Hypothesis / aims of study
Shortage of tissue in reconstructive surgery to treat hypospadias and urethral stricture disease leads towards regenerative medicine, more specific tissue engineering. To generate tissue for urethral reconstruction, all components of the male urethra need to be recreated in vitro. Here we focus on the epithelial component. The objective of this study is to use organoid technology to recapitulate all the aspects of the structure and function of the urethral epithelium. The advantage of organoids is that cells grow in a tissue context and that the adult stem cell population can self-renew and differentiate. Organoids can potentially grow indefinitely, whereas cells on plastic stop after a few divisions. These aspects make organoids an ideal in vitro model to compare the use of bladder or urethral cells as epithelial source for future tissue engineering. Our ultimate goal is to create patient derived organoids as an epithelial source for autologous urethral grafts.
Study design, materials and methods
Tissue was obtained from piglets after anatomy class in veterinary school or from a surplus male rabbit previously used for operation training purposes (waste material). Epithelium from the penile urethra and the bladder from fresh corpses was isolated and transported to the tissue culture facility at our institute. Previously established procedures for culturing bladder organoids [1] were adapted to isolate primary cells to form organoids. For analysis and comparison, tissues, isolated primary epithelial cells, and organoids were stained for epithelial markers, including cytokeratins (CKs), cytoskeletal markers and tight junction proteins.
Results
Porcine bladder epithelium and urethral epithelial organoids could be established from dividing cells in the tissue and maintained for several months. Rabbit urethral cells (a mixed culture of fibroblasts and epithelial cells) could form epithelial organoids after few passages. Organoids could be stored in liquid nitrogen and thawed again. Morphologically, no clear difference between species, nor between bladder and urethral epithelial organoids were observed y. However, a different CK expression pattern was found. Epithelium in bladder organoids did not differentiate into transitional epithelium as seen in normal urothelium. Urethral organoids showed single layer epithelium with a lumen, like in the native urethra. Both rabbit and porcine urethral organoids showed mostly round morphology, but some organoids had a less round phenotype, with extrusions or elongation.
Interpretation of results
The differences in CK expression confirm that urethral epithelium has a different origin than urothelium, as is published before [2]. Furthermore, the variety in morphology (round, elongated, with or without lumen) indicates that the adult stem cells in the epithelium are not unipotent, but can form several specialized epithelial cells. We were also able to established a procedure to enrich epithelial cells in a culture that was mixed with fibroblasts.
Future experiments will include human samples. As urethral biopsy will induce stricture formation, we will aim to explore noninvasive ways to acquire bladder and urethral cells via, for example, urine isolation. Alternatively, induced differentiation towards urethral epithelium from progenitor, multipotent cells like adipose derived stromal cells or urine derived stem cells can be used.