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International Journal of Urology (2014)

doi: 10.1111/iju.12585

Original Article

Mesenchymal stem cell therapy in treatment of erectile dysfunction: Autologous or allogeneic cell sources? Naside Mangir, Cem Akbal, Tufan Tarcan, Ferruh Simsek and Levent Turkeri Department of Urology, Marmara University School of Medicine, Istanbul, Turkey

Abbreviations & Acronyms ADSC = adipose-derived mesenchymal stem cells BSA = bovine serum albumin CD = cluster of differentiation CN = cavernous nerve ED = erectile dysfunction ICP = intracavernosal pressure ICPmax = maximum intracavernosal pressure MAP = mean arterial pressure MHC = major histocompatibility complex MSC = mesenchymal stem cells nNOS = neuronal nitric oxide synthase PBS = phosphate-buffered saline RT–PCR = reverse transcription polymerase chain reaction

Objective: To compare the efficacy of intracavernosal injection of autologous and allogeneic mesenchymal stem cells as potential treatment of erectile dysfunction in an experimental rat model. Methods: Mesenchymal stem cells were isolated from rat paratesticular fat tissue. Bilateral cavernous nerve injury was carried out followed by immediate intracavernosal injection of either autologous or allogeneic mesenchymal stem cells or mesenchymal stem cell lysates. One month after injection, erectile function was evaluated by means of intracavernosal pressure measurement. All rats were eventually killed, and penile tissues were taken for immunhistochemical and molecular investigation. Results: A total of 36 Sprague–Dawley rats were used. The mean maximum intracavernosal pressure in the sham-operated, autologous and allogeneic mesenchymal stem cell injection groups were significantly better compared with the vehicle injection group (80.5 [3.56], 71.1 [2.9] and 69.2 [3.2] vs 40.33 [4.4], respectively). Mean maximum intracavernosal pressure to mean arterial pressure ratios in the autologous and allogeneic mesenchymal stem cell and mesenchymal stem cell lysate injection groups were not significantly different. Conclusions: Intracavernosal injection of both autologous or allogeneic mesenchymal stem cells improve erectile functions in a rat model of cavernous nerve injury. Allogeneic mesenchymal stem cells might provide clinicians with ready to use, standardized and, in certain cases, more effective products. More studies focusing on long-term immunological aspects of allogeneic mesenchymal stem cells are required.

Correspondence: Naside Mangir M.D., Department of Urology, Marmara University School of Medicine, Fevzi Çakmak M. Mimar Sinan C. no. 41, 34899 Pendik/Istanbul, Turkey. Email: [email protected]

Although nerve regeneration strategies for treatment of neurogenic ED are continuing to evolve, cell-based therapies are emerging as another promising treatment option.1 It has been shown that MSC injection into corpus cavernosum improves erectile functions in diabetic2 and hyperlipidemic3 rat models, as well as in neurogenic ED models.4 Human MSC have been isolated from a large number of adult tissues (bone marrow, adipose tissue, skeletal muscle, etc.); and adipose tissue, being a rich and easily obtainable source of MSC, has been of particular interest in treatment of ED.5 By definition MSC are capable of self renewal and differentiation into various phenotypes,6 but they also exert immunomodulatory, proangiogenic, antiapoptotic, antifibrotic and anti-inflammatory effects mainly through secretion of bioactive trophic factors.7,8 MSC express low levels of MHC class 1, and do not express MHC class 2 molecules, thus they are minimally (or not at all) immunogenic.9 So far, this has led to the use of both allogeneic and autologous sources of MSC in various preclinical and clinical studies, both of which showed promising efficacy and safety data.10 The use of allogeneic MSC in medicine has been triggered by some limitations related to the use of autologous MSC in certain clinical situations where there is no time to isolate and expand patients’ own cells ex vivo (e.g. acute myocardial infarction and stroke),11 and when there is a potential of decreased effectiveness of MSC12 depending on patient status and age.13 Some of these limitations could also be valid for urological applications of MSC. Undoubtedly, autologous transplantation of MSC has a clear advantage in terms of safety, as there is always a risk of re-expression of MHC after interaction with the microenvironment,9 and also there is always some risk of contamination by unknown pathogens.14

Received 8 May 2014; accepted 1 July 2014.

© 2014 The Japanese Urological Association

Key words: adipose derived, allogeneic, autologous, mesenchymal stem cells, neurogenic erectile dysfunction.

