Abstract
Cell-based therapy for the bladder has its beginnings in the 1990s with the successful isolation and culture of bladder smooth muscle cells. Since then, several attempts have been made to artificially implant native cell types and stem cell-derived cells into damaged bladders in the form of single-cell injectables or as grafts seeded onto artificial extracellular matrix. We critically examined in the literature the types of cells and their probable role as an alternative to non-drug-based, non-bowel-based graft replacement therapy in disorders of the urinary bladder. The limitations and plausible improvements to these novel therapies have also been discussed, keeping in mind an ideal therapy that could suit most bladder abnormalities arising out of varied number of disorders. In conclusion, muscle-derived cell types have consistently proven to be a promising therapy to emerge in the coming decade. However, tissue-engineered constructs have yet to prove their success in preclinical and long-term clinical setting.
Introduction
The lower urinary tract (LUT) can be affected by various diseases and disorders arising out of multiple factors, intrinsic or extrinsic to the human body, for example, peripheral neuropathy as in diabetes or alcohol abuse, postsurgical central neuropathy, spinal cord lesions, multiple sclerosis, and many more.
Traditional drug-based therapies have involved the use of cholinergics, antiadrenergics, antimuscarinics, and more. Surgical interventions, including electrical modulation and stimulation, have proven to be of importance in cases of urinary incontinence, urogenital obstruction, and neurogenic bladders of various origins.
An estimated 400 million people in the world suffer from various bladder diseases (24). Many disorders and injuries affect the smooth muscles or the uroepithelial layers of the urinary bladder. Tissue replacement surgeries have been conducted to compensate for the damaged tissue. Many replacements from skin, intestine, polylactic glycolic acid, silicone, and collagen have been widely used in this regard. Bulking agents such as collagen, fat, and Teflon have been employed as injectables at the bladder neck for cases of sphincter–urethral dysfunction (8,28,39). In this literature review, we reviewed cell-based injectable therapies and tissue-engineered graft-based therapies that have been attempted since its inception to current developments covering over two decades.
Cell-based therapy for the bladder could not have been a reality today without the successful isolation and culture of bladder smooth muscle cells (SMCs). This was first exhibited by Baskin et al. (4). Several attempts to artificially implant cells into damaged bladders in the form of single-cell injectables have shown some promising results. Tissue grafts seeded onto artificial extracellular matrix have been pushed as experimental therapies into clinics. We conducted a literature review taking in a few landmark publications under various topics described below and critically analyzed the cell types and their probable role as an alternative to non-drug-based, non-bowel-based graft replacement therapy. For the sake of convenience, we have divided the topic into cell-based injectables and tissue-engineered reconstructive grafts with further subdivisions detailing the different sources of cells. Table 1 summarizes the key developments, outcomes, and critical analysis that have been outlined to give a brief idea about the contents of this review.
The Landmark Developments Describing the Approach, Outcome, and Analysis of Various Attempts at Regenerating the Lower Urinary Tract
Cell-Based Injectables
Smooth Muscle Cells and Their Precursor Cells
Every attempt made at regenerating a defective bladder muscular function invariably aims at developing a technique to replace native smooth muscles with a nondiseased healthy population of SMCs capable of fusing into host tissue. Muscle cell precursor cells and muscle cells have been thought to improve various types of urinary incontinence. This has been experimented in various preclinical and clinical models over the past two decades.
Studies concerning the transplantation of SMCs from autologous and allogenic sources are very few in number. Interestingly, smooth muscle replacement therapies have only been carried out in cardiac and urological tissues in the form of single-cell injections or as tissue-engineered grafts. The idea of SMC replacement therapy through cell transplantation was first introduced in cardiac tissues affected by myocardial infarction that resulted in myocardial scars (31). In the urological tissues, we found only one (our) study (17) that deals with direct use of autologous transplantation of SMCs in a diabetes rat model. Later, various cell types with lineage leading to an SMC have been experimented. Even though the resultant cell type for any transplantation is an SMC, direct SMC transplantation is a neglected area of study. The answer to this might lie in the lower survival rates of transplanted SMCs and the nonavailability of unaffected SMCs from any patient in need of such a therapy.
