Progress and prospect of stem cell therapy for diabetic erectile dysfunction.

Diabetic erectile dysfunction (DED) is a common complication of diabetes mellitus, significantly impairing the quality of life of patients. The conventional clinical treatment still has limitations. Stem cells (SCs), as a type of cells with multidirectional or directional differentiation capability and sustainable self-renewal potential, are widely used in regenerative medicine and tissue engineering. With the continuous update of regenerative medicine theory and the success of animal experiments, SCs as a treatment for male erectile dysfunction, especially DED, have attracted widespread attention because of curable possibility. This review focus on the current progress in the clinical application of SC treatment for DED. Moreover, we summarize the development prospects of SCs in the field of DMED therapy. ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.

Core Tip: Diabetic erectile dysfunction (DED) is a common complication of diabetes. Conventional clinical treatments like phosphodiesterase-5 inhibitors, intracavernosal vasoactive drug injection, negative pressure suction device, and low-intensity shock wave therapy are the conventional methods of clinical treatment. However, none of the above therapies has the potential of curing. Stem cell therapy is currently the only possible cure for DED and can avoid the potential complications of conventional therapies. Here we discuss the current role and progress of stem cells in the treatment of DED.

INTRODUCTION

Diabetes mellitus (DM) is a chronic, non-communicable disease caused by genetic, environmental, and other factors. Over the past three decades, the number of people with DM has more than doubled globally, making it one of the most important public health challenges worldwide[1]. Erectile dysfunction, defined as the inability to achieve and/or maintain an erection sufficient to permit satisfactory sexual intercourse[2], is a major complication of DM[3]. Diabetes is considered the main risk factor for the development of erectile dysfunction, and since the 1970s, the association between diabetes and the development of erectile dysfunction has been documented both in animal models and humans[4]. Increasing attention focuses on erectile dysfunction in men with diabetes due to its multifactorial pathophysiology and the concurrence of the same components as vasculopathy, neuropathy, and depression[5]. Although oral phosphodiesterase-5 inhibitors (PDE5Is) represent as a successful first-line therapy, a considerable proportion of men do not respond to oral PDE5Is[6-8]. Other conventional therapeutics for ED consist of oral medications, intracavernosal injections, vacuum erection devices, and penile implants. However, due to the lack of high quality evidence of efficacy and safety, poor patient compliance, and poor treatment outcomes, the above treatment regimens are still controversial in clinical application[9-13]. Recent 20 years have witnessed the progress of stem cell therapy (SCT), and it is expected to be an alternative option in the treatment of diabetic erectile dysfunctions (DED) and replace the current conventional treatment options.

STEM CELL THERAPEUTIC POTENTIAL AND CLASSIFICATION

The term ‘stem cells’ encompasses various cells with self-renewal and differentiation abilities, and many of them can potentially be used therapeutically[14,15]. In addition to differentiation into mature tissue cells, stem cells can also play a therapeutic role due to their chemotaxis, anti-inflammatory, regenerative, angiogenic, and anti-apoptotic properties[16-21]. A large number of regenerative medicine studies have proved the therapeutic potential of stem cells[22].

There are two major types of stem cells, embryonic stem cells (ESCs) and adult stem cells (ASCs)[14]. ESCs are derived from the inner cell mass of preimplantation embryos. Limited by biological and medical ethics, the clinical application of ESCs in the treatment of ED is restricted[23]. ASCs are ethical and easier to obtain from the host. Therefore, ASCs derived from patients themselves have been favored by researchers in recent years[24-31]. At present, many preclinical studies have used various types of ASCs to confirm the conclusion of improving erectile function, including bone marrow-derived mesenchymal stem cells (BMSCs)[32], adipose-derived stem cells (ADSCs)[33], neuro-derived stem cells[34], mesenchymal stem cell (MSCs)[35] , and urine-derived stem cells (USCs)[36,37]. They are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly, making them a promising candidate for therapeutic use in regenerative medicine.