Introduction

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The aim of the present study was to compare the efficacy of intracavernousal injection of autologous (self) and allogeneic (not self) ADSC in a rat model of neurogenic ED.

Methods Study design A total of 36 male Sprague–Dawley rats (aged 18–20 weeks) were randomly divided into six groups. The animals were maintained on a 12-h light/dark cycle, and had access to standard rat chow and water ad libitum. All rats underwent bilateral paratesticular fat tissue excision at the beginning of the study to isolate ADSC. Four weeks later, sham surgery or bilateral CN injury was carried out in all animals, and PBS, ADSC or ADSC lysates were injected into the corpus cavernosum of rats. Another 4 weeks after the nerve injury and treatment, all animals underwent erectile function evaluation by means of ICP measurement, after which they were killed and penile tissues were harvested for histological examination. All experiments were approved by Institutional Animal Care and Use Committee at Marmara University, Istanbul.

ADSC culture Under anesthesia, all rats underwent paratesticular fat tissue excision. Tissues were used immediately for ADSC isolation and kept in cold PBS until used. All fat tissue was processed in our laboratory according to a standard protocol, which was constructed according to previously described studies with minor modifications.15,16 Briefly, collagenase digestion was carried out with 0.01% Collagenase type 1A (Sigma Aldrich, St. Louis, MO, USA) after tissue was minced. Collagenase was neutralized by media, and filtered through a 70-μm cell strainer. The suspension was then centrifuged at 500 g for 10 min at room temperature. The supernatant was removed, and the pellet was treated with 0.1 mL of 1X RBC lysis buffer (eBioscience, San Diego, CA, USA) for 10 min at room temperature. Cells were washed with PBS and centrifuged at 1000 g for 10 min. Then the precipitate was plated onto T25 tissue culture dishes in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin streptomycin (Invitrogen), and kept at 37°C in a 5% CO2 humidified incubator. At day 4 of cell culture, cells were washed with PBS to remove non-adherent cells. After reaching 80% confluence, cells were passaged and used in the experiment at passages 2–5.

Characterization of ADSC Multilineage differentiation capacity At the end of passage 2, ADSC were cultured on slide flasks (Nunc-Fisher Scientific, Loughborough, UK) for 2–3 weeks for osteogenic and adipogenic differentiation. For osteogenic and adipogenic induction, STEMPRO Osteogenesis and Adipogenesis Differentiation Kits (Invitrogen) were used, respectively.

Flow-cytometric analysis At the end of passage 2, 1 × 105 ADSC were harvested by trypsinization and incubated on ice with 1% BSA in PBS con2

Fig. 1 Photograph showing rat major pelvic ganglion (dashed arrow) and CN (arrow) lying on the posterolateral wall of the rat prostate.

taining conjugated antibodies against the following cell surface markers for 2 h: fluorescein isothiocyanate anti-rat CD44 (Biolegend, San Diego, CA, USA), phycoerythrin anti-rat CD11b (Biolegend), fluorescein isothiocyanate anti-rat CD45 (Biolegend), FITC anti-rat CD90 (Biolegend) and phycoerythrin anti-rat CD34 (Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS three times, ADSC were analyzed using fluorescence-activated cell sorting (cell analyzer; BD Biosciences, San Jose, CA, USA).

Cavernous nerve injury Under anesthesia, a lower abdominal incision was made. The bladder and prostate were exposed. Major pelvic ganglia and the CN were exposed on both sides of the prostate (Fig. 1). In the sham group, the abdomen was then closed without further intervention. In the treatment and vehicle groups, bilateral CN injury was carried out as previously described.17 Briefly, a vascular Bulldog clamp was applied to each CN at a position that was 5 mm distal to major pelvic ganglia. It was applied for 30 s, removed for 30 s and applied for another 30 s. After bilateral CN injury, the lower abdominal incision was extended to the root of the penis, except in the sham group. The penile shaft was exposed, and an elastic band was applied to the root of the penis just before intracavernous injection of PBS, autologous or allogeneic ADSC or ADSC lysates. The elastic band was removed 2 min after injection, and the injection site was compressed for 1 min. The total volume of injection was 200 μL in all groups, and injection was carried out by a 26-G needle. In the autologous or allogeneic ADSC or ADSC lysate groups, 1 million cells or lysates of 1 million cells were suspended in 200 μL of PBS.