Myoblasts are precursor cells that mature to form SMCs. Taking a clue from the myoblast transfer therapy proposed during the late 1980s as a cure for Duchenne muscular dystrophy, which involved skeletal myoblast cells (26), Chancellor et al. attempted to transplant similar skeletal myoblasts from immortalized MDX cell lines into the urinary bladder and descending bladder neck (9). They reported formation of multinucleated myotubes between transplanted cells and also with native cells. Defective adenoviral vectors when used to transfect myoblast cells, expressed the required protein during the formation of myotubes. This property of myoblasts makes them an effective vehicle to transfect muscle cells and continuously express required proteins for an extended period of time. An innovative two-step treatment from another clinical trial (5) involving myoblast injections along with electrical stimulation showed promising results. Although critical analysis of results suggested that improvement in quality of life assessment could be due to a mechanism similar to a bulking agent, there is no evidence to suggest muscle cells act as bulk when transplanted into the urinary tract.
This strategy was persistently applied over many years to develop transplantation of muscle-derived stem cells that exhibited better survival rates than myoblast cells. Successful clinical results demonstrated safety of patients and absence of any major adverse events (6). Patients also saw an improvement in quality of life after being relieved from symptoms of stress urinary incontinence (SUI). This is the first study to demonstrate a dose-related improvement of stress leaks in patients of both genders over a 12-month period. Further clinical studies in Europe with eight (37) subjects have reported the procedure of implantation to be well tolerated with no adverse events. They pointed to decrease in loss of urine followed by insignificant change in parameters in female patients. These female subjects failed to show significant improvement in tested parameters such as rate of urine flow and bladder pressure during micturition. Even though the reasons for failure are not mentioned, we presume that it has to do with greater sphincter control provided by extended urethra of male anatomical structures.
In conclusion, even though mixed results are available with various groups undertaking muscle-derived stem cell transplantation in clinical settings, this methodology of using muscle progenitor and muscle-derived stem cells has proven to be successful in reaching the final stages of patient care.
Nonmuscle Cells and Stem Cells
The hunt for ideal replacement of diseased cells started off with targeting smooth muscle replacement therapy. SMCs, muscle-derived cells, myoblasts, and various precursor cells were found to be the right candidate. Gradually, this success was found to be short-lived because of a lack of a healthy source of cells. For example, in cases of incontinence, spinal cord injury, or diabetes, SMCs get irreversibly damaged due to hypertrophy or were found to lack the endogenous population of fully functional cells for complete regeneration. This led to renewed efforts to find a nonurinary, nonmuscle cell type capable of differentiating into SMCs and thereby pave the way for regeneration (30,32). Here we discuss the use of various types of stem cells from different origins and their scope of success in the bladder environment.
Mesenchymal Stem Cells
Stem cells have proven to exist in every organ of the human body. Even though their presence has been confirmed with successful isolation from various sources, this cell type has never been successfully isolated from the bladder, though there are some suitable candidates that fit the idea of a progenitor SMC. Adult stem cell transplantation has been explored as a replacement for pathologically defective SMCs. Initially, stem cell transplantation studies in myocardial tissues were found to be successful in improving cardiac function. Owing to the similarities in smooth muscle architecture and functions of contraction, pathological bladder models were also transplanted with stem cells in the belief that they would be capable of neurogenic and myogenic differentiation. Most of such attempts have produced positive results and are explained in detail under separate headings.
While most researchers consider bone marrow mesenchymal stem cells (BM-MSCs) as an ideal candidate (38), sources from adipose-derived (ADSCs) and muscle-derived stem cells (MDSCs) have also been found to be of immense differentiation potential and easily available through very minimal surgical intervention. BM-MSCs have exhibited vast differentiation potential into urothelium and SMC lineages (15). In vitro myogenic differentiation of human BM-MSCs was evaluated as a potential treatment for urethral sphincter muscle repair. BM-MSCs were transplanted into bladder tissue and have also been demonstrated to directly home to the region of damaged bladder when injected intravenously (40). While the mechanism behind homing of MSCs remains unclear, it would suffice to mention that such data have been rare even though they hold immense potential in view of clinical benefits associated with nonsurgical, preferred route of transplantation. Transplantation into bladder tissue has also led to various inventions in reconstruction surgery (11). BM-MSCs show a fibroblastic morphology similar to SMCs and readily differentiate into SMCs.
