Production of urothelium from pluripotent stem cells for regenerative applications.

As bladder reconstruction strategies evolve, a feasible and safe source of transplantable urothelium becomes a major consideration for patients with advanced bladder disease, particularly cancer. Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are attractive candidates from which to derive urothelium as they renew and proliferate indefinitely in vitro and fulfill the non-autologous and/or non-urologic criteria, respectively, that is required for many patients. This review presents the latest advancements in differentiating urothelium from pluripotent stem cells in vitro in the context of current bladder tissue engineering strategies.

Fas-associated death domain (FADD) is a negative regulator of T-cell receptor-mediated necroptosis.

Cell death is an important mechanism to limit uncontrolled T-cell expansion during immune responses. Given the role of death-receptor adapter protein Fas-associated death domain (FADD) in apoptosis, it is intriguing that T-cell receptor (TCR)-induced proliferation is blocked in FADD-defective T cells. Necroptosis is an alternate form of death that can be induced by death receptors and is linked to autophagy. It requires the death domain-containing kinase RIP1 and, in certain instances, RIP3. FADD and its apoptotic partner, Caspase-8, have also been implicated in necroptosis. To accurately assess the role of FADD in mature T-cell proliferation and death, we generated a conditional T-cell-specific FADD knockout mouse strain. The T cells of these mice develop normally, but lack FADD at the mature stage. FADD-deficient T cells respond poorly to TCR triggering, exhibit slow cell cycle entry, and fail to expand over time. We find that programmed necrosis occurs during the late stage of normal T-cell proliferation and that this process is greatly amplified in FADD-deficient T cells. Inhibition of necroptosis using an inhibitor of RIP1 kinase activity rescues the FADD knockout proliferative defect. However, TCR-induced necroptosis did not appear to require autophagy or involve RIP3. Consistent with their defective CD8 T-cell response, these mice succumb to Toxoplasma gondii infection more readily than wild-type mice. We conclude that FADD constitutes a mechanism to keep TCR-induced programmed necrotic signaling in check during early phases of T-cell clonal expansion.

Using uniaxial tensile testing to evaluate the biomechanical properties of bladder tissue after spinal cord injury in rat model.

To investigate the biomechanical properties of rat bladder tissue after spinal cord injury (SCI) using uniaxial tensile testing. Evidence suggests the bladder wall undergoes remodeling following SCI. There is limited data describing the biomechanical properties of bladder wall after SCI. This study describes the changes in elastic and viscoelastic mechanical properties of bladder tissue using a rat model after SCI. Seventeen adult rats received mid-thoracic SCI. Basso, Beattie, and Bresnahan (BBB) locomotor testing was performed on the rats 7-14 days after injury quantifying the degree of SCI. Bladder tissue samples were collected from controls and spinal injured rats at 2- and 9-weeks post-injury. Tissue samples underwent uniaxial stress relaxation to determine instantaneous and relaxation modulus as well as monotonic load-to failure to determine Young's modulus, yield stress and strain, and ultimate stress. SCI resulted in abnormal BBB locomotor scores. Nine weeks post-injury, instantaneous modulus decreased by 71.0% (p = 0.03) compared to controls. Yield strain showed no difference at 2 weeks post-injury but increased 78% (p = 0.003) in SCI rats at 9 weeks post-injury. Compared to controls, ultimate stress decreased 46.5% (p = 0.05) at 2 weeks post-injury in SCI rats but demonstrated no difference at 9 weeks post-injury. The biomechanical properties of rat bladder wall 2 weeks after SCI showed minimal difference compared to controls. By week 9, SCI bladders had a reduction in instantaneous modulus and increased yield strain. The findings indicate biomechanical differences can be identified between control and experimental groups at 2- and 9-week intervals using uniaxial testing.

Autologous regeneration of blood vessels in urinary bladder matrices provides early perfusion after transplant to the bladder.

