Presentation Authors: Kotaro Suzuki*, Michiyo Koyanagi-Aoi, Keiichiro Uehara, Nobuyuki Hinata, Masato Fujisawa, Takashi Aoi, Kobe, Japan
Introduction: Patients with bladder diseases often require augmentation or reconstruction of urinary bladder. Although gastrointestinal tissue or autologous bladder cells have been employed in treatments for the regeneration of bladder functions, these approaches often cause various adverse events. Recently, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs. This study aimed to develop a protocol for the directed differentiation of hiPSCs into mature stratified bladder urothelium.
Methods: We evaluated the effect of several small molecules, FGF10 and culture devices on the urothelial differentiation of hiPSCs by assessing markers from initial to terminal differentiation stage as well as formation of stratified structure. The marker gene expression was analyzed by Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. To assess stratification, we employed optical coherence tomography (OCT) imaging.
Results: 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 and PD153035. FGF10 not only increased the expression of uroplakins but also enhanced stratification, and transwell culture further enhanced stratification. OCT showed that the urothelium with transwell culture was generally thicker than in a normal dish. In an immunohistological analysis, stratified uroplakin II-positive epithelium was observed in transwells.
Conclusions: We demonstrated for the first time that several factors enhanced the directed differentiation and stratification of bladder urothelium from hiPSCs. Our stratified urothelium model might be useful in the field of regenerative medicine of the bladder.
Source of Funding: This work was supported by a grant for Research Center Network for Realization of Regenerative Medicine (16817073) from the Japan Agency for Medical Research and Development, AMED.