Biotechnol Lett DOI 10.1007/s10529-015-1816-2

ORIGINAL RESEARCH PAPER

Therapeutic potential of human umbilical cord blood mesenchymal stem cells on erectile function in rats with cavernous nerve injury Jian-Qiang Zhu • Hong-Kai Lu • Zhi-Qiang Cui • Yong-Chuan Wang • Yong-Hui Li • Weixin Zhao • Qiang Fu • Yue-Min Xu • Yong Xu • Lu-Jie Song

Received: 12 January 2015 / Accepted: 12 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Objective To evaluate the therapeutic potential of human umbilical cord blood mesenchymal stem cells (hUCBMSCs) on promoting erectile function in a rat model of bilateral cavernous nerve (CN) crush injury. Results Fifty male Sprague–Dawley rats were randomly assigned to sham ? PBS group (n = 10), BCNI (bilateral cavernous nerve crush injury) ? PBS group

J.-Q. Zhu  Y. Xu Department of Urology, Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, China J.-Q. Zhu  Z.-Q. Cui  Y.-H. Li  Q. Fu  Y.-M. Xu  L.-J. Song (&) Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, China e-mail: [email protected] H.-K. Lu Department of Urology, Weifang People’s Hospital, Shandong 261042, China

(n = 10), BCNI ? hUCBMSCs group (n = 30). At day 28 (n = 10) post-surgery, erectile function was examined and histological specimens were harvested. Compared with BCNI ? PBS group, hUCBMSC intracavernous injection treatment significantly increased the mean ratio of ICP/MAP, nNOS-positive nerve fibers in the dorsal penile nerve, smooth muscle content, and smooth muscle to collagen ratio in the corpus cavernousum. Electron microscopy revealed few CN and major pelvic ganglion (MPG) lesions in the BCNI ? hUCBMSCs group. Injected hUCBMSCs were localized to the sinusoid endothelium of the penis and MPG on day 1, 3, 7, and 28 post-intracavernous injection. Conclusion hUCBMSCs intracavernous injection treatment improves erectile function by inhibiting corpus cavernosum fibrosis and exerting neuroregenerative effects on cell bodies of injured nerves at MPG in a BCNI rat model. Keywords Animal model  Bilateral cavernous nerve crush injury (BCNI)  Erectile dysfunction (ED)  Human umbilical cord blood mesenchymal stem cells (hUCBMSCs)  Radical prostatectomy

Y.-C. Wang Department of Urology, Weifang Traditional Chinese Medicine Hospital, Shandong 261041, China

Introduction W. Zhao Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston Salem, NC 27157, USA

Prostate cancer is a common cancer in men in western countries. It has rapidly increased in Asia in recent

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years. About 94 % prostate cancers are clinically localized, and radical prostatectomy (RP) is often the preferred choice (Akaza 2010). However, erectile dysfunction (ED) is a major complication of RP. Although advancing anatomical knowledge, equipment, and surgical techniques have increased the restoration of erectile function by preserving the integrity of the cavernous nerve (CN), about 20 % patients still experience ED following RP (Ficarra et al. 2012; Salonia et al. 2012a, b). CNs course along the posterolateral aspects of the prostate in humans, and the main function of CNs is to control penile erectile function. CNs can be injured by direct crush or stretching during surgery even when nerve-sparing techniques are employed (Piao et al. 2012). CN injury can cause ED as a result of penile hypoxia, smooth muscle apoptosis, fibrosis, and veno-occlusive dysfunction (Magheli and Burnett 2009). Intracavernosal self-injection or intraurethral administration of alprostadil, vacuum erection devices, and phosphodiesterase type 5 inhibitor (PDE5-I) therapy are the current treatment options for postoperative ED, but they are only partially effective (Salonia et al. 2012a, b). Penile prosthesis implantation is the last choice for ED patients at present (Falcone et al. 2013). Stem cells, such as bone marrow stem cells and adipose-derived stem cells, have the potential to restore erectile function by differentiating into or encouraging regeneration of endogenous endothelial cells and smooth muscle cells in the penile corpora cavernosa. The therapeutic efficacy of stem cells for CN injury is due to stem cell trafficking to the major pelvic ganglion (MPG) Lin et al. (2012). Human umbilical cord blood mesenchymal stem cells (hUCBMSCs) differentiate into neural cells and promote nerve regeneration (Yang et al. 2013; Divya et al. 2012). hUCBMSCs attenuate spinal cord injury and cerebral lesion by differentiating directly into neural cells, as well as their ability to secrete various neurotrophins and regulate immune and inflammatory reactions (Seo et al. 2011; Greggio et al. 2014). But, it is still unknown if hUCBMSCs could promote erectile function after CN injury. Thus, this study was designed to evaluate the therapeutic potential of hUCBMSCs on promoting erectile function in a rat model of CN crush injury.

