to robust regenerative tissue growth would seem to be to use stem cells themselves, but ones that had been treated in such a way that they were no longer tumorigenic [27].

Clinical Experience

Once the tissue is harvested, the stem cells are isolated and proliferated in the lab and the cells have to be reinjected in the patients’ body.

In the literature several different options have been presented:

·  Transurethral injection

·  Peri urethral injection

·  Ultrasound-guided transurethral injection into the striated sphincter,

·  Ultrasound-guided injection followed by electrical stimulation.

The different options are summarized in Table 10.1.

In the past, one major problem and limitation in these therapies, regardless of the source of stem cells, is represented by the poor survival of the injected cells [28]. These problems have partially been solved with new seeding technique such as pre-plating and the use of specific growth factors [29].

The working principles on how stem cells can restore the damaged urethral sphincter function are still not completely clear. For instance, research in animal suggests urethral recovery after injection of bone marrow-derived mesenchymal stem cell secretions as after injection of paracrine factors of the stem cells themselves. Therefore, weather the improvement results from the injection of the stem cells or from their secretion of specific proteins is unclear [30]. This might actually represent a possibility for future treatment of SUI. Some authors are speculating that in the future some patients may have SUI prevented with immediate postpartum injection of “off the shelf” proteins into the urethra, while others who have already developed SUI may undergo injection of genes to reawaken the homing potential of the damaged tissue, or undergo a small biopsy and return few weeks later to for injection of stem cells into the urethra.

The use of growth factor that regulates the proliferation and differentiation of skeletal myoblasts to promote healing seems fascinating in the process of enhancing healing process. However, their short biologic half-lives, the rapid clearance of these molecules from the circulation, and the difficulty to maintain a sufficient concentration in the specific anatomic side is currently preventing from its routine clinical use [29].

Conclusion

Currently, midurethral tapes represent the gold standard in the treatment of SUI. The tapes, regardless of the route through which they are deployed, restore the supporting function of ligaments and fascia structure that stabilize urethra during sudden increase in the intra-abdominal pressure. Despite excellent success rates the tapes are not risk-free. One of the concerns with these procedures is related to the fact that the vast majority of the slings are made of polypropylene and are currently under FDA scrutiny. The stem cells injected in the urethra have the potential to restore the functional, contractile response of the striated sphincter. The potential advantage is enormous as this therapy could treat SUI with a minimal risk. The initial results are promising; however, there are many questions that remain unanswered. Larger and longer clinical trials are necessary to move from a promising cure to the new gold standard and a permanent cure of SUI.

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