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259 INTRACAVERNOSAL STEM CELL THERAPY COMBINED WITH ORAL TADALAFIL ADMINISTRATION PRESERVES ERECTILE FUNCTION AFTER CAVERNOUS NERVE INJURY IN RATS
e106
THE JOURNAL OF UROLOGY姞
injection site 1 week later. . Follow up studies are needed to determine the sequence of events contributing to distribution of MSCs at 1 week. LavaCell® fluorophore dye keeps the proliferation characteristics of hMSCs almost intact. Combination of techniques is needed to effectively track cells.
Source of Funding: This work was supported by University Hospitals of Cleveland Family Medicine Award #P0089 (PI: Hijaz, Adonis) and NIH/NIDDK (5K08DK090134) awarded to A. Hijaz.
258 CULTURING HUMAN MUSCLE PRECURSOR CELLS (MPCS) WITH XENO-FREE MEDIUM: GMP AND CLINICAL APPLICATION PREPARATION Fahd Azzabi, Souzan Salemi, Ria Tauscher, Zurich, Switzerland; Katharina Schallmoser, Dirk Strunk, Graz, Austria; Tullio Sulser, Daniel Eberli*, Zurich, Switzerland INTRODUCTION AND OBJECTIVES: Autologous cell injections are promising therapies for many diseases including urinary incontinence, where sphincter muscle damage provokes urine leakage and significantly reduces life quality. Several animal models showed that injection of precursor or stem cells leaded to the regeneration of sphincter muscle tissue. Currently, all types of cell used in these preclinical and clinical studies are expanded in xenogenic media containing fetal bovine serum (FBS). However, before precursors or stem cells can be applied clinically it is mandatory to reduce the potential immunogenic reaction and infection risk by removing any xenogenic contaminants which is required by the new regulations and good manufacturing practice for cell therapies. METHODS: In this research human MPCs were expanded in xeno-free medium using pooled human platelet lysates (pHPL) or pooled human Serum (HS). We assessed the expansion potential, the differentiation by FACS, western blot and the fibre formation in vitro. Further, we assed the in vivo muscle tissue formation after injection, specific muscle expression by western blot and contractility by organ bath. Cells grown in standard FBS medium served as controls. RESULTS: Our results clearly demonstrate that HS is no substitute for FBS: MPCs isolated from human biopsies were not able to expand. Using pHPL we were able to expand the MPCs while maintaining the myo-phenotype as demonstrated by FACS, WB and IHC for MyoD, desmin and MHC. Expanded MPCs gave rise to contractile muscle tissue after injection in vivo, comparable to tissues grown using cells expanded in FBS. CONCLUSIONS: In conclusion, our results show that pHPL is a suitable substitute for FBS allowing the MPCs to form contractile muscle after injection. pHPL may be used to facilitate the preparation for clinical applications. Source of Funding: The Swiss National Science Foundation (SNSF) University Hospital Zurich
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
259 INTRACAVERNOSAL STEM CELL THERAPY COMBINED WITH ORAL TADALAFIL ADMINISTRATION PRESERVES ERECTILE FUNCTION AFTER CAVERNOUS NERVE INJURY IN RATS Juan I. Martínez-Salamanca*, Mercedes Zurita, Claudio Martínez-Ballesteros, Eduardo Martínez-Salamanca, Argentina Fernández, Pedro Cuevas, Jesús Vaquero, Joaquín Carballido, Madrid, Spain; Carla Costa Costa, Porto, Portugal; Javier Angulo, Madrid, Spain INTRODUCTION AND OBJECTIVES: Since the treatment of erectile dysfunction secondary to radical prostatectomy still remains as an outstanding challenge, this work was aimed to evaluate the efficacy of dual targeted strategy to promote cavernosal tissue preservation using systemic PDE5 inhibition and local stem-cell therapy in a rat model of cavernous nerve (CN) injury. METHODS: Bilateral CN crush injury (BCNI) was produced in anesthetized male Wistar rats. Tadalafil was orally administered at 5 mg/kg/day dose, Bone marrow-derived mesenchymal stem cells (BMSC) were obtained from donor rats and in vitro expanded and characterized before intracavernosal implantation by means of an automated microinjector pump. Ex vivo function of corpus cavernosum (CC) and In vivo intracavernosal pressure (ICP) responses to CN electrical stimulation (CNES) were evaluated 4 weeks after BCNI. Cavernosal apoptosis was determined by TUNEL assays. RESULTS: In CC from BCNI rats, neurogenic contractions driven by adrenergic system were enhanced while nitrergic relaxations were markedly impaired. Oral tadalafil, BMSC or combined strategy improved neurogenic relaxations and prevented cavernosal apoptosis (Table). Relaxations of CC to carbachol or sodium nitrroprusside were not altered by BCNI or any of the treatments. BCNI resulted in marked reduction of erectile responses to CNES which were partially recovered by treatment with tadalafil or by intracavernosal implantation of BMSC. The complete recovery of erectile function was only achieved when both treatments were combined (Table). CONCLUSIONS: BCNI did not impair endothelial or smooth muscle relaxation of CC but selectively blunted nitrergic relaxation which was improved by oral tadalafil and intracavernosal BMSC. Erectile function was recovered only when both therapies were combined. These results support the use of dual therapeutic approaches, cell therapy and oral PDE5 inhibitors, for recovering erectile function in patients undergoing radical prostatectomy.
