Stem cells for urinary tract regeneration.

Regeneration of the urinary bladder is a complicated task, due to organ dimensions and diseases (cancer, interstitial cystitis) when autologous bladder cells cannot be used. Cancer is the most frequent indication for bladder removal (cystectomy). Stem cells can be used with the guarantee of the sufficient cell number for the in vitro construction of the urinary bladder wall. Tissue engineering techniques hold great promise for regeneration of dysfunctional urinary sphincter. Denervation following surgical procedures or injuries results in weakness of the urethral sphincter and stress urinary incontinence. Injectable therapies and the potential of stem cells for sphincter restoration was presented in this review. The aim of this review was to present possibilities of urinary bladder regeneration with the use of stem cells and tissue engineering techniques.

INTRODUCTION

Regeneration and reconstruction of the urinary tract is a very complicated task. There are several parts of the urinary tract, which could be regenerated with the use of tissue engineering techniques. The urinary bladder is the most challenging due to the organ's huge dimensions. There are many diseases when autologous urothelial and bladder muscle cells cannot be used for in vitro construction of the elements of urinary tract, for example: bladder wall for augmentation (cystoplasty) or de novo creation of the whole urinary bladder. These diseases, like cancer or interstitial cystitis, are the most frequent indications for urinary bladder removal (cystectomy) in men. The presentation focuses on the idea of harvesting potentially multipotent stem cells isolated from hair follicle or mesenchymal stem cells isolated from bone marrow and using them for the regeneration of the urinary bladder wall. There is no current clinical practice in bladder regeneration with the use of stem cells. However, there are some studies that suggest the use of cultures enriched with progenitors. These stem cell niches give an opportunity to reduce the invasiveness of cell harvesting. Both epithelial and mesenchymal multipotent stem cells populations within hair follicles or bone marrow raise new possibilities for tissue engineering of the urinary bladder. The hypothesis is that stem cells can be used with the guarantee of the sufficient cell number for in vitro construction of a urinary bladder wall replacement. The future will show if is it a good idea or not.

Currently, invasive surgical management of stress urinary incontinence is associated with morbidity and recurrence. Tissue engineering techniques hold great promise for regeneration of dysfunctional urinary sphincter. Urethral denervation following surgical procedures or injury resulted in dysfunction of the skeletal muscle and partial impairment of smooth muscle contractility. The importance of the middle urethra, treatment of stress urinary incontinence including injectable therapies, and the potential of stem cells for sphincter restoration will be presented. Stem cells have the ability to undergo self-renewal and multidirectional differentiation. There are several potential sources of stem cells for the treatment of stress urinary incontinence, like bone marrow stromal stem cells, muscle derived stem cells, and adipose-derived stem cells. Stem cells injection therapy increased leak point pressure and urethral muscle strip contractility in experimental models of stress urinary incontinence. Introducing these therapies into clinical conditions is questionable. We are still waiting for additional investigations and trials related to this new and experimental method of stress urinary incontinence treatment. The aim of this review was to present the possibilities of urinary bladder regeneration with the use of stem cells.

1. Short history of urinary tract regeneration and tissue engineering

Regeneration and reconstruction of the urinary tract is a very complicated task. The history of the reconstruction of urinary tract is very long. In 1888 Tizzoni and Poggi started to experimental studies on urinary tract reconstruction. In 1917 Neuhof presented human urinary bladder augmentation with fascia [1]. These pioneering works were stopped for a period of 40 years. In the fifties Bricker, Kock and other presented different kinds of urinary diversion techniques. Studer and coworkers in 1985 showed orthotopic urinary bladder constructed from ileum, which became the most popular orthotopic bladder [2, 3]. The orthotopic bladder and ileal conduit are both the most popular diversion techniques, but there are many complications related to bowel segments used for urinary diversion [4]. In 1991, Narem and Vacanti defined tissue engineering, which is a discipline based on biomaterial science and knowledge on cell culture to prepare in vitro living grafts and constructs that can be used for tissue regeneration or replacement [5]. The history of tissue engineering started over one hundred years ago. Experimental works of Harrison, Carrel, and Rous showed the possibility to culture cells outside the body [6, 7, 8]. Tissue engineering techniques were developed and introduced into the clinic by Gallico, Ricordi, Brittberg, and many prominent doctors and scientists [9-12].

There are several parts of the urinary tract, which could be regenerated with the use of tissue engineering techniques. The urinary bladder is the most challenging, due to the organ's huge dimensions. In 1999, Oberpenning and coworkers presented urinary bladder augmentation with cells seeded on scaffold in a canine model [13]. Seven years later Atala et al. replicated this work in a human model. They have presented seven patients with neurogenic bladders augmented with bladder wall constructed from autologous urothelial and muscle cells seeded on PGLA/collagen scaffold [14].

