180DEVELOPMENT OF BLADDER AUGMENTATION METHODUSING SKELETAL MUSCLE-DERIVED MULTIPOTENT STEMCELL SHEET-PELLETS TRANSPLANTATION

Shuichi Soeda*, Isehara, Japan; Tetsuro Tamaki, Akio Hoshi, MakiMasuda, Masahiro Nitta, Yukio Usui, Akira Akatsuka, ToshiroTerachi, isehara, Japan

INTRODUCTION AND OBJECTIVES: Reconstruction of con-tracted bladder by illeocystoplasty using a segment of bowel has got awide acceptance in the urological field. In this case, however, nervouscontrol is disrupted due to the complete section of bladder-wall andreplaced bowel. Thus, conservation of bladder functions such as wallcontractions and sensations is difficult, whereas capacity growth ispossible. Here, we tried to dilate the bladder wall associate withvascular and nerve reconstitutions using transplantation of stem cellsheet-pellets, which is formed by expanded skeletal muscle-derivedmultipotent stem cells (MDSCs) that have a synchronized reconstitu-tion capacity of vascular, muscular and peripheral nervous systems.

METHODS: MDSCs were obtained from green fluorescentprotein (GFP) transgenic mice muscles. Muscles were enzymaticallydissected into several fiber-bundles, and totally cultured for 5 days.Then, cells were suspended by EDTA, and removed fibers and otherdebris. Remaining cells were cultured for 4 days to obtaining confluentcellular sheets. Sheets were softly suspended by EDTA, and correctedby centrifuge (MDSCs sheet-pellets). Wild-type C57/BL mice wereused for recipients. Surface of the bladder-wall was sectioned fromsmooth muscle layers along midline legion maintaining mucosal layer,and tag ends were spread out for a transverse plane. The MDSCssheet-pellets were pasted on the open up thin-walled bladder. Cellularengraftment and differentiation were determined by fluorescence im-munohistochemistry 4 wks after transplantation.

RESULTS: GFP� transplanted cells were actively engrafted onthe bladder-wall and contributed to recover the thickness of the wall(Fig. 1). In addition, transplanted GFP� cells differentiated into vascu-lar cells (pericytes, endothelial cells, and adventitial cells) and nervouscells (Schwann cells and perineurial cells), and contribute to the recon-stitution of blood vessels and peripheral nerves (arrows in Fig. 1).

CONCLUSIONS: Present Bladder augmentation method usingone-half wall section cystoplasty and MDSCs sheet-pellets transplan-tation was potentially useful for the reconstruction of contracted bladderassociate with native bladder functions.

Source of Funding: none

181CELL THERAPY WITH AUTOLOGOUS URINE-DERIVED STEMCELLS FOR VESICOURETERAL REFLUX

Shaofeng Wu, GuiHua Liu, Shantaram Bharadwaj, Steve Hoagie,Anthony Atala, Yuanyuan Zhang*, Winston-Salem, NC

INTRODUCTION AND OBJECTIVES: As an alternative to bulk-ing agents, endoscopic injections of autologous cells have been usedto restore the normal structure and function of the vesicoureteraljunction. Vascularization of the injected graft is critical for enhancementof cell survival and maintenance of the volume of the graft. Recently,we have demonstrated that certain cells in urine possess the charac-teristics of stem cells, including extensive expansion capability andmultipotency. The goal of this study was to evaluate the effect ofvascular endothelial growth factor (VEGF) over-expression on urine-derived stem cell (USC) survival, growth and myogenic differentiationand to determine whether this technique could improve injection ther-apy for the correction of VUR.

METHODS: USC were obtained from 9 urine samples (5healthy individual donors; ages 3 to 27). USC were infected withadenovirus containing the human VEGF gene (USC/Ad-VEGF). USC/Ad-VEGF plus endothelial cells (in total 5106 cells) in 500�l collagen-Igel were subcutaneously implanted into 20 athymic mice. After 28days, the grafts were assessed grossly and with histology and immu-nocytochemistry for smooth muscle markers (�-smooth muscle actin,desmin and myosin) endothelial markers (CD 31 and von Willebandfactor) and peripheral nerve cell markers (S-100, neurofilament, glialfibrillary acidic protein).

RESULTS: The grafts containing both USC/Ad-VEGF and en-dothelial cells were larger and better vascularized compared to non-VEGF expressing controls. Additionally, the total number of implantedcells expressing human nuclear markers was significantly higher, indi-cating increased cell survival in the VEGF-expressing grafts. MoreVEGF-expressing cells expressed CD31 and von Willebrand proteins,as well as smooth muscle markers. Interestingly, more nerve fibers andrevascularization appeared in the USC/Ad-VEGF plus endothelial cellsgroup than the controls in vivo. However, the regenerated nerve cellsdid not stain for human nuclear protein expression, indicating that theseregenerated nerve fibers most likely originated from mouse peripheralnerve rather than from the differentiation of USC.

CONCLUSIONS: VEGF over-expression in grafted USC en-hanced the in vivo survival of these cells and increased neovascular-ization, myogenic differentiation of USC, and nerve regeneration withinthe grafts. This approach might have important clinical implications forurological cell therapy for the treatment of VUR and urinary inconti-nence.

Source of Funding: none

182PROTECTIVE EFFECT OF HUMAN AMNIOTIC FLUID STEMCELLS IN ACUTE TUBULAR NECROSIS

Laura Perin, Sargis Sedrakyan, Stefano Da Sacco*, Gianni Carraro,Liron Shiri, Kevin Lemley, Roger De Filippo, Los Angeles, CA

INTRODUCTION AND OBJECTIVES: Acute Tubular Necrosis(ATN) can cause irreparable damage to the kidney epithelial tubularcells that can be associated with severe renal dysfunction. Stem-cellbased therapies may provide alternative approaches to treatment ofATN. We have previously shown that clonal c-kit positive stem cells,derived from human amniotic fluid (hAFSC) can be induced to a renalfate in an ex-vivo system. Herein, we show for the first time thesuccessful therapeutic application of hAFSC in a mouse model withglycerol-induced rhabdomyolysis and ATN.

METHODS: Clonal c-kit� stem cells, derived from human am-niotic fluid (hAFSC) and labelled with luciferase, were injected intoimmunodeficient mice with glycerol-induced rhabdomyolysis and ATN.Mice were sacrificed at different time points (1, 2, 3, 7, 14 days postinjection) and physiological parameters, kidney morphology, apoptosisand proliferation, and renal cytokine expression were investigated.

Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011 THE JOURNAL OF UROLOGY� e75