Abstract
For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.
Highlights
Patients with bladder congenital anomalies, neuropathic disorder, chronic inflammation, and bladder cancer undergo cystectomies and subsequently often require augmentation or reconstruction of the urinary bladder
Several previous reports have suggested that a high dose of GSK3β inhibitor enhanced the directed differentiation of several human induced pluripotent stem cells (hiPSCs) lines to the posterior DE22–24, the concrete values of the concentrations and durations of treatment varied among the reports, suggesting that the optimal concentration and duration of GSK3β inhibitor treatment differ among cell lines
We succeeded in the directed differentiation of hiPSCs into mature bladder urothelium through the posterior definitive endoderm (DE) and caudal hindgut
Summary
Patients with bladder congenital anomalies, neuropathic disorder, chronic inflammation, and bladder cancer undergo cystectomies and subsequently often require augmentation or reconstruction of the urinary bladder. Gastrointestinal tissue has generally been employed as a substitute for bladder tissue in these cases While this approach does provide a urinary reservoir function, the use of intestinal tissue for this purpose often leads to significant complications, including recurrent infections, bladder stones, metabolic disturbance, and an increased cancer risk[1,2]. Patients are at risk for perioperative complications due to this approach, including bowel obstruction and bowel leak of anastomosis[3]. To overcome these complications, autologous bladder cells have been studied as alternative biomaterials for augmentation or reconstruction of the urinary bladder. The stratified structure is a important attribute of the mature bladder urothelium because the structure plays a critical role in the bladder providing a barrier against pathogens, toxins and waste products in urine[18,19]; no report has demonstrated the derivation of stratified bladder urothelium from hiPSCs
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