There are many diseases when autologous urothelial and bladder muscle cells cannot be used for in vitro construction of the elements of urinary tract, for example: bladder wall for augmentation (cystoplasty) or de novo creation of the whole urinary bladder. These diseases like cancer or interstitial cystitis. Bladder cancer is the most frequent indication for urinary bladder removal (cystectomy) in men. The presentation focuses on the idea of harvesting potentially multipotent stem cells out of hair follicle or mesenchymal stem cells and using them for the regeneration of the urinary bladder wall. Current clinical practice suggests the use of cultures enriched with progenitors. These stem cell niches give an opportunity to reduce the invasiveness of cell harvesting. Both epithelial and mesenchymal multipotent stem cells populations within hair follicles or bone marrow raise new possibilities for tissue engineering of the urinary bladder. The hypothesis is that stem cells can be used with the guarantee of the sufficient cell number for the in vitro construction of a urinary bladder wall replacement. The future will show if is it a good idea or not [15].

2. Tissue engineering and urinary bladder regeneration

Until now it was very difficult to replicate experiments of Atala and co-workers (2006) in the case of subtotal cystectomy (cystectomy with triangle sparing) [14]. This method was not tested on a larger group of patients [16]. We do not know if there is a limit of the graft surface (huge animal model and human) beyond which full regeneration of the bladder wall will never occur [17-20]. One in vitro constructed graft for human urinary bladder augmentation with a surface of 150 cm2 needs 140 Petri dishes each with 15 cm in diameter. In all, 2.5 m2 of cells has to be grown. The biopsy specimen has to be multiplied 25,000 times to prepare adequate graft surface [14]. The most important questions are concerning cell behavior in culture. After such intensive cell proliferation a replicative senescence should occur according to the phenomenon described by Hayflick and Moorhead [21]. Will all cultured cells be stable after transplantation? What about the risk of carcinogenesis? These questions are still unanswered [22-25]. An analysis of the risk of carcinogenesis was elaborated for melanocyte cell culture and presented by Czajkowski and colleagues [26]. It was stated that long and extensive cell culture has not influenced on oncogene status of normal cells, so these cells can be probably safely transplanted into the host. There is no doubt that safety of the cell therapy is one of the most important issues in the field of tissue engineering, but this issue is not well elaborated.

There are a lot of studies that confirm the possible use of only acellular matrices for the urinary bladder reconstruction [27-29]. Bladder Acellular Matrix (BAM) is a kind of natural scaffold rich in growth factors [14, 30, 31] and closely matches the host tissue with regard to size and to mechanical, structural, and genetic properties [32]. However, there is some evidence that bladder regeneration with acellular scaffolds results in a fibrotic reaction and leads to fibrosis and stops the process of remodeling [16, 33]. According to those reports the question if we need cells for regeneration of bladder occurs. Beyond a doubt, the use of stem cells to treat and reconstruct urological organs appears to be a very powerful and promising approach. Although it is not known what is the real influence of transplanted cells on bladder wall reconstruction. There is still a speculation that transplanted cells build urinary bladder wall and lead to reconstruction. A major problem with transplanted cells is their poor viability after transfer. This results in sudden cell death, which occurs within a few hours after the cells are transferred to the recipient. Some authors reported no viable cells after transplantation. That is why recently a main concern is to develop procedures, which will improve the efficiency of cell transplantation. Even after autologous transplantation the low cellular survival rate still exists [34-37]. This can be a limitation for proper bladder regeneration, because cells are the factor that guarantees nerve growth and angiogenesis [38]. Yannas proposed the inductive model of regeneration in which cells are connected with membrane basement. He suggested the dermis regeneration template (DRT), nerve regeneration template (NRT), and meniscus regeneration template (MRT) that led to regeneration of the aforementioned organs [39].

3. Tissue Engineering and Stress Urinary Incontinence treatment

The aforementioned results suggest the potential use of hair follicle and mesenchymal stem cells in urinary tract regeneration, including urinary incontinence. There are more than 200 million people worldwide living with incontinence [40]. The most common type of urinary incontinence is stress urinary incontinence (SUI) [41]. Stress urinary incontinence is a major problem, which affects approximately 20% of women and nearly 50% of elderly women [42, 43]. The number of patients with this urologic health problem will rise as the baby-boomer generation continues to age [44]. It is a condition with involuntary leakage from the urethra, synchronous with exertion, or on sneezing or coughing, associated with a reduced quality of life [45]. The etiology of SUI is multifactorial, involving functional impairment of pelvic muscle, connective tissue and associated nerves that occurs as a result of advancing age, hormonal status, and pelvic floor damage resulting from vaginal childbirth, which seems to be the most important risk factor for lifetime incontinence [46-49].