In 2006, Takahashi and Yamanaka[38] reported a method to reprogram multipotent ASCs to the pluripotent state. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) could induce the fibroblasts to become pluripotent. This new form of stem cells is named induced pluripotent stem cells (iPSCs)[39]. One year later, the experiment also succeeded with human cells[40]. iPSCs opened a new field in stem cell research, because they can proliferate indefinitely and differentiate into any kind of cell with unlimited sources. iPSCs bypass the need for embryos in SCT, because they are obtained from the host patient’s own cells, and they are autologous and no longer generate any risk of immune rejection[41,42] (Table 1).

Table 1

Characteristics of the articles published

Ref.	Treatment factor	Method/transfer/period	Evidence	Outcome
[42]	Rat	Injection with ADSCs (1 × 106), EPCs (1 × 106), and ADSCs/EPCs (0.5 × 106/0.5 × 106)	ADSC/EPC group displayed more significantly enhanced ICP and ICP/MAP than the DED or ADSC or EPC group (P < 0.05). (After 28 d)	Combined transplantation of ADSCs and EPCs has a synergic effect in repairing the endothelial function of DED rats
	ADSCs/EPCs/combined ADSCs and EPCs
[43]	Human USCs	Coculture of USCs and rat corpus (CCECs) treated with AGEs/injection with USCs (1 × 106) in rat DED model	USCs protected CCECs from AGE-induced autophagic dysfunction and cellular damage/ DED rats showed lower ratio of ICP/MAP, reduced expression of endothelial markers, and fewer autophagic vacuoles in the cavernosal endothelium	Intracavernous injection of USCs up-regulates autophagic activity in the cavernosal endothelium
[44]	Combined HGF and ADSCs	Injection with ADSCs alone or combined with HGF	Significant down-regulation of TGFβ1-Smad signaling	HGF enhance the beneficial effects of ADSCs on DED through down-regulation of the TGFb1-Smad signaling pathway
[45]	Combined BMSCs and LESWT	1/3 d after BMSC transplantation, the number of surviving BMSCs in the cavernous body was counted	LESWT favored the survival of transplanted BMSCs in the cavernous body, increased stromal cell-derived factor-1, and enhanced angiogenesis	Combined LESWT and BMSC improve the erectile function of DED rats more effectively than either alone
[46]	HP and human BMSCs	hBMSC (1 × 106 cells/mL) seeded on oxygen-saturated HPs	cGMP and NOS levels rose through prolonged stem cell survival	Stem cell/oxygen-releasing HP hybrid system could further improve erectile function
[48]	ADSCs expressing VEGF	ADSCs expressing large amounts of VEGF through virus transfection	VEGF-ADSCs stimulated endothelial function, and increased the content of smooth muscle and pericytes	VEGF-ADSCs improve erectile function
[57]	ADSC EVs	ADSC EVs through ultracentrifugation and treatment of DED rat model through ICI	ADSC-derived EXOs and ADSCs increased the ratio of intracavernous pressure	ADSC-derived EXOs and ADSCs are able to rescue corpus cavernosum endothelial and smooth muscle cells by inhibiting apoptosis
	USC EVs	USC EVs through ultracentrifugation and treatment of DED rat model through ICI	USC-EVS enhanced the expression of endothelial cell markers in DED rats, reduced collagen deposition, and improved neurogenic erectile response through pro-angiogenic miRNAs	USC-EV transplantation can ameliorate DED in rats via mechanisms that may involve the delivery of proangiogenic miRNAs

ADSCs: Adipose tissue-derived stem cells; EPCs: Endothelial progenitor cells; ICP: Intracavernous pressure; MAP: Mean arterial pressure; DED: Diabetic erectile dysfunction; CCECs: Cavernosal vascular endothelial cells; AGEs: Glycation end products; HGF: Hepatocyte growth factor; BMSCs: Bone marrow mesenchymal stem cells; LESWT: Low energy shock wave; HPs: Oxygen-releasing hollow microparticles; EVs: Extracellular vesicles; USCs: Urine derived stem cells.