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Materials and methods Isolation, culture, and introduction of hUCBMSCs This study had approval from the Institutional Review Board of the Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University. hUCBMSCs were generated according to a previously described method (De Schauwer et al. 2011). Briefly, human full-term umbilical cord blood (UCB) samples were obtained from informed and consenting donors at the Department of Obstetrics and Gynecology, Shanghai Sixth People’s Hospital. All samples were collected in a blood collection bag containing citrate phosphate dextrose as an anticoagulant and processed within 24 h. A fraction of mononuclear cells (MNC) was separated by centrifugation in a Ficoll-Paque Plus gradient (Amersham Biosciences), washed with Hank’s balanced salt solution (HBSS), and resuspended in low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco), 15 % (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, and 1 % antibiotics/antimycotics comprising 100 U penicillin/ml, 100 ug streptomycin/ml, and 25 ug amphotericin B/ml. After 7 days, non-adherent cells were discarded, and adherent cells were cultured with two medium changes per week. FACS To characterize cell surface expression of marker proteins, hUCBMSCs were labeled with the following anti-human antibodies conjugated with phycoerythrin (PE-CY5) or fluorescein isothiocyanate (FITC): CD29-PE-CY5, CD45-PE-CY5, CD34-FITC, CD44FITC, and CD105-FITC. About 10,000 cells were measured using a flow cytometer, and the results were analyzed with Cell Quest software. Similar results were obtained from three independent experiments. Examination of multiple induced potential of hUCBMSCs in vitro hUCBMSCs induced into neural cells Cells were plated at 5 9 103 in a culture plate and cultured with an appropriate amount of pre-culture medium for neural induced differentiation (LDMEM ? 20 % v/v FBS). On the 2 day, the cell

Biotechnol Lett

medium was changed to pre-induction medium for neural induced differentiation (L-DMEM ? 10 ng basic fibroblast growth factor/ml ? 20 % v/v FBS). After 24 h, the cells were cultured with induction medium for neural-induced differentiation (L-DMEM ? 200 lM butylated hydroxyanisole) for 6 h. Cells were fixed, cryopreserved, and expression of glial fibrillary acidic protein (GFAP), nestin, and neurofilament (NF) was detected by immunofluorescent staining. Induced hUCB-MSCs into smooth muscle cells and identification Cells were plated on a culture plate. When the cells were 80 % confluent, the cells were treated with an appropriate amount of culture solution for muscle cells to induce differentiation (L-DMEM ? 10 % v/v FBS ? 10 uM 5-aza). After 48 h, the cells were treated with a culture solution to induce muscle cell differentiation (L-DMEM ? 10 % v/v FBS). Then, the cells were cultured with maintenance media for 22 days. Finally, the cells were fixed, cryopreserved, and expression of desmin and smooth muscle actin (SMA) were detected by immunofluorescent staining. Animal experiments Fifty 12-week-old, male, Sprague–Dawley rats were assigned to 3 groups. Group 1 received an abdominal incision with intracavernous injection (ICI) of PBS (n = 10). Group 2 received bilateral CN crush injury (BCNI) with an ICI of PBS (n = 10). Group 3 received BCNI with ICI of hUCBMSCs (n = 30). BCNI rat model and hUCBMSCs ICI All rats were anesthetized with sodium pentobarbital (40 mg/kg) by an intraperitoneal injection. An abdominal incision was adopted to expose the urinary bladder and prostate gland. The animals in Group 1 underwent a lower abdominal incision and no additional surgical manipulation was performed on major pelvic ganglions (MPGs and CNs. In Groups 2 and 3, bilateral CNs were compressed 5 mm from the origin of CN at the MPG using mosquito forceps (12.5 cm, the first teeth were buckled) for a duration of 1 min (Fig. 2I). The abdomen was closed and the skin overlying the penis was incised to expose it. Prior to