sham 9
BCNI 9
BCNI⫹ TAD 5
BCNI⫹ BMSC 6
BCNI⫹ TAD⫹ BMSC 5
Emax adrenergic (% of K)
102.9Â⫾34.2
214.5Â⫾25.0*
117.8Â⫾38.2†
218.8Â⫾47.3
157.5Â⫾20.3
Emax nitrergic (%)
64.4Â⫾10.0
20.4Â⫾5.8*
36.3Â⫾19.1
39.3Â⫾9.9
45.4⫾6.5†
Apoptotic cells/field
1.6Â⫾0.4
15.8Â⫾0.8***
4.0Â⫾0.7*†††
5.5Â⫾0.8**†††
4.6Â⫾1.0*†††
100.0Â⫾6.3
45.9Â⫾5.7***
76.3⫾11.1†
62.2⫾14.8*
92.4Â⫾4.8††
n
max ICP (% of sham)
†
*p⬍ 0.05, **p⬍0.01, ***p⬍0.001 vs. sham, p⬍0.05, BCNI.
††
p⬍0.01,
†††
p⬍0.001 vs.
Source of Funding: None
260 THE ROLE OF MIR-135A VIA REGULATION OF FOXO1 GENE EXPRESSION IN MAINTENANCE OF SPERMATOGONIAL STEM CELLS Yoshinobu Moritoki*, Nagoya, Japan; Yoshiyuki Kojima, Fukushima, Japan; Kentaro Mizuno, Yasuhiro Shibata, Hideyuki Kamisawa, Makoto Imura, Satoshi Kurokawa, Akihiro Nakane, Toshiki Kato, Tetsuji Maruyama, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Spermatogonial stem cells (SSCs) function as a resource for spermatogenesis throughout
THE JOURNAL OF UROLOGY姞
injection site 1 week later. . Follow up studies are needed to determine the sequence of events contributing to distribution of MSCs at 1 week. LavaCell® fluorophore dye keeps the proliferation characteristics of hMSCs almost intact. Combination of techniques is needed to effectively track cells.
Source of Funding: This work was supported by University Hospitals of Cleveland Family Medicine Award #P0089 (PI: Hijaz, Adonis) and NIH/NIDDK (5K08DK090134) awarded to A. Hijaz.
258 CULTURING HUMAN MUSCLE PRECURSOR CELLS (MPCS) WITH XENO-FREE MEDIUM: GMP AND CLINICAL APPLICATION PREPARATION Fahd Azzabi, Souzan Salemi, Ria Tauscher, Zurich, Switzerland; Katharina Schallmoser, Dirk Strunk, Graz, Austria; Tullio Sulser, Daniel Eberli*, Zurich, Switzerland INTRODUCTION AND OBJECTIVES: Autologous cell injections are promising therapies for many diseases including urinary incontinence, where sphincter muscle damage provokes urine leakage and significantly reduces life quality. Several animal models showed that injection of precursor or stem cells leaded to the regeneration of sphincter muscle tissue. Currently, all types of cell used in these preclinical and clinical studies are expanded in xenogenic media containing fetal bovine serum (FBS). However, before precursors or stem cells can be applied clinically it is mandatory to reduce the potential immunogenic reaction and infection risk by removing any xenogenic contaminants which is required by the new regulations and good manufacturing practice for cell therapies. METHODS: In this research human MPCs were expanded in xeno-free medium using pooled human platelet lysates (pHPL) or pooled human Serum (HS). We assessed the expansion potential, the differentiation by FACS, western blot and the fibre formation in vitro. Further, we assed the in vivo muscle tissue formation after injection, specific muscle expression by western blot and contractility by organ bath. Cells grown in standard FBS medium served as controls. RESULTS: Our results clearly demonstrate that HS is no substitute for FBS: MPCs isolated from human biopsies were not able to expand. Using pHPL we were able to expand the MPCs while maintaining the myo-phenotype as demonstrated by FACS, WB and IHC for MyoD, desmin and MHC. Expanded MPCs gave rise to contractile muscle tissue after injection in vivo, comparable to tissues grown using cells expanded in FBS. CONCLUSIONS: In conclusion, our results show that pHPL is a suitable substitute for FBS allowing the MPCs to form contractile muscle after injection. pHPL may be used to facilitate the preparation for clinical applications. Source of Funding: The Swiss National Science Foundation (SNSF) University Hospital Zurich
