Stem Cells in Functional Bladder Engineering.

Conditions impairing bladder function in children and adults, such as myelomeningocele, posterior urethral valves, bladder exstrophy or spinal cord injury, often need urinary diversion or augmentation cystoplasty as when untreated they may cause severe bladder dysfunction and kidney failure. Currently, the gold standard therapy of end-stage bladder disease refractory to conservative management is enterocystoplasty, a surgical enlargement of the bladder with intestinal tissue. Despite providing functional improvement, enterocystoplasty is associated with significant long-term complications, such as recurrent urinary tract infections, metabolic abnormalities, stone formation, and malignancies. Therefore, there is a strong clinical need for alternative therapies for these reconstructive procedures, of which stem cell-based tissue engineering (TE) is considered to be the most promising future strategy. This review is focused on the recent progress in bladder stem cell research and therapy and the challenges that remain for the development of a functional bladder wall.

Predifferentiated Smooth Muscle-Like Adipose-Derived Stem Cells for Bladder Engineering.

Introduction: All organs of human body are a conglomerate of various cell types with multidirectional interplay between the different cells and the surrounding microenvironment, leading to a stable tissue formation, homeostasis, and function. To develop a functional smooth muscle tissue, we need to simulate and create a multicellular microenvironment. The multilineage adipose-derived stem cells (ADSCs), which can be easily harvested in large numbers, may provide an alternative cell source for the replacement of smooth muscle cells (SMCs) in cell-based detrusor bioengineering therapeutic approaches. The aim of this study was to investigate whether predifferentiated smooth muscle-like ADSC (pADSC) can support SMCs to generate stable smooth muscle tissue through remodeling of extracellular matrix (ECM) and factor secretion. Methods: Rat SMC and pADSC were mono- and cocultured in the cell ratios 1:1, 1:2, 1:3, and 1:5 (SMC-pADSC) and grown for up to 2 weeks in vitro. The expression of the SMC-specific markers alpha-smooth muscle actin, calponin, myosin heavy chain 11 (MyH11), and smoothelin was assessed, and cell proliferation and contractility were analyzed. Proteomic analysis of the secretome (cell-cell contact was compared with a noncontact transwell 1:1 coculture) and the cell pellets was performed, with the focus on ECM deposition and remodeling, integrin expression and growth factor secretion. Results: SMC and pADSC were strongly positive for all smooth muscle markers. After 1 and 2 weeks of culture, the 1:1 cell ratio developed a significantly higher number of smooth muscle organoids and improved contractility. These organoids were highly structured, consisting of an SMC core surrounded by a pADSC layer. The deposition of various EMC proteins, such as collagens 1a1, 1a2, 2a1, 3a1, 5a2, 6a2, 12a1, and fibrillin 1, was significantly increased. A decreased matrix metalloproteinase 3 (MMP3), MMP9 and MMP13 secretion, as well as increased tissue inhibitors of metalloproteinase 1 (TIMP1) and TIMP2 secretion were found in the contact coculture compared with the monoculture controls. Conclusion: SMC-pADSC 1:1 cocultures exhibit an improved cell proliferation, contractility, and organoid formation compared with all other ratios and monoculture, while retaining a stable phenotype that is comparable with the SMC monoculture. These effects are mediated by increased ECM deposition and tight ECM remodeling by the secreted MMP and TIMP. Impact statement Harvesting smooth muscle cells (SMCs) from diseased bladders represents a significant limitation for clinical translation of bladder Tissue Engineering. Our results suggest that autologous predifferentiated smooth muscle-like adipose-derived stem cell can substitute SMCs, and may be used in combination with SMCs to generate contractile detrusor muscle tissue for patients suffering from end-stage bladder diseases. We demonstrate a beneficial effect when using these cells in a 1:1 ratio with improved deposition of extracellular matrix (ECM) molecules and superior remodeling of the ECM by matrix metalloproteinases and decreased tissue inhibitors of metalloproteinase activity.

Detrusor bioengineering using a cell-enriched compressed collagen hydrogel.

OBJECTIVE: The gold standard for bladder regeneration in end-stage bladder disease is the use of intestinal tissue, which is however associated with significant long-term complications. Our study aims to bioengineer functional detrusor muscle combining bladder smooth muscle cells (SMC) and SMC-like adipose-derived stem cells (pADSC) in compressed collagen (CC) hydrogels and to investigate biocompatibility and tissue regeneration of such detrusor-equivalents in a rat detrusorectomy model. METHODS: Compressed collagen hydrogels seeded with 1 × 106 or 4 × 106 SMC alone or in combination with pADSC in a 1:1 ratio were investigated. Morphology, phenotype, and viability as well as proteomic secretome analysis were assessed in the 1:1 co-cultures and the respective monocultures. The hydrogels were implanted into rat bladders after partial detrusorectomy. Bladders were harvested 8 weeks after transplantation, and assessed for tissue morphology, detrusor regeneration, neo-vascularization and -innervation. RESULTS: Co-cultured cells exhibited native SMC morphology, high viability and proliferated to form microtissues in vitro. The pro-angiogenic factors angiogenin, vascular endothelial growth factor (VEGF)-A and -D were increased in the secretome of the pADSC samples. After 8 weeks of in vivo, the regenerated bladder wall showed a multilayered structure containing all bladder wall components. The overall performance of the bladder wall regeneration of CC seeded with 4 × 106 cells was significantly better than with 1 × 106 cells and the combination SMC:pADCS performed slightly better than SMC alone. CONCLUSION: Compressed collagen possesses an adequate regenerative potential to promote regeneration of bladder wall tissue in vivo. Seeded with a combination of pADSC and SMC this may well be the first step towards a functional bladder reconstruction especially in patients suffering of end-stage bladder diseases.

Bladder regeneration through stem cell therapy.

INTRODUCTION: Impaired bladder function in children and adults often causes lifelong morbidity, as functional therapeutic approaches in this field are nonexistent. If reconstructive procedures are required, intestinal tissue is used as a gold standard for bladder repair. As this procedure is associated with significant long-term complications there is a strong clinical need for alternative sources of stable and reliable bladder tissue, of which stem cells are considered most promising. AREAS COVERED: This review focusses on the recent development in stem cell use for bladder bioengineering. Further, we discuss the importance of the microenvironment in stem cell differentiation, function and tissue regeneration and its effect on the development of functional bladder tissue. EXPERT OPINION: Functional bladder bioengineering requires a complex approach that involves the development of a multi-layered scaffold, with each layer offering a specific microenvironment for the generation and support of the respective cell type used in its redevelopment. The formation of cellular cross-talk within and between the layers is the key in this process. While autologous stem cells may provide a viable source of tissue for bladder reconstruction, their in situ activation combined with repair of the diseased microenvironment may offer better, more lasting solutions for functional bladder regeneration.