Autologous urothelial cells are often obtained via bladder biopsy to generate the bio-engineered urethra or bladder, while urine-derived stem cells (USC) can be obtained by a non-invasive approach. The objective of this study is to develop an optimal strategy for urothelium with permeability barrier properties using human USC which could be used for tissue repair in the urinary tract system.
USC were harvested from six healthy adult individuals. To optimize urothelial differentiation, five different differentiation methods were studied. The induced cells were assessed for gene and protein expression markers of urothelial cells via RT-PCR, Western blotting, and immunofluorescent staining. Barrier function and ultrastructure of the tight junction were assessed with permeability assays and transmission electron microscopy (TEM). Induced cells were both cultured on trans-well membranes and small intestinal submucosa, then investigated under histology analysis.
Differentiated USC expressed significantly higher levels of urothelial-specific transcripts and proteins (Uroplakin III and Ia), epithelial cell markers (CK20 and AE1/AE3), and tight junction markers (ZO-1, ZO-2, E-cadherin, and Cingulin) in a time-dependent manner, compared to non-induced USC. In vitro assays using fluorescent dye demonstrated a significant reduction in permeability of differentiated USC. In addition, transmission electron microscopy confirmed appropriate ultrastructure of urothelium differentiated from USC, including tight junction formation between neighboring cells, which was similar to positive controls. Furthermore, multilayered urothelial tissues formed 2 weeks after USC were differentiated on intestine submucosal matrix.
The present study illustrates an optimal strategy for the generation of differentiated urothelium from stem cells isolated from the urine. The induced urothelium is phenotypically and functionally like native urothelium and has proposed uses in vivo urological tissue repair or in vitro urethra or bladder modeling.
Stem cell research & therapy. 2018 Nov 08*** epublish ***
Qian Wan, Geng Xiong, Guihua Liu, Thomas D Shupe, Guanghui Wei, Deying Zhang, Dan Liang, Xiongbing Lu, Anthony Atala, Yuanyuan Zhang
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA., Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Child Urogenital Development and Tissue Engineering, Department of Urology, Children's Hospital of Chongqing Medical University, Chongqing, China. ., State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China., Department of Urology, The Second Affiliated Hospital at Nanchang University, Nanchang, China., Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA. .
Go Beyond the Abstract and Read the Commentary by the Author, Yuanyuan Zhang