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
259 INTRACAVERNOSAL STEM CELL THERAPY COMBINED WITH ORAL TADALAFIL ADMINISTRATION PRESERVES ERECTILE FUNCTION AFTER CAVERNOUS NERVE INJURY IN RATS Juan I. Martínez-Salamanca*, Mercedes Zurita, Claudio Martínez-Ballesteros, Eduardo Martínez-Salamanca, Argentina Fernández, Pedro Cuevas, Jesús Vaquero, Joaquín Carballido, Madrid, Spain; Carla Costa Costa, Porto, Portugal; Javier Angulo, Madrid, Spain INTRODUCTION AND OBJECTIVES: Since the treatment of erectile dysfunction secondary to radical prostatectomy still remains as an outstanding challenge, this work was aimed to evaluate the efficacy of dual targeted strategy to promote cavernosal tissue preservation using systemic PDE5 inhibition and local stem-cell therapy in a rat model of cavernous nerve (CN) injury. METHODS: Bilateral CN crush injury (BCNI) was produced in anesthetized male Wistar rats. Tadalafil was orally administered at 5 mg/kg/day dose, Bone marrow-derived mesenchymal stem cells (BMSC) were obtained from donor rats and in vitro expanded and characterized before intracavernosal implantation by means of an automated microinjector pump. Ex vivo function of corpus cavernosum (CC) and In vivo intracavernosal pressure (ICP) responses to CN electrical stimulation (CNES) were evaluated 4 weeks after BCNI. Cavernosal apoptosis was determined by TUNEL assays. RESULTS: In CC from BCNI rats, neurogenic contractions driven by adrenergic system were enhanced while nitrergic relaxations were markedly impaired. Oral tadalafil, BMSC or combined strategy improved neurogenic relaxations and prevented cavernosal apoptosis (Table). Relaxations of CC to carbachol or sodium nitrroprusside were not altered by BCNI or any of the treatments. BCNI resulted in marked reduction of erectile responses to CNES which were partially recovered by treatment with tadalafil or by intracavernosal implantation of BMSC. The complete recovery of erectile function was only achieved when both treatments were combined (Table). CONCLUSIONS: BCNI did not impair endothelial or smooth muscle relaxation of CC but selectively blunted nitrergic relaxation which was improved by oral tadalafil and intracavernosal BMSC. Erectile function was recovered only when both therapies were combined. These results support the use of dual therapeutic approaches, cell therapy and oral PDE5 inhibitors, for recovering erectile function in patients undergoing radical prostatectomy.
sham 9
BCNI 9
BCNI⫹ TAD 5
BCNI⫹ BMSC 6
BCNI⫹ TAD⫹ BMSC 5
Emax adrenergic (% of K)
102.9Â⫾34.2
214.5Â⫾25.0*
117.8Â⫾38.2†
218.8Â⫾47.3
157.5Â⫾20.3
Emax nitrergic (%)
64.4Â⫾10.0
20.4Â⫾5.8*
36.3Â⫾19.1
39.3Â⫾9.9
45.4⫾6.5†
Apoptotic cells/field
1.6Â⫾0.4
15.8Â⫾0.8***
4.0Â⫾0.7*†††
5.5Â⫾0.8**†††
4.6Â⫾1.0*†††
100.0Â⫾6.3
45.9Â⫾5.7***
76.3⫾11.1†
62.2⫾14.8*
92.4Â⫾4.8††
n
max ICP (% of sham)
†
*p⬍ 0.05, **p⬍0.01, ***p⬍0.001 vs. sham, p⬍0.05, BCNI.
††
p⬍0.01,
†††
p⬍0.001 vs.
Source of Funding: None
260 THE ROLE OF MIR-135A VIA REGULATION OF FOXO1 GENE EXPRESSION IN MAINTENANCE OF SPERMATOGONIAL STEM CELLS Yoshinobu Moritoki*, Nagoya, Japan; Yoshiyuki Kojima, Fukushima, Japan; Kentaro Mizuno, Yasuhiro Shibata, Hideyuki Kamisawa, Makoto Imura, Satoshi Kurokawa, Akihiro Nakane, Toshiki Kato, Tetsuji Maruyama, Yutaro Hayashi, Kenjiro Kohri, Nagoya, Japan INTRODUCTION AND OBJECTIVES: Spermatogonial stem cells (SSCs) function as a resource for spermatogenesis throughout