stain, and transmission electron microscopy (TEM). Finally, differenti- ated (4.510 5 cells) and non-induced USC (4.510 5 cells) were seeded on collagen-coated inserts for barrier function assessmenton day 7 and 14. RESULTS: Morphologically, urothelial differentiated USC formed cobblestone-like cells. These differentiated USCs expressed transcripts and proteins of urothelial-specific (Uroplakin-III, -Ia) and general epithelial cell markers (CK7, CK13, CK20 and AE1/AE3) in a time-dependent manner. Expression of these markers also depended on the amount of EGF in the media. In an assay of barrier function, urothelial differentiated USC expressed tight junction gene and protein markers (ZO-1 and E-cadherin). TEM demonstrated the ultrastructural aspects of urothelial differentiated USC, including tight junction forma- tion between neighboring cells. Using a fluorescent tracer on differen- tiated cells cultured on inserts, at least a 50% decrease in leakage of the tracer across the insert occurred after 3h in vitro, indicating that these cells could perform a barrier function similar to the normal urothelial cells. CONCLUSIONS: USC can be efficiently differentiated into functional urothelial cells with barrier microstructures, and these cells may be a potential cell source for tissue-engineered urethras. Source of Funding: None 20 TISSUE ENGINEERING SMOOTH MUSCLE FROM ADIPOSE TISSUE DERIVED STEM CELLS FOR GENITOURINARY RECONSTRUCTION Hazem Orabi*, Assiut, Egypt; Tom F Lue, San Francisco, CA INTRODUCTION AND OBJECTIVES: Cell therapy with differ- entiated smooth muscle cells (SMC) offer alternative treatment modal- ities for diseases that involve smooth muscle cells (SMC) pathology in the urogenital organs. Adipose derived stem cells (ADSCs), being easily and efficiently harvested with considerably less donor morbidity, represent an ideal source for differentiated SMC. The aim of the study was to investigate whether ADSCs can be differentiated into SMC, whether these differentiated cells can be transported in various forms including dissociated cells, cell sheets and cell seeded scaffolds and if these induced cells can survive after in vivo transplantation. METHODS: Rat and Human ADSCs were isolated and ex- panded. They were induced to SMC using DMEM with 10% fetal bovine serum and TGF-1. The growth and morphology of the cells were followed up for 4 weeks. The phenotype of induced cells were checked for SMC markers; smooth muscle actin(SMA), calponin and smooth muscle myosin(SM myosin) through immunohistochemistry(IHC) and western blot. Then, the cells were used to construct SMC sheets and SMC grafts by seeding induced cells on small intestinal submucoa (SIS) for one week. The cell sheets were stained with H&E and Masson Trichrome and checked for SMA, calponin, myosin, phalloidin and collagen IV. The induced cells were labeled with 5-ethynyl-2-deoxyuri- dine (EdU). These cell labeled seeded grafts were implanted subcuta- neously in rats, and evaluated after 1 week for cell survival, proliferation and phenotype. RESULTS: The induced cells showed positive staining for 3 smooth muscle markers indicating their SMC phenotype after 4 weeks in rat cells and 3 weeks in human cells. The western blot and regular microscopy confirmed their phenotype conversion. The cells sheets could be detached easily in intact form constantly. The sheets were formed of 2-5 cell layers that showed positive staining for SMC mark- ers. They also showed positive staining for collagen VI indicating the formation of extracellular matrix. The seeded SIS grafts showed cell viability, attachment, proliferation and maintenance of SMC phenotype. After in vivo implantation, the seeded cells survived and contributed to blood vessel formation. CONCLUSIONS: These results showed the ability of ADSCs to form SMC and comprise a renewable source for SMC cellular therapy. The cell sheets made of differentiated SMC form a new technology for repair of muscle deficiency or dysfunction in urogenital organs. Cell- seeded grafts constitute a viable option in case of whole organ replace- ment. Source of Funding: None Urodynamics/Incontinence/Female Urology: Basic Research (1) Moderated Poster Session 2 Saturday, May 4, 2013 1:00 PM-3:00 PM 21 RECEPTORS INVOLVED IN PUDENDAL INHIBITION OF THE MICTURITION REFLEX Abhijith Mally*, Yosuke Matsuta, Fan Zhang, Bing Shen, Jicheng Wang, James Roppolo, William de Groat, Changfeng Tai, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: To determine the role of opioid and metabotropic glutamate 5 receptors (mGluR5) in pudendal inhibition of bladder overactivity. METHODS: Cystometrograms (CMGs) were performed in 11 cats under alpha-chloralose anesthesia by slowly infusing the bladder with saline or 0.25% acetic acid (AA). Pudendal nerve stimulation at intensities of multiple times the threshold (T) to induce observable anal twitching was applied during CMGs to inhibit the bladder overactivity induced by AA irritation. Naloxone (0.1, 0.3, and 1 mg/kg, i.v.) was administered to block opioid receptors followed by MTEP (3 and 10 mg/kg, i.v.) administration to block mGluR5 receptors. After each dose of drug, pudendal inhibition of bladder overactivity was examined during CMGs. RESULTS: AA irritated the bladder, induced bladder overactiv- ity, and significantly (P0.0001) reduced bladder capacity to 23.62.7%% of saline control capacity. Pudendal nerve stimulation at 1-1.5T and 4T suppressed bladder overactivity and significantly in- creased the capacity to 57.58.1% (P=0.0005) and 10615% (P=0.0002), respectively, of the saline control capacity. Naloxone had no effect on pudendal inhibition, but MTEP eliminated the inhibition induced by low intensity stimulation and significantly (P0.05) reduced the inhibition induced by high intensity stimulation. Neither naloxone or MTEP altered baseline bladder overactivity. CONCLUSIONS: Opioid receptors are not involved in pudendal inhibition of bladder overactivity, but mGluR5 receptors are partially involved. Understanding neurotransmitter mechanisms could improve the efficacy of neuromodulation therapy for overactive bladder (OAB) treatment, and identify molecular targets for development of new drugs for treating OAB. Source of Funding: This study is supported by the NIH under grants DK-068566, DK-090006, and DK-091253. 22 MICTURITION REFLEX INHIBITED WITH COMBINATION OF TRAMADOL AND TRANSCUTANEOUS FOOT STIMULATION Abhijith Mally*, Fan Zhang, Yosuke Matsuta, Bing Shen, Jicheng Wang, James Roppolo, William de Groat, Changfeng Tai, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: To determine whether transcutaneous electrical foot stimulation in combination with a low dose of tramadol can completely suppress bladder overactivity. METHODS: Repeated cystometrograms (CMGs) were per- formed in 18 alpha-chloralose anesthetized cats by infusing the bladder with saline or 0.25% acetic acid (AA). Transcutaneous electrical stim- ulation (5 Hz) of the cat hind foot at 2-4 times the threshold (T) intensity e8 THE JOURNAL OF UROLOGY Vol. 189, No. 4S, Supplement, Saturday, May 4, 2013