ICI, an elastic band was placed at the base of the penis to block blood flow so that more hUCBMSCs could adhere to the cavernous sinus of the penis. In Groups 1 and 2, 200 ll PBS was injected into both corpora cavernosums at the mid-penile level. In Group 3, 2 9 106 hUCBMSCs (8th passage) diluted in 200 ll PBS were injected into both corpora cavernosums at the mid-penile level. For the purpose of cell tracking, when 70 % confluency was reached, the hUCBMSCs were cultured with 10 lM 5-bromo-2-deoxyuridine (BrdU) for 48 h before ICI. A proper pressure was given at the injection site for 3 min to prevent backflow of treatment suspension. Three minutes after injection, the elastic band was released. Then, the skin overlying the penis was closed in layers. All rats were housed with a 12-h light/dark cycle in a temperaturecontrolled facility and had access to standard rat chow and water during the entire experimental period. Erectile hemodynamic evaluation Four weeks after surgery, ten rats from each group were anesthetized. The CN and MPG lateral to the prostate were exposed and isolated through a repeat lower midline abdominal incision. A butterfly needle (23 gauge) with heparin (250 U/ml) was inserted into the left crus of the penis and connected to a pressure transducer (Utah Medical Products, Midvale) to measure intracavernosal pressure (ICP). A bipolar stainless steel hook electrode connected to an electrical pulse stimulator was used for stimulating CN 1–2 mm proximal to the injury site with stimulus parameters of 1.5 mA, 20 Hz, 0.2 ms pulse width, and duration of 60 s. Mean artery pressure (MAP) was recorded when the CN was stimulated. Immunohistochemistry and Masson’s trichrome staining of penis After ICP evaluation, the rats were sacrificed. The middle part of the penis of each rat was fixed, dehydrated, and embedded in paraffin prior to sectioning, followed by immunohistochemical staining. Tissue sections (5 lm) were cut for histological evaluation of nitric oxide synthase expression (nNOS, 1:200), a-smooth muscle actin expression (a-SMA, 1:200), and Masson’s trichrome staining. Sections stained with nNOS, a-SMA, and Masson’s trichrome

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were evaluated by two independent observers using a fluorescent microscope. BrdU staining of penis and MPG At day 1, 3, 7, and 28 (n = 5) post-surgery, specimens from the BCNI ? hUCBMSCs group were harvested and fixed with 2 % (v/v) formaldehyde and 0.002 % picric acid in 0.1 M PBS for 24 h, followed by immersion in 30 % (w/v) sucrose in PBS overnight at 4 °C. The fixed tissue was then embedded in optimal cutting temperature compound, cut into 5 lm thick sections, and mounted on glass slides. The slides were then placed in 0.3 % H2O2 for 10 min, washed twice in PBS and incubated with 3 % (v/v) horse serum in PBS/0.3 % Triton X-100 for 30 min at room temperature. After draining this solution from the tissue section, the slides were pre-treated with 2 M HCI for 10 min, followed by incubation overnight at 4 °C with rabbit anti-BrdU antibody (BrdU, 1:40). Control tissue sections were similarly prepared, except no primary antibody was added. The tissue was then incubated with secondary antibody conjugated with TRITCconjugated goat anti-rat IgG. Nuclei were stained with 40 ,6-diamidino-2-phenylindole for 5 min, and washed twice in PBS. Stained tissues were examined by fluorescence microscopy.

the nerve in pixels was calculated at a magnification of 9400, and all nerve branches of the dorsal nerve on each slide were included in the analysis. For analysis of smooth muscle content and smooth muscle to collagen ratio, the corpus cavernosum was photographed at a magnification of 9100, and smooth muscle content and the ratio of smooth muscle/collagen within the tunica albuginea was used for statistical analysis. For statistical analysis of the number of BrdU ? hUCBMSCs/field in the penis and MPG, the corpus cavernosum was photographed at a magnification of 9 200. All images were analyzed using ImagePro Plus 5.1 software (Media Cybernetics, Bethesda, MD, USA). Statistical analysis The mean averages of maximum ICP/MAP ratios, nNOS staining, a-SMA staining, and smooth musclecollagen ratios were used to calculate mean ± standard deviation. Differences among groups were evaluated using analysis of variance and post hoc analysis. SPSS 17.0 was used for results analysis, and statistical significance was set at P \ 0.05.