STEM CELL THERAPY IN DIABETIC ERECTILE DYSFUNCTION MODELS

Stem cell-based therapies have been investigated in the field of DED. The overall aim is to repair the underlying corpus cavernosum cellular damage. Yang et al[43] injected ADSCs and endothelial progenitor cells (EPC) alone or in combination in DMED rat models and found that on the 28th day after injection, the mean arterial pressure (MAP) ratio were significantly higher in the ADSC group, EPC group, and ADSC combined with EPC group than in the DED group, and in the ADSC combined with EPC group than in the DED group, ADSC group, and EPC group (P < 0.05). It is revealed that both ADSCs and EPCS can improve erectile function, and the combined transplantation of ADSCs and EPC can synergistically improve the endothelial function in DMED rats. The therapeutic effect is better than the treatment with ADSCs or EPCs alone. Zhang et al[44] explored the therapeutic mechanism of USCs, using advanced glycation end products to treat rat cavernous endothelial cells to simulate a diabetic environment. Then, the two cells were co-cultured to evaluate the protective effect of USCs in vitro. Finally, they concluded that autophagy dysfunction is related to cavernous endothelial function and erectile dysfunction in DMED rats. In addition, USCs can up-regulate the autophagy activity of sponge endothelial cells, which improves endothelial sponge dysfunction and is ultimately expected to improve erectile dysfunction caused by diabetes. These stem cell-based preclinical studies have emphasized the improvement of the erectile function of DED animals after stem cell treatment and explained the underlying mechanism.

Some researchers also explored the combination of stem cells with conventional therapy. Liu et al[45] conducted related studies and used hepatocyte growth factor (HGF) combined with ADSCs to treat DMED rat models. One month later, treatment with ADSCs alone can significantly improve the erectile function of diabetic rats. However, the combined application with HGF has a more significant effect, making the erectile function even close to the normal level. It shows that HGF can significantly enhance the beneficial effects of ADSCs on the erectile function of diabetic rats, and its mechanism may be closely related to the down-regulation of the TGF-β1-Smad signaling pathway. Shan et al[46] combined BMSCs and low energy shock wave (LESWT) to treat a DMED rat model. On days 1, 3, and 28 after BMSC transplantation, they observed the number of surviving BMSCs in the cavernous body. The result showed that combined application of LESWT and BMSC transplantation maintained more BMSCs in cavernous body and improved the erectile function of diabetic rats more effectively than LESWT or BMSC transplantation alone. This may promote the expression of stromal cell-derived factor-1 in the diabetic cavernous tissue, thus promoting angiogenesis. In another study, Jung et al[47] used an oxygen-releasing hollow microparticle (HP) system with human BMSC to attempt to overcome certain limitations of SCT, including insufficient nutrient and oxygen supplies for transplanted stem cells. The results demonstrated that a stem cell/oxygen-releasing HP hybrid system could further improve erectile function, cyclic guanosine monophosphate level, and nitric oxide synthase (NOS) level in a bilateral cavernous nerve injury rat model through prolonged stem cell survival. Their data suggest that a stem cell/oxygen-releasing HP system is a promising clinical treatment option for postprostatectomy erectile dysfunction. Furthermore, this system may be relevant in different disease therapies and regenerative medicine.

Next-generation stem cells may serve as therapeutic agents by overexpressing neurotrophic factors, chemokines, anti-inflammatory molecules, angiogenic factors, and other molecules that aid in recovery and endogenous repair of tissues[48]. Modified stem cells may repair the damaged signaling pathway in DED that causes endothelial dysfunction, confirmed by various studies. We have prepared ADSCs expressing large amounts of vascular endothelial growth factor (VEGF) through virus transfection[49], and found that these ADSCs could stimulate endothelial function, increase the content of smooth muscle and pericytes, and significantly improve the erectile function of DED rats. Kizub et al[50] also conducted similar experiments in MSCs, and MSCs expressing VEGF also showed the same advantages. In addition, ADSC injection combined with insulin therapy can produce better therapeutic effects in a DMED rat model[51]. The above experiments show that stem cell injection can help restore the erection and other physiological functions of the penis.