Preperation of ADSC lysate ADSC lysate was prepared by osmotic rupture of cells in a centrifuge tube with 200 μL of deionized water for 30 min. Then three freeze thaw cycles using a −80°C freezer were carried out to further dissociate the lyzed cell sediments. An absence of viable cells was confirmed by trypan blue staining of © 2014 The Japanese Urological Association

MSC in treatment of ED

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CD45 FITC-A Population Fig. 2 Flow cytometric analysis showed that cultured MSC expressed CD90 in 89% and CD44 in 92.6%, but did not express CD34 in 99.9%, CD11b in 98.6% and CD45 in 97.7%.

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lysate samples. After centrifugation at 1000 g, the supernatant was collected for ADSC lysate injection.

ICP measurement One month after ADSC injection, ICP measurement was carried out in all rats. A transverse neck incision was made, and the left internal carotid artery was isolated. It was then cannulated with a heparinized polyethylene-50 tube, which was connected to a pressure transducer and an amplifier unit (COMMAT Pharmacology & Physiology Instruments, Ankara, Turkey). The amplifier was connected to a data acquisition module (MP 35 data acquisition system, Ankara, Turkey). This allowed the MAP to be recorded on a computer with the Biopac Student Lab PRO recording software (Biopac Systems, Gotela, CA, USA). Each animal’s penis was circumcised and denuded of skin. The ICP was measured by inserting a 24-G needle into the right crus of the penis. This needle was connected to a transducer through a heparinized polyethylene-50 tube in a similar fashion as that of the MAP measurement. The CN was located bilaterally as carried out during the initial surgery. A stainless steel bipolar electrode with parallel hooks (1-mm apart) was placed around the nerve just distal to the ganglion, but proximal to the point of the nerve injury. The electrode cable was attached to a STPTO2-A stimulator (COMMAT Pharmacology & Physiology Instruments) and stimulated (i.e. with the parameters of 1.5 mA, 20 Hz, pulse width 5, 35 ms delay, at 7.5 V) for 60 s each. Both nerves were stimulated in this manner. The maximal ICP/MAP ratio was calculated by dividing the highest ICP recorded during stimulation by the corresponding MAP and presented as a percentage.

RT–PCR For quantitative analysis of nNOS mRNA expression from the corpus cavernosum of rats, RT–PCR was carried out by monitoring the increase in fluorescence of the SYBR Green dye (Light Cycler-DNA Master SYBR Green I; Roche, Indianapolis, IN, USA) in real time using a Light Cycler quantitative PCR system (Roche) and a commercial cytokine control kit (Light Cycler Control Kit RNA; Roche). In brief, all RNA obtained using a “High Pure RNA Isolation Kit (Roche)” were denatured and reverse transcribed using random hexanucleotides according to the instructions from the manufacturer (Roche). cDNA were analyzed by electrophoresis on 2% agarose gels and were © 2014 The Japanese Urological Association

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% Parent 0.1 3.1 7.7 89.0

visualized using ethidium bromide staining. Quantitative RT–PCR was carried out with published primers for nNOS, as follows: ACCTGAAGAGCACACTGGAAAC (sense) and GATGGCCGACCTGAGATTC (antisense).18

Histology and immunohistochemistry Tissue samples were fixed for 12 h in a paraformaldehyde-PBS solution (i.e. 4% [w/v] paraformaldehyde with 100 mmol/L phosphate buffer [pH 7.4]) at 4°C, dehydrated in an ethanol series and xylene, and embedded in paraffin wax. Then, 5-μm sections from the mid-shaft of the penis were dewaxed and rehydrated through an alcohol series and finally in PBS. The sections were incubated in 3% hydrogen peroxide and washed in PBS. Non-specific protein binding was blocked using a 3% BSA–PBS solution. Sections were incubated overnight in a humidified chamber at 4°C with nNOS antibody (Novus Biologicals, Littleton, CO, USA). Detection was carried out using the streptavidin-peroxidase-diaminobenzidine system.

Statistical analysis The results were analyzed using SPSS version 17.0 (SPSS, Chicago, IL, USA). Treatment and sham groups were compared with a control group using the Mann–Whitney U-test. A P-value of

Mesenchymal stem cell therapy in treatment of erectile dysfunction: autologous or allogeneic cell sources?

To compare the efficacy of intracavernosal injection of autologous and allogeneic mesenchymal stem cells as potential treatment of erectile dysfunctio...
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