Results

Transmission electron microscopy (TEM)

Identification and introduction of hUCBMSCs

TEM was performed as previously described (Tang et al. 1998). The segment of the distal part of CN from the crush injury and MPG of each rat were clipped by microsurgical scissors and cut into 1 mm3 cubes, then fixed in 2.5 % (v/v) glutaraldehyde for 6 h. All fixed tissues were then post-fixed in 1 % (w/v) osmium tetroxide, dehydrated, and embedded in plastic, sectioned into 1 lm thick sections, and stained with 2 % (w/v) uranyl acetate and 3 % (w/v) lead citrate. Electron microscopy was performed using an H-7500 TEM to observe changes in the CN and MPG.

When observed by an inverted-phase contrast microscope, cells presented with spindle-shaped adherent growth. The majority of expanded cells was positive for CD29 (99.95 %), CD44 (97.2 %), and CD105 (90 %), and were negative for CD45 (0.05 %) and CD34 (0.73 %). (Fig. 1I). Positive results of GFAP, nestin, and NF immunofluorescent staining for neural cells and desmin/ SMA immunofluorescent staining for smooth muscle cells confirmed the induced potential of hUCBMSCs to neural cells and smooth muscle cells. (Fig. 1II).

Digital analysis of sections

Maximal ICP/MAP ratio

For image analysis, three mid-penile tissue sections from each animal were photographed for statistical analysis. For statistical analysis of neural nitric-oxide synthase (nNOS) content, the ratio of the number of nNOS-positive fibers per nerve over the total area of

At day 28 post-surgery, the maximal ICP/MAP was significantly less in the BCNI ? PBS group than in the BCNI ? hUCBMSCs group and sham ? PBS group (31.2 ± 3.5 %, 61.8 ± 4.6 % vs. 81.9 ± 5.5 %; P \ 0.05). Maximal ICP/MAP in the BCNI ? hUCBMSCs

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Fig. 1 I. a Morphology of hUCBMSCs. b–f representative flow cytometry results of hUCBMSCs. The cells staining for the CD marker of CD29-PE-CY5, CD44-FITC, and CD105-FITC are strongly positive, and the CD markers of CD45-PE-CY5 and CD34-FITC are negative; II. hUCBMSCs were induced to

neural cells and muscle cells. (Scale bars for GFAP, nestin, and NF = 20 um; Scale bars for desmin and SMA = 10 um). Representative pictures of GFAP, Nestin, and NF immunofluorescent staining for nerve cells, and desmin and SMA for smooth muscle cells

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Fig. 2 I. The anatomy of MPG and CN. a The MPG and CN are located in the green box (right side); b MPG and CN are seen on the dorsolateral lobes of the prostate (right side); MPG receives inputs of hypogastric nerve and pelvic nerve, and one of the outputs is the CN. c CN crush injury animal model (left side). (DD deferent duct, BL bladder, PR prostate, PE penile, PN pelvic nerve, MPG major pelvic ganglion, CN cavernous nerve);

II. Representative recording of ICP to electrostimulation of CN at 4 weeks; histogram shows ratios of intracavernous pressure/ mean arterial pressure (ICP/MAP), which were detected during CN electrical stimulation for each group. Each bar shows the mean values (±standard deviation) from n = 10 rats per group. (asterisk indicates P \ 0.05)

group was significantly increased compared with the BCNI ? PBS group (P \ 0.05). (Figure 2II).

compared with BCNI ? PBS group model rats, (P \ 0.05) (Fig. 3a–d).

Histological studies nNOS positive nerve fibers in the dorsal penile nerve

a-SMA expression in the corpus cavernousum

At day 28 postsurgery, nNOS expression in the dorsal penile nerve of the BCNI ? PBS group (0.48 ± 0.12 %) and BCNI ? hUCBMSCs group (0.98 ± 0.16 %) were significantly less than the sham ? PBS group (1.44 ± 0.10 %), respectively, (P \ 0.05). The rats treated with hUCBMSC ICI significantly increased nNOS expression in the dorsal penile nerve

a-SMA expression in the corpus cavernosum was significantly decreased in the BCNI ? PBS group (3.35 ± 0.52 %) compared with the sham ? PBS group (5.7 ± 0.7 %), (P \ 0.05). After treatment with hUCBMSC ICI, a-SMA expression significantly increased to 4.7 ± 0.55 % compared with the BCNI ? PBS group, (P \ 0.05) (Fig. 3e–h).

