Hydrogen sulfide (H(2)S), which is a well-known toxic gas, has recently been recognized as a biological messenger that plays an important role in physiological and pathophysiological conditions. Relatively high levels of H(2)S have been discovered in mammalian tissues. It is mainly synthesized by 2 enzymes, including cystathionine β-synthase and cystathionine γ-lysase, which utilize L-cysteine as substrate to produce H(2)S. H(2)S has been demonstrated to exhibit potent vasodilator activity both in vitro and in vivo by relaxing vascular smooth muscle. Recently, H(2)S has been discovered in penile tissue with smooth muscle relaxant effects. Furthermore, other effects of H(2)S could play a role in the physiology of erection. Understanding H(2)S in the physiology of erection might provide alternative erectile dysfunction strategies for those patients with poor or no response to type 5 phosphodiesterase inhibitors. This review intends to present the H(2)S pathway in penile tissue and the potential role of H(2)S in the physiology of erections.

Mesenchymal stem cells (MSCs) are well known for their immunomodulatory capabilities. In particular, their immunosuppressive property is believed to permit their allogeneic or even xenogeneic transplantation into immunocompetent recipients without the use of immunosuppressants. Adipose-derived stem cell (ADSC), owing to its ease of isolation from an abundant tissue source, is a promising MSC for the treatment of a wide range of diseases. ADSC has been shown to lack major histocompatibility complex-II expression, and its immunosuppressive effects mediated by prostaglandin E2. Both preclinical and clinical studies have shown that allogeneic transplantation of ADSCs was able to control graft-versus-host disease. In regard to xenotransplantation a total of 27 preclinical studies have been published, with 20 of them performed with the investigators' intent. All 27 studies used ADSCs isolated from humans, possibly due to the wide availability of lipoaspirates. On the other hand, the recipients were mouse in 13 studies, rat in 11, rabbit in 2, and dog in 1. The targeted diseases varied greatly but all showed significant improvements after ADSC xenotransplantation. For clinical application in human medicine, ADSC xenotransplantation offers no obvious advantage over autotransplantation. But in veterinary medicine, xenotransplantation with porcine ADSC is a practical alternative to the costly and inconvenient autotransplantation.

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is a form of energy transfer that is of lower intensity (<0.2mJ/mm²) relative to traditional Extracorporeal Shock Wave Lithotripsy (ESWL) used for management of urinary stones. At this intensity and at appropriate dosing energy transfer is thought to induce beneficial effects in human tissues. The proposed therapeutic mechanisms of action for Li-ESWT include neovascularization, tissue regeneration, and reduction of inflammation. These effects are thought to be mediated by enhanced expression of vascular endothelial growth factor, endothelial nitric oxide synthase, and proliferating cell nuclear antigen. Upregulation of chemoattractant factors and recruitment/activation of stem/progenitor cells may also play a role. Li-ESWT has been studied for management of musculoskeletal disease, ischemic cardiovascular disorders, Peyronie's Disease, and more recently erectile dysfunction (ED). The underlying mechanism of Li-ESWT for treatment of ED is incompletely understood. We summarize the current evidence basis by which Li-ESWT is thought to enhance penile hemodynamics with an intention of outlining the fundamental mechanisms by which this therapy may help manage ED.

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34(-) is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34(-) HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34(+) bone marrow cells as immunogen. Thus, neither MSC being CD34(-) nor HSC being CD34(+) is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34(+) fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.

Background

Thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs.

Methods

EdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay.

Results

EdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express β-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1.

Discussion

At the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.

Introduction

While phosphodiesterase type 5 inhibitors (PDE5is) and others are used to treat Erectile dysfunction (ED), many patients are either unresponsive or resistant to it. Stem cell therapy (SCT) is a promising alternative approach. Numerous preclinical trials have demonstrated improved erectile function in animal models using SCT, although the number of clinical trials investigating SCT for men with ED is limited. Nonetheless, findings from human clinical trials suggest that SCT may be a useful treatment option.

Areas covered

Biomedical literature, including PubMed, ClinicalTrials.gov, and European Union Clinical Trials Registry, were analyzed to summarize and synthesize information on stem cell therapy for ED in this narrative review. The achievements in preclinical and clinical evaluations are presented and critically analyzed.

Expert opinion

SCT has demonstrated some benefits in improving erectile function, while further studies are urgently needed. Such studies would provide valuable insights into the optimal use of stem cell therapy and its potential as a therapeutic option for ED. Taking advantage of different mechanisms of action involved in various regenerative therapies, combination therapies such as SCT and low-energy shock waves or platelet-rich plasma may provide a more effective therapy and warrant further research.