In addition to BM-MSCs, a similar type of MSCs derived from amniotic fluid (AF-MSCs) have shown that in models of cryoinjured bladders, both BM-MSCs and AF-MSCs fail to show greater morphogenesis and integration into native smooth muscle bundles (12). Upon quantitative analysis, a large population of transplanted cells remained undifferentiated, probably owing to reduced availability of growth factors such as hepatocyte growth factors and transforming growth factor (TGF)-β. These factors related to low differentiation in vivo, prompting researchers to either process the BM-MSCs to dedifferentiate in vitro or adopt stem cells from other sources. Use of AF-MSCs in an interesting approach to ameliorate bladder dysfunction in an animal model of Parkinson's disease was first detailed by Soler et al. (36). Even though the cells were not directly transplanted into the urinary tract, the urodynamic improvement was observed as a result of a paracrine effect at the site of injury (brain). AF-MSCs remain an untapped and promising source for direct cell grafting and indirect regeneration of LUT in neurogenic models via unclear pathways. Their clinical benefits, however, are beginning to ascertain the usefulness of this niche.
MDSCs offer higher survival and integration rates when compared to muscle cells and muscle precursor myoblast cells (5). Interestingly, MDSCs are the only cell types that were found to develop myotubes with native host SMCs. This unique property has so far made it an ideal candidate for bladder neck and midurethral repair. MDSCs are the only cell types to successfully make it to the stage of single-cell injectable therapy in clinical trial. Encouraging results of clinical trials have begun to emerge from a mixture of procedures using MDSC transplantation and traditional SUI techniques such as vaginal tapes and slings in cases of SUI and prostatectomyrelated sphincter damage (7,16,18).
Adipose-Derived Stem Cells (ADSCs)
Bladder SMCs are derived from bladder mesenchyme. Hence, multipotent cells from the mesenchyme are well poised to develop into myogenic lineage. ADSCs were found to be capable of multilineage differentiation. These cells have specifically been tested in models of SUI and have shown myogenic differentiation in vivo (19,23,42). The issue with this cell type is the presence of mixed cell types in culture. Some researchers used the whole component of lipoaspirate cells without further selection while most researchers have been successful in isolating ADSCs in transplants to bladder neck and midurethra. Further standardization of this technique is necessary to ascertain specific cell types and destiny of transplanted cells. While there have been consistent reports of SMC differentiation over longer periods posttransplantation, natural neurogenic differentiation of ADSCs and one report of such differentiation in vivo raise questions about the safety of these source of cells in regenerative urologic treatment (23). Also, the bulky nature of adipose tissue cells makes them natural alternatives to artificial bulking agents such as collagen and polytetrafluoroethylene. Their role as bulking agents must clearly be negated while assessing cell integration and functional improvement in diseased animals and clinical studies.
Adipose tissues has been a useful source of mesenchymal stem cells because of their vestigial nature and ease of access to this tissue. Their ability to differentiate into SMC phenotype via the TGF-β pathway has made them a preferred choice in various diseases of the LUT (22,32). While mode of transplantation and delivery remain important factors influencing the success of any cell transplantation, Zhang et al. report amelioration of bladder dysfunction in 40% of test animals of diabetes mellitus where the cells were introduced via the intravenous route, and direct homing of ADSCs was observed into affected bladder, probably with the expression of certain homing factors. Direct transplantation of these cells into the bladder tissue resulted in an improvement in voiding function in 60% of treated animals (42).
ADSCs also showed equally appreciable improvements resulting in lower micturition frequency combined with higher voided volume in a hyperlipidemic model of overactive bladder (19). Lipoaspirate cells have hence been proven to have greater potential, but further studies and its clinical implementation have not been established. It is also unclear if use of ADSCs sourced from metabolically compromised cases of diabetes or hyperlipidemia would be of any use considering effect of these disorders on defective signaling and growth pathways.
Tissue-Engineered Reconstructive Grafts
Autologous and allogenic cells from various sources used as single-cell injectables for bladder regeneration have been discussed in the previous segment. However, the biggest breakthroughs in regenerative medicine in urology, as described in detail below, have been from using these cells as an alternate material to mimic the native host tissue and finally act as the best replacement for defective tissue. Tissue engineering of urological tissues such as urethra, bladder wall, and ureters have provided short-term success in clinical setup, which would be impossible to achieve through traditional conservative management options such as drugs and surgical interventions. Cell types native to the bladder have today been successfully implanted as graft tissue and have shown the way to replace enterocystoplasty using bowel tissues. Several groups have attempted to design such a biological analog tissue for grafting into urinary bladder (10,29).
Cells From the Urinary Tract for Tissue-Engineered Grafts
The credit for creating the first autologous tissue-engineered bladder goes to Atala and his team currently at the Wakeforest Institute for Regenerative Medicine (2).
