**4. Myogenic differentiation of USCs**

Our previous studies demonstrated that USCs can be efficiently induced to differentiate into smooth muscle cells [23, 24]. After myogenic differentiation with TGF-1 (2.5 ng/ml) and PDGF-BB (5.0 ng/ml), induced USCs displayed spindle-shape morphology, and expressed smooth muscle-specific gene and protein markers (such as smoothelin, myosin, and desmin). In addition, these cells exhibited a contractile pattern similar to bladder smooth muscle cells. In our recent study, we demonstrated that USCs can give rise to skeletal muscle-like cells with myotube formation in standard myogenic differentiation media (i.e. SkGM2/Differentiation Medium. USCs are culture in Sk-MC growth Medium (SkGM-2, Lonza) for 2 weeks followed by Differentiation medium (DMEM:F-12 [1:1], 2% Horse serum and 1% Penecillin/streptomy‐ cin). Differentiated USCs expressed specific skeletal muscle cell transcripts and protein markers (myoD, myogenin, myosin, and Myf5). *In vitro* calcium labeling studies showed that skeletal myogenic differentiated USCs resulted in increased calcium efflux. After the induced cells (1x106 ) mixed with 2% collagen-I gel (100 μl) were implanted into the tibialis anterior muscle of nude mice for one month, the grafted cells maintained expression of skeletal muscle cell markers *in vivo*. The USCs differentiated into a skeletal muscle cell lineage, which could be used as a potential source for cell injection therapy in the treatment of urethral sphincter dysfunction.

#### **5. Paracrine factors of USCs**

We have recently demonstrated that USCs are able to secrete several angiogenic growth factors, such as VGEF, PDGF, FGF-1, IGFBP-1 and 3, MMP-9, and angiogenin when UCSs in culture media were assessed by ELISA. *In vivo* studies showed that USCs can stimulate the resident cells to migrate from the host into the graft tissue. More cells with no human nuclear protein expression were observed in the sites where USCs were implanted, compared to a cellfree injection sites.

There are several potential advantages to using USCs as a cell source for cell therapy, including the following: 1) USCs can be easily harvested by a non invasive method and grown in culture; 2) they do not require enzyme digestion or culture on a layer of feeder cells to support cell growth; 3) they start as one single-cell colony in the initial culture, which creates more homogeneous cells after differentiation; 4) since they possess telomerase activity, they can generate more cells and provide a longer life span for tissue regeneration; 5) they possess higher plasticity and more efficiency when induced to differentiate into other functional cells, including endothelial and muscle cells; and 6) as autologous cells, USCs do not raise ethical issues or cause immune reactions to engineered implants. Therefore, obtaining and using stem cells from urine could be an attractive alternative to the standard urological tissue biopsies currently used in cell therapy and tissue engineering. Additionally, myogenic differentiated USCs might be used in other diseases involved in sphincter dysfunction via an endoscopic procedure, including muscle sphincter-deficient diseases such as vesicoureteral reflux, gastroesophageal reflux disease [25,26], and anal incontinence [27].