Ectopic overexpression of transcription factors has been used to reprogram cell fate. For example, virus-mediated overexpression of four transcription factors OCT4, SOX2, MYC, and KLF4, known as Yamanaka factors, can convert somatic cells to induced pluripotent stem (iPS) cells. However, gene-specific switch-on of endogenous gene production without the use of foreign DNA remains a challenge. The small RNA machinery that comprised small RNAs and Argonaute proteins is known to silence gene expression, but can be repurposed to activate gene expression when directed to gene promoters, a phenomenon known as RNA activation or RNAa. By screening of dsRNAs targeting OCT4 promoter, we identified a small activating RNA (saRNA) that activated OCT4 expression in several types of human mesenchymal stem cells (MSCs). We found that saRNA-induced OCT4 activation can be further enhanced by a histone deacetylase inhibitor, valproic acid. Furthermore, introducing OCT4 saRNA in combination with viruses encoding the remaining three Yamanaka factors (SOX2, MYC, and KLF4) into MSCs led to the derivation of partially reprogrammed iPS cells. Findings from this study suggest that, with further optimization, RNAa can be a powerful tool to reprogram cell fate by inducing the expression of endogenous genes.

Introduction.  Low-energy shockwave therapy (LESWT) has been shown to improve erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). However, the underlying mechanism remains unknown. Aim.  The aim of this study is to investigate whether LESWT can ameliorate DM-associated ED in a rat model and examine the associated changes in the erectile tissues. Methods.  Newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous mesenchymal stem cells (MSCs). Eight weeks later, eight of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group), whereas another eight rats were subject to shockwave (SW) treatment (DM+SW group). Each rat in the DM+SW group received 300 shocks at energy level of 0.1 mJ/mm(2) and frequency of 120/minute. This procedure was repeated three times a week for 2 weeks. Another 2 weeks later, all 24 rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Main Outcome Measures.  Erectile function was measured by ICP. Neuronal nitric oxide synthase (nNOS)-positive nerves and the endothelium were examined by immunofluorescence staining. Smooth muscle and MSCs were examined by phalloidin and EdU staining, respectively. Results.  STZ treatment caused a significant decrease in erectile function and in the number of nNOS-positive nerves and in endothelial and smooth muscle contents. These DM-associated deficits were all partially but significantly reversed by LESWT. MSCs (EdU-positive cells) were significantly more numerous in DM+SW than in DM rats. Conclusion.  LESWT can partially ameliorate DM-associated ED by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis. These beneficial effects appear to be mediated by recruitment of endogenous MSCs. Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, and Lin C-S. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2013;10:738-746.

INTRODUCTION: Radiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED). AIM: To investigate whether injection of adipose-derived stem cells (ADSCs) can ameliorate RT-associated ED. METHODS: Thirty male rats were divided into three groups. The control + phosphate-buffered saline (PBS) group received tail-vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail-vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail-vein injection of ADSCs, which were labeled with 5-ethynyl-2-deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining. MAIN OUTCOME MEASURES: Erectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification. RESULTS: Radiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU-positive cells were visible in MPG. CONCLUSIONS: Radiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration.

Spatiotemporal mechanobiology resulting in penile pathologies continues to be investigated using small scale animals models such as mice. However, species-dependent functional biomechanics of a mouse penis, is not known. In this study, spatial mapping of a mechanosensitive transcription factor, scleraxis (Scx), at ages 4, 5, 6 weeks, and 1 year were generated to identify mechanoactive regions within penile tissues. Reconstructed volumes of baculum collected using micro X-ray computed tomography illustrated significantly increased baculum length with decreased porosity, and increased mineral density (p < 0.05) with age. The bony-baculum was held centrally in the Scx positive corpus cavernosum glandis (CCG), indicating mechanoactivity within the struts in a 6 week old mouse. The struts also were stained positive for fibrillar proteins including collagen and elastin, and globular proteins including protein gene product 9.5, and α-smooth muscle actin. The corpus cavernosum penis (CCP) contained significantly (p < 0.05) more collagen than CCG within the same penis, and both regions contained blood vessels with equivalent innervation at any given age. Comparison of volumes of flaccid and erect penile forms revealed functional characteristics of the CCP. Results of this study provided insights into biomechanical function of the CCG; in that, it is a high-pressure chamber that stiffens the penis and is similar to the human corpus cavernosum.