The use of stem cell-derived extracellular vesicles (EVs) avoids the risks of any undesired stem cell growth or potential for tumor-promoting effects, which has been noted for MSCs in certain situations[52]. At present, stem cells are widely used as biomaterials for the regeneration of tissue defects. The treatment mechanism is based on the differentiation into specific target cells after implantation and the paracrine release effect of EVs[53,54]. EVs secreted by stem cells are believed to play a major role in the treatment of DED. The paracrine release effect of EVs means that the exosomes after injection can repair damaged tissues by delivering cytoprotective molecules, anti- inflammatory molecules, and anti-apoptotic molecules. Many studies support this view, proposing the direct use of stem cell EV therapy to restore the function of damaged organs and tissues and achieve the direction of cell-free SCT[55-57]. Chen et al[58] acquired ADSC EVs through ultracentrifugation and treated a DED rat model through intracavernous injection (ICI). The results show that, like ADSCs, exosomes derived from ADSCs can increase the number of sponge endothelial cells and smooth muscle cells by inhibiting cell apoptosis, thereby promoting the recovery of erectile function in type 2 diabetic rats. Ouyang et al used the same method to obtain the EVs of USCs[59]. Their results showed that USC-EVs enhance the expression of endothelial cell markers in DED rats, reduce collagen deposition, and improve neurogenic erectile response. The transport of pro-angiogenic microRNAs secreted by EVs may play an important role in endothelial repair. EVs secreted from engineered stem cells are considered an alternative cell-free next-generation approach for delivering vascular repair factors, anti-apoptosis agents, and other tissue-repairing agents.

HUMAN CLINICAL TRIALS OF SCT

Although SCT has been proved effective in multiple animal ED models, there are only a few human clinical trials of SCT, thus we include clinical trials of other types of ED. Bahk et al[60] recruited seven patients with type 2 DED in the study, all of whom were insensitive to drug treatment for more than 6 mo and needed to wait for penile prosthesis implantation. The researchers injected 1.5 × 1.7 human umbilical cord blood cells into the cavernous body and recorded their International Erectile Function Index-5, Sexual Encounter Profile, Global Assessment Question, erectile diary, blood glucose diary, and medication dosage. The results showed that three of the six patients recovered the morning erection reaction after 1 mo, and it rose to five after 3 mo. At the 6 mo follow-up, two of the patients completed their sexual life and reached orgasm under the premise of oral PDE5i. After 9 mo, all five patients felt an increase in libido. During the entire follow-up process, two patients quit to implant a penile prosthesis, and four had recovered erectile function. And no side effect of SCT was reported. Levy et al[61] injected placental matrix-MSCs to treat patients with ED into eight patients with ED who had failed to take oral medications, and MSC treatment was followed at 6 wk, 3 mo, and 6 mo to assess peak systolic velocity (PSV), end diastolic velocity, stretched penile length, penile width, and erectile function status based on the International Index of Erectile Function questionnaire. The results showed that at 6 wk, two patients developed spontaneous and maintained erections; at 3 mo, one patient can erect spontaneously. The average PSV values of patients at 3 and 6 mo were significantly higher than those before treatment. However, the measured end-diastolic speed changes, extended penis length, penis width, and erectile function international index score were not statistically significant. In a phase 1 clinical trial[62], 12 patients who had ED after radical resection of prostate cancer (drug treatment failed) were given ICI of BMSCs four times, and the injection dose of BMSCs was increased gradually (2 × 107, 2 × 108, 1 × 109, and 2 × 109). The results showed that 9 out of 12 patients exhibited significant improvement in erectile function under the premise of oral PDE5i. It was found that as the injection dose increased, the incidence of spontaneous erection increased, and the study did not report any serious adverse reactions after injection. In phase II clinical trial of the same study, six more patients were recruited, and the best dose (1 × 109) of BMSCs determined in phase 1 was injected. The results showed that the sexual satisfaction scores of 18 patients in the IIEF-5 index and the erectile function scores were significantly improved compared to those before treatment. However, the six patients who participated in the second stage had lower erectile function scores than the 12 patients who participated in the first stage. In the first phase, no prostate cancer recurrence was detected[63]. In the phase I clinical trial conducted by Demour et al[64] in 2018, human BMSCs were used for the first time to treat DED. They included four patients with DED who had failed medical treatment and injected the BMSCs extracted from the patients into the penile sponge twice. The patients’ tolerability was evaluated immediately after injection and 24 h later. The effectiveness was evaluated by IIEF-5 and EHS scores 1 year later, and safety was evaluated 2 years later. The results showed that all patients tolerated the operation well, and there was no obvious adverse reaction. After 12 mo of follow-up, the patients' IIEF-15, EHS, erectile function, libido, sexual satisfaction, and overall satisfaction scores were significantly improved compared to those before treatment. Haahr et al[65] discussed the effectiveness and safety of ADRCs in the treatment of patients with ED after RP. The researchers injected 21 patients' own ADSCs into the cavernous body at one time, followed them for 1 year, and evaluated and recorded adverse events, IIEF-5 score, and EHS score. The results showed that no serious adverse events occurred during the follow-up process, but eight cases had reversible adverse reactions related to liposuction, including three cases of redness and swelling in the penis and five cases of mild hematoma in the abdomen, all of which occurred within a short period of time after treatment and subsided without any sequelae. Eight of the 21 participants (38%) recovered sufficient erectile function during the 12-mo observation period. These eight patients had poor erection assistance effects before receiving stem cell treatment. After treatment, three men could complete sexual intercourse without erectile assistance. Six of all participants had erectile dysfunction before RP, and these six patients did not recover their erectile function after receiving SCT.