Large animal testing and clinical trials using bioengineered bladder for augmentation have revealed that large grafts fail due to insufficient blood supply. To address this critical issue, an in vivo staged implant strategy was developed and evaluated to create autologous, vascularized bioengineered bladder tissue with potential for clinical translation. Pig bladders were used to create acellular urinary bladder matrices (UBMs), which were implanted on the rectus abdominus muscles of rats and pigs to generate cellular and vascular grafts. Rectus-regenerated bladder grafts (rrBGs) were highly cellularized and contained an abundance of CD31-positive blood vessels, which were shown to be functional by perfusion studies. Muscle patterns within grafts showed increased smooth muscle formation over time and specifically within the detrusor compartment, with no evidence of striated muscle. Large, autologous rrBGs were transplanted to the pig bladder after partial cystectomy and compared to transplantation of control UBMs at 2 weeks and 3 months post-transplant. Functional, ink-perfused blood vessels were found in the central portion of all rrBGs at 2 weeks, while UBM grafts were significantly deteriorated, contracted and lacked central cellularization and vascularization. By 3 months, rrBGs had mature smooth muscle bundles and were morphologically similar to native bladder. This staged implantation technique allows for regeneration and harvest of large bladder grafts that are morphologically similar to native tissue with functional vessels capable of inosculating with host bladder vessels to provide quick perfusion to the central area of the large graft, thereby preventing early ischemia and contraction.

Combination product of dermal matrix, preconditioned human mesenchymal stem cells and timolol promotes wound healing in the porcine wound model.

A combination product of human mesenchymal stem/stromal cells (MSCs) embedded in an extracellular matrix scaffold and preconditioned with hypoxia and the beta-adrenergic receptor antagonist, timolol, combined with sustained timolol application post implantation, has shown promising results for improving wound healing in a diabetic mouse model. In the present study, we extend those findings to the more translatable large animal porcine wound model and show that the combined treatment promotes wound reepithelialization in these excisional wounds by 40.2% and increases the CD31 immunostaining marker of angiogenesis compared with the matrix control, while maintaining an accumulated timolol plasma concentration below the clinically safe level of 0.3 ng/mL after the 15-day course of topical application. Human GAPDH was not elevated in the day 15 wounds treated with MSC-containing device relative to wounds treated with matrix alone, indicating that the xenografted human MSCs in the treatment do not persist in these immune-competent animals after 15 days. The work demonstrates the efficacy and safety of the combined treatment for improving healing in the clinically relevant porcine wound model.

In Vitro Differentiation and Propagation of Urothelium from Pluripotent Stem Cell Lines.

Bioengineering of bladder tissue, particularly for those patients who have advanced bladder disease, requires a source of urothelium that is healthy, capable of significant proliferation in vitro and immunologically tolerated upon transplant. As pluripotent stem cells have the potential to fulfill such criteria, they provide a critical cell source from which urothelium might be derived in vitro and used clinically. Herein, we describe the in vitro differentiation of urothelium from the H9 human embryonic stem cell (hESC) line through the definitive endoderm (DE) phase via selective culture techniques. The protocol can be used to derive urothelium from other hESCs or human-induced pluripotent stem cells.

The Current Use of Stem Cells in Bladder Tissue Regeneration and Bioengineering.

Many pathological processes including neurogenic bladder and malignancy necessitate bladder reconstruction, which is currently performed using intestinal tissue. The use of intestinal tissue, however, subjects patients to metabolic abnormalities, bladder stones, and other long-term sequelae, raising the need for a source of safe and reliable bladder tissue. Advancements in stem cell biology have catapulted stem cells to the center of many current tissue regeneration and bioengineering strategies. This review presents the recent advancements in the use of stem cells in bladder tissue bioengineering.

Inosculation of blood vessels allows early perfusion and vitality of bladder grafts--implications for bioengineered bladder wall.

