Volume 182, Issue 1, Pages 355-365 (July 2009) Mature, Adipocyte Derived, Dedifferentiated Fat Cells Can Differentiate Into Smooth Muscle-Like Cells and Contribute to Bladder Tissue Regeneration  Takahiro Sakuma, Taro Matsumoto, Koichiro Kano, Noboru Fukuda, Daisuke Obinata, Kenya Yamaguchi, Toshio Yoshida, Satoru Takahashi, Hideo Mugishima  The Journal of Urology  Volume 182, Issue 1, Pages 355-365 (July 2009) DOI: 10.1016/j.juro.2009.02.103 Copyright © 2009 American Urological Association Terms and Conditions

Figure 1 For DFAT cell preparation small piece of adipose tissue was digested with 0.1% collagenase. After centrifugation unilocular adipocytes were isolated from floating layer. Isolated cells were cultured in culture flasks filled with DMEM containing 20% FBS for 7 days. During culture cells attached and flattened to upper flask surface, followed by conversion to fibroblast-like DFAT cells. Flasks were turned upside down and DFAT cells were incubated using conventional culture method. Cultures showed log phase of rapid growth. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 2 Morphological changes from mature adipocytes to DFAT cells by ceiling culture method. Approximately 97% of cells represented isolated, lipid filled, mature adipocytes positive for AdioRed with single nucleus (a) compared to AdipoRed negative cell morphology (b). Bar represents 50 μm. Adipocyte morphology on ceiling culture day 3 showed cells attached and flattened to upper flask surface, followed by production of fibroblast-like DFAT cells (c), while fluorescence microscopy (d) reveals that adipocytes were divided asymmetrically (top) or released large lipid droplet (bottom), generating DFAT cells. Bar represents 50 μm. Morphology of adipocytes on ceiling culture day 7 shows that DFAT cells proliferated and formed colony (e). Bar represents 100 μm. DFAT cells demonstrated multilineage differentiation potential when cultured under appropriate culture conditions (f to i). Cells stained positive for lipid vacuole with AdipoRed (f), alkaline phosphatase activity (g), alizarin red S (h) and aggrecan (i), indicating adipogenic (f), osteogenic (g and h) and chondrogenic (i) differentiation. Bar represents 100 μm. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 3 DFAT cells differentiated into smooth muscle-like cells in response to low FCS concentration. Real-time RT-PCR of mature adipocytes (MA) and DFAT shows that DFAT cells expressed ASMA mRNA before SMC differentiation induction (a). Values represent mean ± SD of triplicate dishes. Asterisk indicates Mann-Whitney U test p <0.05 vs mature adipocytes. Human DFAT cells were grown in growth medium of DMEM with 20% FCS for 2 days and cultured for 7 days in DMEM containing indicated concentrations of FCS (b to g). Cells were immunostained for ASMA and nuclei were stained with Hoechst 33342. Photomicrographs show cells cultured in DMEM containing 20% FCS before induction (b) and after culture for 7 days in DMEM containing 1% (c), 3% (d), 5% (e) and 10% FCS (f). Bar represents 50 μm. Percent of ASMA positive cells was quantified (g). Most efficient induction was observed when cells were cultured in DMEM containing 5% FCS. Values represent mean ± SD of triplicate dishes. Asterisk indicates Mann-Whitney U test p <0.05 vs before induction. Similar results were obtained in 3 individual experiments. After induction with DMEM containing 5% FCS cells were immunostained for ASMA and β-actin (h) or ASMA and vinculin (i). Bar represents 50 μm. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 4 TGF-β1 effect on smooth muscle-like cell differentiation in human DFAT cells grown in growth medium of DMEM containing 20% FCS for 2 days and cultured for 7 days in DMEM containing 5% FCS with indicated TGF-β1 concentrations. Cells were immunostained for ASMA and nuclei were stained with Hoechst 33342. Photomicrographs reveal cells cultured in DMEM containing 5% FCS with 1 (a), 5 (b) and 50 (c) ng/ml TGF-β1. Bar represents 100 μm. Percent of ASMA positive cells was quantified (d). Most efficient induction was observed when cells were cultured in DMEM/5% FCS containing 5 ng/ml TGF-β1. Values represent mean ± SD of triplicate dishes. Asterisk indicates Mann-Whitney U test p <0.05 vs absent TGF-β1. Similar results were obtained in 3 individual experiments. FACS analysis of cells cultured in DMEM containing 5% FCS and 5 ng/ml TGF-β1 (e). The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 5 TGF-β signaling blockade inhibited smooth muscle-like cell differentiation in human DFAT cells pretreated with neutralizing antibody for TGF-β1 and/or specific inhibitor for TGF-β receptor 2 SB431542 and cultured for 7 days in DMEM/5% FCS with or without 5 ng/ml TGF-β1 in presence of neutralizing antibody and/or SB431542. Cells were immunostained for ASMA and nuclei were stained with Hoechst 33342. Percent of ASMA positive cells was quantified. Values represent mean ± SD of triplicate dishes. −, negative. +, positive. Asterisk indicates Mann-Whitney U test p <0.05. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 6 Real-time RT-PCR analysis of human DFAT cells during SMC differentiation culture. Cells were cultured for 7 days with DMEM containing 5% FCS and 5 ng/ml TGF-β1. Total RNA was extracted at indicated time points and real-time RT-PCR was performed. ASMA and SM-MHC mRNA expression increased on day 3 of induction culture but PPARγ expression decreased during culture. Values represent mean ± SD of triplicate dishes. Asterisk indicates Mann-Whitney U test p <0.05. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions

Figure 7 DFAT cell transplantation promoted smooth muscle regeneration in mouse cryo-injured bladder model. GFP-DFAT cells or saline was injected into mouse bladder tissue injured by chilled aluminum rod. Comparison of normal uninjured and cryo-injured bladder tissue 7 days after injury showed that smooth muscle fiber disappeared in injured tissue (a). H&E, bar represents 200 μm. Immunostaining of cryo-injured bladder tissue 30 days after DFAT cell transplantation or saline injection reveals distribution of GFP positive DFAT cells at injured area (b) and number of DFAT cells expressing ASMA (b and c). Bar represents 50 (b) and 10 (c) μm. Quantification of ASMA positive area in injured bladder tissue sections demonstrates significantly larger SMA positive area in 5 DFAT transplanted (D) than in 5 saline injected (S) control mice (d). Bars represent mean ± SD. Asterisk indicates Mann-Whitney U test p <0.05. The Journal of Urology 2009 182, 355-365DOI: (10.1016/j.juro.2009.02.103) Copyright © 2009 American Urological Association Terms and Conditions