Volume 70, Issue 6, Pages 1032-1041 (December 2016) Adipose-derived Stem Cells Counteract Urethral Stricture Formation in Rats  Fabio Castiglione, Karel Dewulf, Lukman Hakim, Emmanuel Weyne, Francesco Montorsi, Andrea Russo, Luca Boeri, Trinity J. Bivalacqua, Dirk De Ridder, Steven Joniau, Maarten Albersen, Petter Hedlund  European Urology  Volume 70, Issue 6, Pages 1032-1041 (December 2016) DOI: 10.1016/j.eururo.2016.04.022 Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 1 Urethra stricture rat model. (A) Instruments: 1=polyethylene catheter marked with methylene blue, 2=polyethylene catheter, 3–4=syringes. (B) Catheter introduction, (C) site of penile injection and injuries, and (D) injection of human adipose stem cells. European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 2 Representative microultrasound images of penile urethras of a sham rat and rats from the urethra stricture group treated with vehicle (urethral stricture; US) or with human adipose stem cells (hADSCs). Upper panels are longitudinal sections and lower panels are cross-sections from the same rat. Dotted lines depict the position of cross-sectional registration. White arrows indicate sites of hyperechogenic tissue of the urethras of rats from the US and the hADSC group. Note the more extensive areas of hyperechogenic tissue and narrowing of the urethral lumen in the stricture of the US rat. Vertical white scale bars (upper right of each image)=2mm. cs=corpus spongiosum; u=urethral lumen. European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 3 Functional organ bath investigations of isolated bladder tissue from sham control rats, vehicle-treated urethral stricture rats (US) and human adipose stem cell (hADSC)-treated US rats (eight preparations from four rats per group). Frequency-response curves during transmural activation of nerves. Values are given as percent of a 60mM potassium control contraction of each preparation normalized to the weight of each preparation. All values are given as mean standard error of the mean. Note the lower contractions of bladder preparations from US rats compared with sham and hADSC. Included p-values indicate trends for differences between US and hADSC rats (analysis of variance Student Newman-Keuls). * p<0.05 versus sham. European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 4 Histology. Representative photomicrographs of Masson's trichrome and hematoxylin and eosin (H & E) staining in midshaft sections of rat penises at magnification 10×, 20×, and 40×. (A) Masson's trichrome on sections from a sham rat and (B) corresponding H & E staining on an adjacent section from the same rat. (C) Masson's trichrome on sections from a vehicle-treated urethral stricture rat (US) and (D) corresponding H & E staining on an adjacent section from the same rat. (E) Masson's trichrome sections from a human adipose stem cell (hADSC)-treated US rat and (F) corresponding H & E staining on an adjacent section from the same rat. The sham urethra is lined by a layer of pseudo-stratified columnar epithelium that lies on a basement membrane (*). Beneath the basement membrane (§) there is a connective tissue layer containing vascular sinusoids of the corpus spongiosum and smooth muscle fibers. In the US rats, there is deposition of amorphic extracellular matrix material with scattered high numbers of cells (black arrowhead) and fewer vascular sinusoids of the corpus spongiosum are observed (°). Also note the increase of and severe disorganization of thickened collagen bundles as indicated by larger confluent and intense dark blue stainings (white arrowheads). In the hADSC rat, tissue changes were less extensive than those of US rats with preserved wave-like orientation of collagen fibers (#) and visible vascular sinusoids of the corpus spongiosum (v). European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 5 Western blot. Analysis on the midshaft penises for collagen III, elastin, collagen I, inducible nitic oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS; six rats per group). (A) Representative chemiluminescence images of blotted membranes containing protein extracts of all three groups. Double bands are due to binding of antibodies to glycosylated and no glycosylated forms of the antigens probed. (B) Summarized protein expression levels for collagen III, elastin, and collagen I, iNOS, and eNOS. * p<0.05 versus sham group in analysis of variance with posthoc Student-Newman-Keuls analysis. ** p<0.05 versus both sham and human adipose stem cell-treated urethral stricture groups in analysis of variance with posthoc Student-Newman-Keuls analysis. *** p<0.05 versus both sham and vehicle-treated urethral stricture groups in analysis of variance with posthoc Student-Newman-Keuls analysis. GAPDH=glyceraldehyde 3-phosphate dehydrogenase; hADSC=human adipose stem cell; US=urethral stricture. European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 6 Fibrosis-associated gene expression (three rats per group). The expression of each gene in the vehicle-treated urethral stricture rat (US) and human adipose stem cell (hADSC)-treated US groups was reported as a fold increase of the mean expression of the same gene of the sham group. Differences in gene expression were considered significant when p ≤0.05 by analysis of variance. * p<0.05 versus sham and urethral stricture. ** p<0.05 versus both sham and human adipose stem cell groups. *** p<0.05 versus sham. **** p<0.05 versus urethral stricture. Ctgf=connective tissue growth factor; Eng=endogolin; Faslg=Fas ligand; Mmp14=matrix metallopeptidase 14; Pdgf-beta=platelet derived growth factor-beta; Plau=plasminogen activator; Snai1=snail family zinc finger 1; Tgfb1=transforming growth factor-beta. European Urology 2016 70, 1032-1041DOI: (10.1016/j.eururo.2016.04.022) Copyright © 2016 European Association of Urology Terms and Conditions