Urothelium with barrier function differentiated from human urine-derived stem cells for potential use in urinary tract reconstruction

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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 in vivo urological tissue repair or in vitro urethra or bladder modeling.

Keywords: Barrier function; Bladder diseases; Tight junctions; Tissue engineering; Urine-derived stem cells; Urothelium.

Fig. 1

Cell surface markers and morphology of USC and UC-induced USC. Human USC expressed MSC surface marker profiles (CD73, CD90, CD 105, and CD146), but not hemoprotein stem cell markers (CD 31, CD34, and CD45) assessed by flow cytometry

Fig. 2

Permeability barrier function test of urothelial differentiation of USC. a In vitro permeability test model of UC-induced USC in dynamic culture condition vs. static culture condition. b Urothelial induction of USC in different conditions of differentiation culture for 2 weeks. c Effect of time-dependent induction on urothelial differentiation of USC on weeks 1 and 2, respectively. d Impact of dynamic culture on permeability barrier function of USC 2 weeks after urothelial induction. Notes: Foot numbers in graphs c and d represent: (1) USC, (2) UC, (3) USC + UC/CM, (4) USC + EGF, (5) USC + SMC/CM. Abbreviations: USC = urine-derived stem cells, UC = urothelial cells, SMC = smooth muscle cells, CM = conditioned medium, UC/CM = urothelium-conditioned medium SMC/CM = smooth muscle cell-conditioned medium EGF = epidermal growth factor. Single seeding = seeding cells only once, Triple Seeding = seeding cells each at first 3 days

Fig. 3

UC-induced USC expressing urothelial cell gene and protein markers. Expression level of urothelial cell transcripts and protein markers (Uroplakin Ia/III, AE1/AE3, and CK20) under different induction culture conditions assessed by: a real-time PCR, b Western blotting, and c immunofluorescence staining 14 days after urothelial differentiation. Cell morphology of USC, UC, and induced USC are also illustrated. Notes: Foot numbers in graphs (a) and (b) represent: (1) USC, (2) UC, (3) USC + UC/CM, (4) USC + EGF, (5) USC + SMC/CM. Abbreviations: UPIa = Uroplakin Ia, UPIII = Uroplakin III. USC = urine-derived stem cells, UC = urothelial cells, SMC = smooth muscle cells, CM = conditioned medium, UC/CM = urothelium-conditioned medium SMC/CM = Smooth muscle cells conditioned medium EGF = epidermal growth factor. Scale bar = 50 μm

Fig. 4

UC-induced USC expressed gene and protein markers of tight junction markers 14 days after differentiation. Tight junction markers (E-cadherin and Cingulin, ZO1, ZO2) displayed on cell membrane boundaries between cells in the urothelially induced USC and UC, but not in USC, assessed by a real-time PCR, b Western blotting, and c immunofluorescent staining assessed under confocal microscope. Scale bar = 40 μM. d Tight junctions or desmosomes between neighboring cells in the induced USC and UC groups, but not in non-induced USC, examined by transmission electron microscopy. LM scale bar = 500 nm, HM scale bar = 100 nm. Notes: Foot numbers in graphs a and b represent (1) USC, (2) UC, (3) USC + UC/CM, (4) USC + EGF, (5) USC + SMC/CM. Abbreviations: USC = urine-derived stem cells, UC = urothelial cells, SMC = smooth muscle cells, CM = conditioned medium, UC/CM = urothelium conditioned medium SMC/CM = Smooth muscle cells conditioned medium EGF = epidermal growth factor. LM = low magnitude, HM = high magnitude

Fig. 5

Cell proliferation curves of urothelially induced USC at different seeding cell concentrations. Cell growth pattern of single seeding was similar with that of triple seeding pattern. Dotted square showed at the 14th day the cell proliferation curve goes to almost same metric. Both groups were urine-derived stem cells cultured in urothelial-conditioned medium under dynamic culture environment. Notes: Single seeding = seeding cells only once, Triple seeding = seeding cells each at first 3 days

Fig. 6

Effect of nature collagen matrix on multilayer formation of urothelium of UC-induced USC. Urothelially induced USC developed urothelium with five to seven cell layers stained positive for AE1/AE3 onto a SIS matrix under dynamic culture, which is similar to urothelial cells. In contrast, USC treated with EGF or SMC/CM grew thinner layer and USC alone formed a single layer. In addition, the induced USC penetrated into the porous SIS matrix to a great depth to form well adhesion structure of cell-matrix. Scale bar = 50 μm. Abbreviations: USC = urine-derived stem cells, UC = urothelial cells, SMC = smooth muscle cells, CM = conditioned medium, UC/CM = urothelium conditioned medium SMC/CM = smooth muscle cell-conditioned medium EGF = epidermal growth factor