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.

Epithelium-free bladder wall graft: epithelial ingrowth and regeneration--clinical implications for partial cystectomy.

PURPOSE: Most patients who need a bioengineered bladder wall have bladder cancer. A graft made with autologous urothelium would not be safe. To investigate the feasibility of providing bioengineered tissue for patients with partial cystectomy we evaluated the host and graft response after transplanting an epithelium-free graft. MATERIALS AND METHODS: De-epithelialized bladder wall grafts from male rats were transplanted on syngeneic female rat bladders after partial cystectomy. Urothelial morphology, vessel density, inflammation, stromal thickness and uroplakin expression were evaluated 1, 3, 6 and 9 months after surgery. Cell gender was distinguished by fluorescent in situ hybridization using unique X and Y chromosome probes. RESULTS: There was no significant graft contraction at any time. Male graft urothelial morphology and uroplakin expression were similar to those of controls at all time points. The donor bladder had decreased vessel density at early time points while the host had increased vascularity, which normalized in each by 6 months. Graft inflammation and edema normalized by 9 months. There was no muscular hypertrophy. Fluorescence in situ hybridization revealed early ingrowth of host female urothelium and a small fraction of male urothelial cells, which appeared between 1 and 3 months. CONCLUSIONS: Within 9 months de-epithelialized grafts appeared histologically as normal bladder, surprisingly faster than an equivalent model with full-thickness grafts. The safety and function of an epithelium-free graft must be determined in a large animal model. These early data in a small animal model substantiate the feasibility and equivalency of using grafts without epithelium, which would allow application in patients with cancer.

Bladder wall transplantation--long-term survival of cells: implications for bioengineering and clinical application.

Current bioengineered bladder wall substitutes include acellular scaffolds and grafts seeded with autologous cells. The transplanted cells on a seeded graft may regenerate and/or be replaced by cells of the patient's bladder. This may or may not be advantageous depending upon the underlying pathology. A theoretically perfect bioengineered graft would be intact bladder wall. To determine if such a graft is feasible and to study the cellular changes, we transplanted full-thickness bladder grafts from male inbred rats onto bladders of female syngeneic rats. Bladders were harvested at 1, 3, 6, 12, and 16 months after surgery and evaluated for histologic changes. Cell origin (male donor vs. female host) was determined with fluorescent in situ hybridization with unique probes for rat X and Y chromosomes. Urothelial hyperplasia, inflammation, and increased stromal thickness subsided down to control values by 6 months after surgery. At 16 months, graft muscle demonstrated persistence of male cells. On the other hand, graft urothelium was partially replaced by female host cells with a pattern suggestive of a hematogenous route rather than ingrowth from the host bladder. Bladder wall transplantation is feasible. The slow replacement of the transplanted urothelium and persistence of muscle may imply the same fate for engineered grafts.