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Fig. 3 a–c Neuronal nitric oxide (nNOS) immunohistostaining in the dorsal penile nerve of each group. (Scale bars = 10 um; red arrows point to nNOS positive neurons); graph d showing result of nitric oxide synthase (nNOS) quantification expressed as number of nNOS-expressing fibers/area of the nerve. e–g asmooth muscle actin (a-SMA) immunohistostaining in the corpus cavernosum tissue of each group (scale bars = 20 um); graph h shows smooth muscle contents as ratio of the area

stained positive for a-SMA to total area within the tunica albuginea. i–k Masson’s trichrome staining of the corpus cavernosum tissue of each group (Scale bars = 20 um; smooth muscle is stained red and collagen fibers are stained blue); graph l shows smooth muscle to collagen ratio within the tunica albuginea. Each bar shows the mean values (±standard deviation) from n = 10 rats per group. (asterisk indicates P \ 0.05)

Smooth muscle/collagen ratio in the corpus cavernousum

hUCBMSCs group model rats compared with BCNI ? PBS group model rats, (P \ 0.05) (Fig. 3i–l).

The average smooth muscle/collagen ratios in the sham ? PBS, BCNI ? PBS, and BCNI ? hUCBMSCs groups were 9.75 ± 1.4 %, 4.6 ± 0.65 % and 7.2 ± 0.9 %, respectively. The average smooth muscle/collagen ratio was significantly increased in the BCNI ?

hUCBMSC tracking On day 1, 3, 7, and 28 post-ICI, hUCBMSCs were visible in the penis and MPG by immunofluorescence staining of BrdU. The numbers of Brdu ? hUCBMSCs

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in the penis per field (9200 magnification) at four time points were 42.2 ± 1.84, 21.2 ± 1.44, 11.2 ± 1.04, and 5.2 ± 0.64, respectively (P \ 0.05). In the MPG, the number of Brdu ? hUCBMSCs per field (9200 magnification) at four time points as 6.6 ± 0.88, 15 ± 0.8, 4.4 ± 0.88, and 1.2 ± 0.32, respectively (P \ 0.05) (Figs. 4, 5). Ultrastructural changes of MPG and CN Electron microscopy identified abundant mitochondrial structural changes in the MPG cells of BCNI ? PBS group rat models (920,000), including mitochondrial crista fracture and mitochondrial vacuolar degeneration. However, very little of these mitochondrial changes were observed in the BCNI ? hUCBMSCs group model rats. CN nerve fibers were also observed by electron microscopy (95000), and medullated nerve fibers revealed significant changes (medulla sheath thickening, segregation, disintegration, or dissolved) in the BCNI ? PBS group model rats. The rats treated with hUCBMSC ICI showed slight changes in CN nerve fibers compared with BCNI ? PBS group model rats. Additionally, the medulla sheath of many medullated nerve fibers in the

Fig. 4 I. Presence of BrdU ? hUCBMSCs in the penis on day 1, 3, 7, and 28 after cavernous-nerve crush and intracavernous injection. (BrdU, red; DAPI, blue; Scale bars = 50 um); II.

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Fig. 5 The histogram I and II show the number of BrdU ? hUCBMSCs/field in the penis and MPG on day 1, 3, 7, and 28 after cavernous-nerve crush and intracavernous injection, respectively. Each bar shows the mean values (±standard deviation) (asterisk indicates P \ 0.05)

BCNI ? PBS group and BCNI ? hUCBMSCs group were much more thickened than rat models in the sham ? PBS group (Fig. 6).