DISSCUSSION

According to the current multiple preclinical studies and a small number of clinical studies for the treatment of organic ED with SCT, the effectiveness and safety of SCT are considerable, and it is also considered to be a very promising way to treat organic ED in the future. A meta-analysis included ten preclinical studies on SCT in ED rat models, with a total of 302 rats. The results showed that SCT improved erectile function in diabetic rats (standard mean difference = 4.03, 95% confidence interval: 3.22-4.84, P < 0.001)[66]. The contents of vascular smooth muscle and endothelial cells in the stem cell treatment group were significantly higher than those in the control group. The expression of endothelial NOS and neural NOS, smooth muscle/collagen ratio, and VEGF secretion were significantly increased. In addition, stem cell treatment can reduce apoptotic cells. Subpopulation analysis showed that modified stem cells were more effective than unmodified stem cells. These results suggested that SCT can significantly improve the erectile function of diabetic rats. Some specific modifications, especially the genetic modification of growth factors, can improve the effectiveness of SCT. SCT may become an effective strategy for the treatment of diabetic ED.

There are only nine clinical trials reporting the efficacy and safety of stem cell treatment in men with ED, and the trial sample size is less than 100 cases. Based on the results of each study, the overall penile hemodynamic and erectile function scores of patients are significantly improved. No major adverse reactions was observed.

After years of exploration in many preclinical trials, the current mechanism of SCT for organic ED has been clarified. There are two major hypotheses about SCT. First, SCT can repair and replace cavernous nerves. SC differentiate into cavernous nerve cells, smooth muscle cells, and endothelial cells[67]. Second, SCT provides penile tissue support through the paracrine effect of EVs, delivering proangiogenic, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties[68]. Studies have shown that the release of paracrine repair cytokines is an endogenous mechanism independent of the differentiation of stem cells into different cell types[69].