Prior to the early 1990s, natural and artificial materials were explored to replace intestinal bowel tissues, which were being used in enterocystoplasty procedures. Seromuscular tissues, omentum flaps, Teflon, and other synthetic meshes were being used. These turned out to be lithogenic, mucous-producing, and immunogenic substances. Inventions in cosmetic surgery to treat burn patients led to discovery of cell-seeded engineered tissues capable of supporting natural regeneration of damaged skin. This discovery was applied to finding an ideal replacement for urological tissues.
Initially, degradable (resorbable) polymers of polyglycolic acid were used as lattice and coated with urothelial cells (UCs) grown in serum-free media (3). Sustained research led to invention of a completely de novo reconstructed urinary bladder successfully transplanted in canine models of cystectomy. Later, the first reported clinical trials in patients with myelomeningocele announced an era of reengineered tissues that could support any ailment arising from urological structures (2). These bladders used the scaffold of collagen–PGA polymer and were seeded with urothelial and SMCs in layers. Patients postoperatively demonstrated a dramatic increase in bladder compliance, bladder capacity, and increased leak point pressures. Renal and bladder functions were preserved and showed normal recovery during the 5-year follow up. A recently concluded similar clinical trial involving spina bifida patients over a 36-month follow-up suggested an unsuccessful outcome with serious adverse effects including bladder rupture during later stages and bowel obstruction (20). Although two more multicentric clinical trials involving pediatric spina bifida cases and adult spinal cord injury patients have been initiated by Atala's group (41), results of these studies are yet to be published, but the subsequent licensing of this technology with a private company (Tengion Inc., Winston-Salem, NC, USA) has provided much less information regarding the progress made in this field ever since.
Critical evaluation of the abovementioned approach indicates several points that need to be answered in subsequent publications and scientific communications. 1) Long-term results of fully functional neobladders need to be assessed to find problems that have previously been associated with enterocystoplasty. 2) Reinnervation of the entire bladder seems to be a problem that has never been addressed. Although preclinical studies in large canine models with no neurogenic bladder condition and pathologically normal bladders showed normal reinnervation capable of supporting regeneration, such an effect in clinical case studies needs to be assessed. This is especially important when pathological bladders have been used as a source for procuring cells and grafting three fourths of neobladders with one fourth from patients' native tissue. Whole bladder substitutions have always remained doubtful. 3) Effect of neobladder constructs on other cases of end-stage bladder disease such as bladder cancer, congenital bladder defects, and neurogenic bladders arising out of various pathological diseases. Apart from these medical queries, technical problems involving high costs and challenging human expertise make it improbable to implement it in regular clinics in the near future.
Moving ahead from engineered neobladders, it became clear that the cells originating from pathological bladders have remained irreversibly changed in their signaling and growth pathways. Hence, the ultimate goal of regenerative medicine would be to use cells from an allogenic source or from autologous nonurinary organs. As discussed in the previous topic dealing with introduction of stem cells, these cell types were the alternative source of cells differentiating into native SMCs and UCs. Pluripotent and multipotent stem cells from hair follicles, adipose tissue, bone marrow, muscle biopsies, Wharton's jelly, and amniotic sac were tested. Myogenic and uroepithelial differentiation could be achieved with cells from MSCs (1) and embryonic stem cells (25).
Cells From Non-Urinary Tract Tissues for Tissue Engineering
Apart from the multipotent MSCs used as cell-based injectables in bladder regeneration, pluripotent stem cells have been used in various other cardiovascular, gastrointestinal, and neurological dysfunctions. SMCs derived from pluripotent hair follicle stem cells were first reported by Liu et al. (21), which were channelized to bladder research by Drewa et al. (14). Under a specific microenvironment related to urinary SMCs and UCs, pluripotent stem cells derived from hair follicles were cultured on bladder acellular matrix and anastomosed to defective bladders of rats.
Interestingly, the pluripotent nature of hair follicle stem cells (HFSCs) allows us to use only a single cell type that serves as progenitor to various types of lineages in the urinary bladder. Myogenic, urothelial, neuronal, and vascular cells seem to originate from HFSCs. The same property of pluripotency is also a disadvantage. Controlled differentiation into the above-mentioned lineages is a matter of concern. Long-term effects of these induced differentiation in vitro and in vivo need to be observed. Also a matter of concern is the use of exogenous agents to induce lineage-specific differentiation. Most transplant procedures in cell therapy have always preferred autologous trophic factor-driven in vivo differentiation, whereas this is the only case where induced differentiation of cells have been experimented (13). The choice of biomaterials plays an important part in assisting the regeneration of aug mented cell types. Some of the materials proved detrimental to the growth of native tissue over the transplanted ones. Revascularizing the tissue became an inevitable strategy to lengthen the survival of graft tissue. Two of the latest materials to have shown a greater degree of success are PGA and electrospun nanofibers. Many studies have begun to show that these materials allow greater movement of growth factors and available nutrition from native host tissue and hold a promising future in resolving the challenges in tissue engineering. One such study (35) describes the use of these nanoscale fibers seeded with MSCs, which showed formation of muscle bundles and partial regeneration of bladder in partially cystectomized rats.
