Abstract
BackgroundAutologous 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.MethodsUSC 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.ResultsDifferentiated 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.ConclusionThe 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 in vivo urological tissue repair or in vitro urethra or bladder modeling.
Highlights
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
Fluorescence activated cells sorting (FACS) analysis showed that USC consistently expressed stem cell surface markers (CD73, CD90, CD105) (Fig. 1), but did not express hematopoietic stem cell markers (CD31, CD34, CD45) or endothelial cell markers (CD31) as described previously [13,14,15,16]
We have demonstrated that stem cells present in urine can efficiently differentiate into urothelial cells with robust barrier function that form multilayered structures similar to normal urothelium
Summary
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. Urothelial cells (UC) are classified as transitional epithelium, and they cover almost the entire luminal surface of the urinary tract. This includes the renal pelvis, ureters, bladder, and the proximal segment of the urethra. Urothelium provides a robust permeability barrier across the urinary tract. Compromised urothelium leads to several common urologic diseases such as recurrent urinary tract infection, urethral injuries or stricture, interstitial cystitis, overactive bladder, and bladder cancer
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