| Cell based therapy provides a promising alternative for urethra tissue reconstruction. Stem cells are found in human urine and these cells can efficiently differentiate into urothelial and smooth muscle cells. These urine derived stem cells (USCs) can be obtained via a simple, non-invasive and low-cost approach. In addition, human USCs enhanced regeneration in tissues including kidney, corpus cavernous penis, and urethra sphincter in an athymic rat model. However, rat is not a good animal model for urethral reconstruction due to their smaller body size. To eliminate the immune response to xenograft, it would be highly desirable to use autologous stem cells for urethral tissue repair in a non-rodent models,abbits are commonly used as an animal model in urethra tissue repair. However, it is unknown whether there are rabbit urinary stem cells that can be isolated and cultured.OBJECTIVES:The goal of this study is to characterize rabbit urine-derived stem cells (rUSCs) and induce these cells to differentiate into urothelial and smooth muscle cells for potential use in lower urinary tract tissue regeneration in a rabbit model.METHODS:Urine was collected from six adult male rabbits (weight 2.0-2.5 kg) through a F8 catheterization. Cells were isolated from the urine, cultured with mixed medium of KSFM and EFM and extensively expanded in vitro. Growth curves, cell surface markers,karyotype, urothelial and smooth muscle cell lineage differentiation of rUSCs were assessed in vitro.RESULTS:We successfully cultured rUSCs in 14/24 urine samples, about 1-2 cell clones per 30 ml urine. The stem cells appeared rice grain-like in morphology. Mean population doubling and average doubling time of rUSCs were 48.5±6.2 hrs and 25.7±8.4 hrs, respectively. A single of rUSC clone can generates about 4×1014 cells (i.e. PD 48.5= 248.5 i.e.3.98×1014cells) within 51.6 ± 0.5 days. Cell growth curve of rUSCs display normal patter in cell proliferation assessment. The rUSCs at p3 were positive for CD29, CD90 and CD 105, but negative for CD31, CD34, CD45 in flow cytometry. When exposed to TGF-β1 and PDGF-BB, these cells could differentiate into spindle-like cells, expressing smooth muscle-specific proteins, including a-smooth muscle actin, desmin, and myosin. Urothelially-differentiated rUSCs expressed urothelial-specific proteins, i.e. uroplakin-Ia and-Ⅲ, AE1/AE3, when exposed to epidermal growth factor.CONCLUSIONS:Rabbit urine-derived stem cells can be easily isolated and cultured in vitro. These cells possess strong proliferative ability, and are capable of differentiating in urothelial and myogenic lineages. Thus, they are a potential alternative autologous cell source for lower urinary tract repair in a rabbit model. |
PDF Full Text Request