MSCs from the bone marrow have the potential to differentiate into smooth muscle and urothelial lineages. MSCs subjected to coculture and conditioned medium environments readily differentiate into the required cell type (38). This principle was used in an in vivo transplantation of induced MSCs cultured on a nanofibrous poly-l-lactic acid scaffold. The in vitro induced differentiation, as well as the in vivo studies, showed expression of smooth muscle and urothelial markers such as α-smooth muscle actin, myosin, uroplakins, and pancytokeratins. However, no functional studies were performed on the animal models undergoing an augmentation procedure. Also, the question regarding the permanent nature of SMC and UC dedifferentiation or further differentiation into unknown phenotypes remains a matter for further studies.
MSCs have also been used in experimental augumented cystoplasty procedures wherein they were seeded onto small intestinal submucosa and grafted into bladder (34). Improvements in bladder capacity and compliance values in nonhuman primate animal models provided excellent proof of better performance of cell-seeded grafts against acellular mimetic tissues. A more recent development in the same technique from Sharma et al. (33) goes further to address the crucial lacunas of reinnervation and revascularization in tissue-engineered constructs. With the BM-MSCs seeded onto an elastomeric scaffold that mimics the native bladder tissue, the group introduces a combinational approach of using cluster of differentiation 34-positive (CD34+) hematopoietic stem progenitor cells. These secondary treatments showed enhanced vascularization and innervation of triple-layered in vivo graft tissue. Such a combination of treatments shows a promising future for engineered urological tissues.
An encompassing analysis of 131 cases of clinical outcomes of tissue-engineered bladders (27) detailing the success and critical analysis of drawbacks states that the core issues affecting the tissue-engineering approaches remain unsolved. Innervation and vascularization strategies by omental flaps, endothelial cells along with Schwann cell-seeding strategies have been analyzed. An observation is also made regarding the use of healthy animal models in testing such new strategies and the lack of testing in diseased models whose impaired regeneration capability might prove to be an overlooked matter of utmost importance. On the whole, they provide an insight into an optimistic future for tissue engineering of the urinary tract in the days to come.
Conclusion
A consensus of sorts seems to be emerging between the use of cell injectables and artificially reconstructed bladders and their replacement. Injectable autologous cells capable of pluripotency and multipotency are becoming a natural way of replacing and regenerating some of the damaged cell functions of bladders and LUT tissue. However, artificial tissues and tissue replacements have had their fair share of problems yet to be overcome.
In the coming decade, native cell types as well as specialized stem cells isolated from vestigial parts of the human body with very little effort may become a better choice for the abovementioned disorders. Similar views from our previous work (17) as well as the continuing works from various scientists leading this effort seem to converge at the same point. It is now clear that the bladder and lower organs undoubtedly possess an ability to regenerate after cell grafting because of mechanisms either due to trophic bystander effect or due to active cell replacement phenomena. Further research on the mechanisms, as well as long-term safety in the clinical setting needs to be ascertained. Certainly, an unaffected, nonurinary cell type with clear potential to differentiate into urinary tract cell types would be an ideal choice.
Tissue replacements and artificially de novo constructed tissues have shown a lot of promise in the past. Unfortunately, insufficient data regarding the mechanism of action responsible for the regeneration of cellular structures along with reinnervation, combined with lack of long-term safety data seem to suggest a bleak future for this strategy until proven otherwise.
Footnotes
Acknowledgments
We would like to acknowledge and thank Prof. Karl Erik Andersson for reviewing this manuscript. This work was supported by a fellowship from Marie Curie Initial Training Networks Urology TRUST (Grant No. 238541) under which Chaitanya Gopinath holds a Ph.D. studentship. Peter Ponsaerts is a postdoctoral fellow of the Fund for Scientific Research – Flanders (FWO-Vlaanderen). The authors declare no conflicts of interest.