Discussion Results from the present study have demonstrated a therapeutic potential for hUCBMSCs on erectile

Presence of BrdU ? hUCBMSCs at the MPG on day 1, 3, 7, and 28 after cavernous-nerve crush and intracavernous injection. (BrdU, red; DAPI, blue; scale bars = 50 um)

Biotechnol Lett

Fig. 6 a–c Electron micrographs of CN (scale bars = 5.50 um). The yellow arrows in the image show normal medullated nerve fibers. The red arrows in the image b and c show medulla sheath thickening, segregation, or dissolved nerve fibers; d–f electron micrographs of MPG (Scale

bars = 1.00 um). The purple arrows in image d show normal mitochondria. The blue arrows in image e show mitochondrial vacuolar degeneration. The green arrows in image f show mitochondrial disrupted crista

function after bilateral CN crush injury. We found that the ratio of ICP/MAP significantly increased, accompanied with increased nNOS-positive nerve fibers in the dorsal penile nerve, reduced fibrosis in the corpus cavernosum, and reduced ultrastructure damage in CNs and MPG of the animals in the BCNI ? hUCBMSCs group compared with animals in the BCNI ? PBS group. General mechanisms of how stem cells injected into corpus cavernousum facilitate recovery of erectile function have been proposed. For instance, ICI of stem cells inhibit apoptosis of smooth muscle cells, neural cells, and other cell types; stem cells secrete neurotrophins to modulate neuronal growth and survival via paracrine pathways; stem cells also move to the injury site to initiate neuroregeneration (Kendirci et al. 2009; Fall et al. 2009; Fandel et al. 2012). Compared with other stem cells, hUCBMSCs are associated with few ethical issues and are less immunogenic. Furthermore, human umbilical cord blood (CB) will become the most abundant stem cell source as the annual global human birth rate exceeds

more than 100 million a year, and CB banks are being established all over the world. In addition, the isolation and use of hUCBMSCs in clinical therapy is more convenient. The procedures of CB collection only take about 5 min and pose no risk to mother or baby (ArienZakay et al. 2010). Kao et al. (2007) reported that hUCBMSCs cells are beneficial for restoring function of the hindlimb by stimulating production of both glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF) in an SCI model. Additionally, a previous study showed that hUCBMSCs are capable of differentiating into neural cells (Harris and Rogers 2007). On the basis of these results, we adopted hUCBMSC ICI to restore CN crush injury in this study. To the best of our knowledge, the current study is the first to show the therapeutic effects of hUCBMSCs on BCNI model rats. Although the cells we used were harvested from humans and were injected into the rat penis, no immunogenic rejection reaction was observed. hUCBMSCs have been successfully and efficiently used to treat cerebral ischemia and spinal cord injury

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in rat models, and the safety of hUCBMSC ICI is attributed to its immunosuppressive properties and low immunogenicity. hUCBMSCs have immunosuppressive properties and can suppress proliferation of stimulated immune cells; hUCBMSCs exhibit low immunogenicity and express an immunosuppressive isoform of the human leukocyte antigen (HLA), HLAG6 (Weiss et al. 2008). Following CN injury, a-SMA expression and smooth muscle/collagen ratio in the corpus cavernousum was significantly reduced in the BCNI ? PBS group model rats. The reduction of smooth muscle cells prevented perfusion of oxygenated blood in the corpus cavernosum, resulting in corpus cavernosum fibrosis. Podlasek et al. (2007) discovered that expression of sonic hedgehog protein (SHH) significantly decreased in the corpora cavernosa, leading to increased activity of caspase 3 and inhibition of the G1 to S transition, which further leads to smooth muscle apoptosis. The BCNI model rats treated with hUCBMSC ICI showed significantly increased a-SMA expression and smooth muscle/collagen ratio in the corpus cavernousum compared with the BCNI ? PBS group, but still less than the sham ? PBS group. These results may reflect the effectiveness of hUCBMSCs in inhibiting smooth muscle apoptosis and corpus cavernosum fibrosis. The deficiency of nNOS in the penis caused by CN injury is believed to underlie a low response to PED5-I in ED patients after RP (Tanget et al. 1998). In this study, animal models treated with hUCBMSCs exhibited more nNOS-positive nerve fibers in the dorsal penile nerve than models treated with PBS. This could be the result of hUCBMSCs mediating a rehabilitative effect on nitrergic neuronal axons and ganglia. Additionally, improved erectile function indirectly improved dorsal nerve nNOS by increasing inflow of oxygenated blood and growth factors. According to electron microscopy, CN crush injury induced mitochondrial crista fracture and vacuolar degeneration in the MPG, as well as medulla sheath thickening, segregation, and dissolution in the CN. The numbers of medullated nerve fibers of CN significantly decreased after CN injury in the BCNI ? PBS group and BCNI ? hUCBMSCs group, but more medullated nerve fibers were conserved in the BCNI ? hUCBMSCs group than BCNI ? PBS group. By contrasting the ultrastructural changes between BCNI ? PBS group and BCNI ?

