Characterization of rabbit urine-derived stem cells for potential application in lower urinary tract tissue regeneration

Tissue-engineered urethra with autologous cells seeded on biodegradable scaffolds offers an alternative for lower urinary tract reconstruction. Rabbit is most commonly used as an animal model in urethra and bladder tissue repair. The goal of this study is to characterize rabbit urine-derived stem cells (rUSC) and induce these cells to differentiate into urothelial and smooth muscle cells as an autologous cell source for potential use in lower urinary tract tissue regeneration in a rabbit model. We successfully cultured rUSC from 12 urine samples and 13 bladder wash samples of six rabbits. rUSC colonies appeared more in the bladder wash solution (2–4/15 ml) than those in the urine samples (1–2 clones/15 ml urine). The cells displayed rice grain-like in morphology. Mean population doubling of rUSC was 48.5 ± 6.2 and average doubling time was 25.7 ± 8.4 h, indicating that a single of rUSC clone generated about 4 × 1014 cells in 50 days. The rUSC were positive for CD29, CD90 and CD105 but negative for CD31, CD34 and CD45 in flow cytometry. When exposed to PDGF-BB and TGF-β1, these cells could differentiate into spindle-like cells, expressing smooth muscle-specific proteins, including α-smooth muscle action, desmin and myosin. Urothelially differentiated rUSC expressed urothelial-specific proteins, i.e., AE1/AE3 and E-cadherin when exposed to epidermal growth factor (EGF). Osteogenic-differentiated rUSC expressed osteogenic marker, i.e., alkaline phosphatase when exposed to serum containing DMEM low-glucose medium with osteogenic supplements. In conclusion, rUSC can be isolated from bladder wash or urine samples and cultured in vitro. There stem cells possess strong proliferative ability and are capable of differentiating in urothelial, myogenic and osteogenic lineages. Thus, rUSC are a potential alternative autologous cell source for lower urinary tract repair with tissue engineering technology in a rabbit model.