Bioengineered urothelium provides a valuable tool for both urinary tract reconstruction and the study of urothelial dysfunction. Compromised urothelium leads to several urological diseases such as interstitial cystitis, overactive bladder, recurrent urinary tract infection, and bladder cancer. An in vitro intact urothelium with barrier function and tight junction construction is required for disease modeling, drug development, and urothelial tissue repair. Traditionally, urothelial cells are often obtained by invasive bladder tissue biopsy. In addition, several adult stem cell types have been used for urothelial differentiation potential; however, this remains challenging. Thus, an alternative autologous stem cell source obtained with non-invasive procedures is desirable.
In this study, we have demonstrated that stem cells present in urine, called urine-derived stem cells (USC), possess beneficial regenerative properties, including robust proliferation potential and multipotent differentiation potential. Differentiated USC expressed significantly higher levels of urothelial-specific transcripts and proteins (Uroplakin-III, -Ia), epithelial cell markers (Ck-7, -13, -20 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, multi-layered urothelial tissues formed over time when USC has differentiated on the intestine submucosal matrix.
There are several advantages of human USC use over other stem cells. USC can be easily obtained from healthy individuals or patients by non-invasive and low-cost procedures that generate high-quality cells that can be expanded extensively. Up to 140 USC clones per 24-hour urine collection were consistently obtained from a single healthy individual1. Thus, a 24-hour urine sample can provide >1x108 cells over three passages, a number sufficient for a majority of the intended applications. In addition, cell viability is preserved during isolation as the method used does not require enzymes (such as collagenase) for tissue dissociation. Furthermore, USC is a safe cell source for potential clinical use as no evidence of oncogenic potential has been identified over several in vivo studies.
Urothelium generated from patient-derived USC would provide an excellent platform for the study of mechanisms underlying urological diseases including interstitial cystitis, overactive, neurogenic or obstructive bladder, and provide a testbed for the development of therapies to treat these diseases. In addition, induced urothelium can be used in evaluating the impact of pharmacological treatment on urothelial barrier function in both normal bladders and in the bladder that has been compromised by diseases. Furthermore, multiple layer urothelium formed on a natural collagen-based matrix may be used for the reconstruction of urological tissues that have been damaged by trauma or disease. Taken all together, we optimized a strategy to induce human USC differentiation into functional urothelial cells with barrier function and appropriate cellular 3D architecture. These cells would represent a powerful tool for urological tissue research and urological regenerative medicine.
Yuanyuan Zhang, MD, Ph.D., Associate Professor, Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States
1. Lang R, Liu G, Shi Y, et al. Self-renewal and differentiation capacity of urine-derived stem cells after urine preservation for 24 hours. PloS one 2013; 8(1): e53980.
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