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hUCBMSCs group model rats, the potential for hUCBMSCs to maintain the ultrastructure or promote nerve regeneration of CN and MPG was confirmed. On day 1, 3, 7, and 28 post-ICI, hUCBMSCs were tracked in the penis and MPG. On day 1 post-ICI, most of intracavernously injected BrdU ? hUCBMSCs were found in the penis (42.2 ± 1.84), and only a few (6.6 ± 0.88) were found in MPG. In the penis, the number of BrdU ? hUCBMSCs showed a timedependent decline and only 5.2 ± 0.64 cells, which localized to the sinusoid endothelium, were observed at day 28 post-ICI. However, in the MPG, there were more BrdU ? hUCBMSCs at day 3 post-ICI than at day 1, and these numbers gradually reduced by day 7 and day 28 post-ICI. This finding is analogous to previously reported findings (Fandel et al. 2012; Lin et al. 2011). The corpus cavernosum is composed of endothelium-lined sinusoids that are anatomically and physiologically similar to arteries and veins. Thus, cells injected by ICI can be transported by blood to distant locales, including target tissue (Lin et al. 2011). Fandel et al. demonstrated transient homing of ADSCs to the MPG after CN crush injury, and elevated stromal cell-derived factor-1 (SDF-1) secretion at the MPG appeared to be responsible for this observation (Fandel et al. 2012). These considerations point to the possibility that hUCBMSCs could be transported and recruited to the MPG through the blood stream and then play a therapeutic role via paracrine pathways. Our study has some limitations. Only one time point was observed in this study, and the mechanisms for how hUCBMSCs restore CN injury remain unknown. In the future, different time points should be studied in longterm studies to explore the therapeutic efficacy of hUCBMSCs on CN-injured animal models.

Conclusions To the best of our knowledge, the current study is the first to show therapeutic potential of hUCBMSCs for BCNI model rats. At 4 weeks of observation, we found therapeutic effects of hUCBMSCs in BCNI model rat by functional and morphological experiments. Our data support the hypothesis that hUCBMSC ICI is a potential choice for ED treatment following radical prostatectomy. However, further studies are needed to assess the feasibility of using this therapy for post-prostatectomy ED.

Biotechnol Lett Acknowledgments We would like to thank Professor ShengMing Zhang, Department of Histology and Embryology at Weifang Medical College, for electron microscopy assistance. This work was supported by the National Natural Science Foundation of China (30901489); Scientific Research Foundation for Returned Scholars, Ministry of Education of China (2014); Shanghai Jiaotong University Biomedical Engineering Cross Research Foundation (YG2014MS15); Shandong Science and Technology Development Plan (2011GGH21813) and Shanghai Jiaotong University Morningstar Scholars Program (2012). Conflict of interest All authors declare no competing financial interests.

References Akaza H (2010) Aim of the working group on the Asian perspectives on cancer control: Asian perspective on prostate cancer prevention. Jpn J Clin Oncol 40(Suppl 1):i2–i6 Arien-Zakay H, Lazarovici P, Nagler A (2010) Tissue regeneration potential in human umbilical cord blood. Best Pract Res Clin Haematol 23:291–303 De Schauwer C, Meyer E, Cornillie P, De Vliegher S, van de Walle GR, Hoogewijs M, Declercq H, Govaere J, Demeyere K, Cornelissen M, Van Soom A (2011) Optimization of the isolation, culture, and characterization of equine umbilical cord blood mesenchymal stromal cells. Tissue Eng Part C Methods 17:1061–1070 Divya MS, Roshin GE, Divya TS, Rasheed VA, Santhoshkumar TR, Elizabeth KE, James J, Pillai RM (2012) Umbilical cord blood-derived mesenchymal stem cells consist of a unique population of progenitors co-expressing mesenchymal stem cell and neuronal markers capable of instantaneous neuronal differentiation. Stem Cell Res Ther 3:57 Falcone M, Rolle L, Ceruti C, Timpano M, Sedigh O, Preto M, Gonella A, Frea B (2013) Prospective analysis of the surgical outcomes and patients’ satisfaction rate after the AMS Spectra penile prosthesis implantation. Urology 82:373–376 Fall PA, Izikki M, Tu L, Swieb S, Giuliano F, Bernabe J, Souktani R, Abbou C, Adnot S, Eddahibi S, Yiou R (2009) Apoptosis and effects of intracavernous bone marrow cell injection in a rat model of postprostatectomy erectile dysfunction. Eur Urol 56:716–725 Fandel TM, Albersen M, Lin G, Qiu X, Ning H, Banie L, Lue TF, Lin CS (2012) Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury. Eur Urol 61:201–210 Ficarra V, Novara G, Ahlering TE, Costello A, Eastham JA, Graefen M, Guazzoni G, Menon M, Mottrie A, Patel VR, Van der Poel H, Rosen RC, Tewari AK, Wilson TG, Zattoni F, Montorsi F (2012) Systematic review and metaanalysis of studies reporting potency rates after robot-assisted radical prostatectomy. Eur Urol 62:418–430 Greggio S, de Paula S, Azevedo PN, Venturin GT, Dacosta JC (2014) Intra-arterial transplantation of human umbilical