Bioengineered bladder tissue is needed for patients with neurogenic bladder disease as well as for cancer. Current technologies in bladder tissue engineering have been hampered by an inability to efficiently initiate blood supply to the graft, ultimately leading to complications that include graft contraction, ischemia, and perforation. To date, the biological mechanisms of vascularization on transplant have not been suitably investigated for urologic tissues. To better understand the mechanisms of neovascularization on bladder wall transplant, a chimeric mouse model was generated such that angiogenesis and vasculogenesis could be independently assessed in vivo. Green fluorescence protein (GFP) transgenic mice received bone marrow transplants from ß-galactosidase (LacZ) transgenic animals and then subsequent bladder wall transplants from wild-type donor mice. Before euthanization, the aorta was infused with fluorescent microbeads (fluorospheres) to identify perfused vessels. The contributions of GFP (angiogenesis) and LacZ (vasculogenesis) to the formation of CD31-expressing blood vessels within the wild-type graft were evaluated by immunohistochemistry at different time points and locations within the graft (proximal, middle, and distal) to provide a spatiotemporal analysis of neovascularization. The GFP index, a measure of angiogenic host ingrowth, was significantly higher at proximal versus mid or distal regions in animals 2-16 weeks post-transplant. However, GFP index did not increase over time in any area. Within 7 days post-transplant, perfusion of primarily wild-type, donor blood vessels in the most distal areas of the graft was observed by intraluminal fluorospheres. In addition, chimeric host-donor (GFP-wild type) blood vessels were evident in proximal areas. The contribution of vasculogenesis to vascularization of the graft was limited, as LacZ cells were not specifically associated with the endothelial cells of blood vessels, but rather found primarily in areas of inflammation. The data suggest that angiogenesis of host blood vessels into the proximal region leads to inosculation between host and donor vessels and subsequent perfusion of the graft via pre-existing graft vessels within the first week after transplant. As such, the engineering of graft blood vessels and the promotion of inosculation might prevent graft contraction, thereby potentiating the use of bioengineered bladder tissue for transplantation.

Induction of human embryonic and induced pluripotent stem cells into urothelium.

In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.

Constitutive phosphorylation mutation in Fas-associated death domain (FADD) results in early cell cycle defects.

Fas-associated death domain (FADD) is an adaptor molecule for the death receptor subfamily of the tumor necrosis factor receptor superfamily, but it is also required for cell proliferation. Cell cycle-specific regulation of FADD phosphorylation plays an important role in FADD proliferative function since mice with a mutant form of FADD mimicking constitutive phosphorylation at serine 191 (FADD-D) exhibit defective T cell proliferation. Here we characterized these mice in detail and found that T cell development in 2-4-week-old mice is relatively normal, although mature FADD-D T cells manifest defective G(0) and G(1) to S transition with abnormalities in regulation of p130, p27 degradation, retinoblastoma protein phosphorylation, and CDK2 kinase activity. These downstream defects are further associated with the failure to up-regulate the forkhead box M1 cell cycle transcription factor, FoxM1. FADD-D protein is also mislocalized during cell cycle progression. Thus, regulation of FADD phosphorylation is crucial for proper cell cycle entry.

Functional role for I kappa BNS in T cell cytokine regulation as revealed by targeted gene disruption.

Triggering of the TCR by cognate peptide/MHC ligands induces expression of I kappa BNS, a member of the I kappa B family of NF-kappaB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of I kappa BNS in TCR triggering, we created a targeted disruption of the I kappa BNS gene. Surprisingly, mice lacking I kappa BNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, I kappa BNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-gamma than wild-type cells. Transfection analysis demonstrates that I kappa BNS and c-Rel individually increase IL-2 promoter activity. The effect of I kappa BNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-kappaB rather than the CD28RE site; mutation of the NF-kappaB site extinguishes the induction of transcription by I kappa BNS in transfectants and prevents association of I kappa BNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-gamma-linked transcripts in I kappa BNS knockout T cells. Collectively, our findings demonstrate that I kappa BNS regulates production of IL-2 and other cytokines induced via "strong" TCR ligation.

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities.

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

Peptide-induced negative selection of thymocytes activates transcription of an NF-kappa B inhibitor.

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.