Newly emerging technology that may provide a novel method for the treatment of SUI is tissue engineering. The deficiency of muscle and connective tissue that results in SUI can be regenerated by stem-cell therapy, which is currently at the forefront of incontinence research [50]. Stem cells injected into the middle urethra can potentially restore the contractility of the striated muscles and rhabdosphincter.

Carr and coworkers have made the clinical therapy with muscle derived stem cells (MDSCs) biopsy from lateral thigh muscle [51]. Eight patients were included in the first trial using either a periurethral or transurethral MDSC injections into the middle urethra and external urethral sphincter (EUS). The measurable improvement was observed in two patients who underwent periurethral injection and two patients who received transurethral injections using a 10-mm needle. Two patients with initial injections using an 8-mm needle had no benefit. Five of eight patients who followed up for more than one year reported significant improvement. That is why these results are the potential for pure cellular therapy in treating stress urinary incontinence and emphasize the importance of proper cell placement in resulting effectiveness.

4. Cell populations for urological structures regeneration

There are many urinary tract diseases in which autologous cells cannot be used for in vitro construction of the urinary bladder wall for augmentation (cystoplasty). The most common indication for bladder excision is urothelial cell cancer and muscle invasive bladder cancer. These conditions disqualify the use of urinary bladder autologous cells in clinical application. That is why a new source of easily accessible cells with high proliferation rate and plasticity potential are required. Stem cell research is expanding at an extremely rapid pace with new developments and potential applications in medicine. Stem cells are undifferentiated cells that are defined by their abilities of self-renewal and differentiation, producing mature progeny consisting of both non-renewing progenitors and terminally differentiated effector cells [52]. One of the most interesting features of stem cells is their plasticity, which enables converting one cell type to another, even with change of cell lineage. Somatic stem cells seem to be ideal candidates for tissue engineering purposes. Embryonic stem cells are another attractive cell source for regeneration. Human embryonic stem cell lines were successfully generated in 1998. ESCs can potentially be maintained in an undifferentiated state in vitro and theoretically can be directed to differentiate to any cell type of the body. Although the potential of ESCs in regenerative medicine is clear, several methodological problems and issues of control of differentiation following transplantation need to be solved before they can be used in human regenerative therapies [25]. Less controversial, but promising, are somatic stem cells that probably have a much wider differentiation potential than was previously thought. Referring to the previous results of urinary bladder regeneration by stem cells we decided to regenerate epithelium and muscles by hair stem cells and bone marrow mesenchymal stem cell, respectively [53, 54]. Hair follicle and bone marrow stem cells may serve as a source of relatively easily accessible multipotent stem cells for therapeutic approaches.

4. 1. Hair follicle and bone marrow mesenchymal stem cells in urology

Hair follicles are mini organs, which undergo cyclic regeneration through their lifetime. The hair follicle boasts two kinds of stem cell niches, (1) epithelial (bulbar region) and (2) mesenchymal (dermal papilla and fibrotic capsula) cells [55].

These cells were differentiated into keratinocytes, muscle cells, neurons, glia, and melanocytes. Our previous studies showed that different isolation and culture methods influence the phenotype of a hair follicle stem cell while indicating their heterogeneity [56]. The hair follicle epithelial stem cells do not express cytokeratins specific for bladder (CK 7, 8 and 18) and are highly positive for cytokeratin 15. These cells can be converted into urothelial-like phenotype when cultured in media collected from cultures of normal urothelial cells [57]. It was not proven yet that transdifferentiation will be permanent, but it shed a new light on the novel stem cell source for urinary tract regeneration.

Bone marrow mesenchymal stem cells (MSCs) are well accessible and can differentiate into many different cell types, including: adipocytes, chondrocytes, osteoblasts, cardiomyoctes, tenocytes, and skeletal muscle cells in vitro. This stem cell population is characterized by CD44, SH-4 (CD73), CD90, SH-2 (CD105), CD117 (c-kit), SH-3 (CD166), Sca-1, and STRO-1 expression and the lack of CD11b, CD14, CD31, CD 34, and CD45 antigens [58].

It was proven that different conditioned media provide a convenient source of inductive signals to initiate cell reprogramming and their transdifferentiation. TGF-β1 and 5'azacytidine supplemented media as well as conditioned media obtained from H9C2, L6, and primary skeletal muscle cell lines and co-culture system (consisted of MSCs and skeletal muscle cells) induced MSCs transdifferentiation into myogenic (skeletal and smooth muscle) phenotype [59]. Transdifferentiation of stem cells harvested form different niches seems to be a promising option, which can be used in the future for urinary tract regeneration avoiding urinary bladder cells.

CONCLUSIONS

Adult stem cells can be used in urinary tract regeneration, especially hair follicle and bone marrow mesenchymal stem cells. These stem cell populations show high plasticity potential and are able to differentiate into urothelium and muscle layer in vitro under defined culture conditions.