Although SCT seems to have good prospects in the treatment of DED, there are still many unresolved challenges. First, there is still a lack of large-scale clinical studies to verify its effectiveness and safety. Although SCT has achieved great success in some animal experiments, due to the huge anatomy and physiology gap between animal DED model and human DED[70], it is difficult to clarify whether the successful results of animal experiments will still work in human DED due to the lack of high-quality and large-scale human trials of SCT. Most of existed clinical trials are phase I studies, with a small sample size and lack of blinding method, as well as ambiguous inclusion and exclusion criteria, and primary and secondary outcome indicators. The inclusion of negative studies makes these studies insufficient to provide convincing evidence.

Second, SCT may have safety issues. Although previous studies have not found serious complications after treatment, some studies reported that MSCs could penetrate into prostate cancer cells, including prostate tissue[71], and SCs may act as a tumor promoter to affect its occurrence and development[72].

Third, the cell source selection of SCT warrants further investigation. Existing studies have found that both autologous and foreign SCs can improve the erectile function in ED. As for the trade-off between the pros and cons of choosing autologous or allogeneic SCs, only one study has explored this issue, but it still does not directly compare the application of autologous cells and allogeneic cells[73]. It is believed that choosing autologous stem cells will bring obvious safety advantages and it is convenient to obtain materials because it can overcome the antigenic problem of allogeneic cell transplantation. However, recent studies have found that allogeneic ADSCs can induce a locally suppressed microenvironment by secreting intraoperative cytokines to regulate the function of natural killer cells and T cells, thereby avoiding allogeneic immune rejection[67]. Meanwhile, in another study on a rat model of acute myocardial infarction treated with SCs, it was found that allogeneic transplantation of MSCs only maintained their ability to overcome allogeneic immune rejection in a short period of time. As the treatment time prolonged, MSCs differentiated in myocardial tissue and lost their immune privilege status, thereby affecting the therapeutic effect[74]. Autologous stem cells are not suitable for all patients. For example, for elderly patients, the proliferation and differentiation ability of stem cells in the body decreases significantly as the body ages[75]. In addition, for cancer patients, the use of autologous stem cells for transplantation is prohibited[76].

Fourth, treatment methods have not yet been standardized. Multiple studies have demonstrated the presence of MSCs in the lung, immediately after injection[77-80]. However, the majority of cells are, however, cleared within the first days of treatment[81]. It is reported that less than 1% of stem cells injected intravenously in SCT actually reached the target tissue, but the treatment effect was not affected[82]. Hence, there is a demand for retaining stem cells in the corpus cavernosum after ICI for ED therapy. ADSCs incubated with NanoShuttle were magnetic and maintained in the corpus cavernosum. This improved the adipose-derived SCT of ED in a CNI rat model[83]. Nevertheless, the cell type, the injection concentration, the course of treatment, and the evaluation endpoint of the treatment effect have not yet been determined.

Fifth, there are ethical issues. SCs constitute one of the most promising tools for regenerative medicine. Thus, it seems morally compelling to explore all the sources that might provide us with them. However, some of these sources, such as somatic cell nuclear transfer, embryo destruction, or even induced pluripotency obtained by reprogramming, have raised serious ethical issues[84]. For the use of cell, tissue, and stem-cell products in treating patients, the US Food and Drug Administration[85], the European Medicines Agency guidelines, and the International Society for Stem Cell Research have also developed or updated several specific guidelines for SCT with the help of stem cell experts from all around the world[86]. According to these guidelines, the most important topics of SCT related to ethical, legal, and social considerations of cell therapy include: (1) Manufacturing conditions and characterization of clinical-grade cells; (2) genetic material and confidential personal information; (3) informed consent; (4) genetic manipulation of the cells; and (5) intellectual property and patents, along with some other important issues[87].

CONCLUSION

SCT for DED has broad development prospects. However, many problems need to be solved to achieve an effective and safe clinical treatment plan. In addition to the need to further clarify its specific mechanism of action, effectiveness, and safety, it is also necessary to clarify the optimal treatment plan to solve the immunogenicity and heterogeneity of SCT, and improve its high cost and low efficiency in application. In the future, more phase II and phase III clinical trials are needed to make full preparations for the transformation of SC treatment of ED from preclinical to clinical trials and translate into clinical applications.