cord blood mononuclear cells in neonatal hypoxic-ischemic rats. Life Sci 96(1–2):33–39 Harris DT, Rogers I (2007) Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine. Curr Stem Cell Res Ther 2:301–309 Kao C-H, Chen S-H, Chio C-C, Lin M-T (2007) Human umbilical cord blood-derived Cd34? cells may attenuate spinal cord injury by stimulating vascular endothelial and neurotrophic factors. Shock 29:49–55 Lin G, Qiu X, Fandel T, Banie L, Wang G, Lue TF, Lin CS (2011) Tracking intracavernously injected adipose-derived stem cells to bone marrow. Int J Impot Res 23:268–275 Lin Ching-Shwun, Xin Zhong-Cheng, Wang Zhong, Deng Chunhua, Huang Yun-Ching, Lin Guiting, Lue TomF (2012a) Stem cell therapy for erectile dysfunction: a critical review. Stem Cell Dev 21(3):343–351 Lin CS, Xin ZC, Wang Z, Deng C, Huang YC, Lin G, Lue TF (2012b) Stem cell therapy for erectile dysfunction: a critical review. Stem Cells Dev 21:343–351 Magheli A, Burnett AL (2009) Erectile dysfunction following prostatectomy: prevention and treatment. Nat Rev Urol 6:415–427 Piao S, Kim IG, Lee JY, Hong SH, Kim SW, Hwang TK, Oh SH, Lee JH, Ra JC, Lee JY (2012) Therapeutic effect of adipose-derived stem cells and BDNF-immobilized PLGA membrane in a rat model of cavernous nerve injury. J Sex Med 9:1968–1979 Podlasek CA, Meroz CL, Tang Y, McKenna KE, McVary KT (2007) Regulation of cavernous nerve injury-induced apoptosis by sonic hedgehog. Biol Reprod 76:19–28 Salonia A, Burnett AL, Graefen M, Hatzimouratidis K, Montorsi F, Mulhall JP, Stief C (2012a) Prevention and management of postprostatectomy sexual dysfunctions. Part 1: choosing the right patient at the right time for the right surgery. Eur Urol 62:261–272 Salonia A, Burnett AL, Graefen M, Hatzimouratidis K, Montorsi F, Mulhall JP, Stief C (2012b) Prevention and management of postprostatectomy sexual dysfunctions part 2: recovery and preservation of erectile function, sexual desire, and orgasmic function. Eur Urol 62:273–286 Seo Y, Yang SR, Jee MK, Joo EK, Roh KH, Seo MS, Han TH, Lee SY, Ryu PD, Jung JW, Seo KW, Kang SK, Kang KS (2011) Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice. Cell Transpl 20:1033–1047 Tang Y, Rampin O, Calas A, Facchinetti P, Giuliano F (1998) Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat. Neuroscience 82:241–254 Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, Troyer D, McIntosh KR (2008) Immune properties of human umbilical cord Wharton’s jellyderived cells. Stem Cells 26:2865–2874 Yang S, Sun H-M, Yan J-H, Xue H, Wu B, Dong F, Li WS, Ji FQ, Zhou DS (2013) Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells. J Neurosci Res 91:978–986

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