Potential therapeutic applications of mesenchymal stem cells for erectile dysfunction in diabetes mellitus: From preclinical/clinical perspectives

Diabetic ED involves nerve damage, endothelial injury, and cavernosal muscle fi brotic alterations (Figure 1) [3,8-10]. Complications due to diabetes can limit blood fl ow into the penis if atherosclerotic damage corresponds to major blood vessels in the vascular system [11]. Advanced glycation endproducts (AGE) cause microvascular complications as pathogenesis of ED in streptozotocin-induced diabetic rats[12]. AGE and its receptor (RAGE) interaction develop a metabolic memory [1315]. Insulin therapy for glycemic control can reduce AGE and RAGE and control the corresponding infl ammatory response in the penis[14,16].


Penile erection and diabetic ED
Erectile function is a multifaceted neurovascular phenomenon that necessitates the healthy coordination of endothelial, nerve, and smooth muscle cells [22,23]. NO is formed by an enzymatic pathway involving both endothelial NO synthase [24] and neuronal (n) NOS and plays a crucial role in a normal erection [23,25]. For instance, the main cavernous nerve of the rat branches into the dorsal and intracavernous nerves. There is a loss of nNOS nitrergic fi bers due to damage to both locations of the cavernous nerve [26,27]. Cavernous nerve integrity is critically important for normal erectile physiology.
Burke, et al. [28] documented that nearly 50% of diabetic men aged 40-79 years suffer from ED. Additionally, many men seeking help for their ED are unaware they have diabetes [8]; however, clinical studies demonstrate that early metabolic The tight control of glycemia is an essential fi rst step in the management of diabetes-related ED. In a recent study of ED in a rat model of type 1 diabetes, islet transplantation with improved hyperglycemic status allowed for smooth muscle cell regeneration, and reduced CC fi brosis to its normal state in rats  intracavernosal pressure (ICP)/ mean arterial pressure (MAP) and erections in diabetic ED rats [64].
Recently, BM-MSCs with a signifi cantly lowered maternally expressed gene (MEG)-3 were implanted intracavernous and improved ED of diabetic rats [65]. FOXM1 protein can be degraded by MEG3, and the differentiation of the endothelium is ultimately regulated by BM-MSCs [65].
A signifi cant aspect of diabetic complications is an injury caused by oxidative stress due to hyperglycemia; therefore, future studies of the antioxidant capacity of MSCs are warranted. Endothelial-progenitor cells (EPCs) from the bone marrow can be mobilized to counter diabetes-induced oxidative stress. In 2012, Qui et al. documented that treatment with melatonin promoted EPC mobilization and thereby preserved erectile function in type-1 diabetic rats [66]. ED was improved by transplantation of VEGF165-transfected EPCs into the CC of diabetic rats [67]. In a previous study, tissue repair and angiogenesis were synergistically promoted by the combined transplantation of MSCs and EPCs [68].
There are many unanswered questions, e.g., the question of single versus multiple injections. A single injection of MSCs may be insuffi cient for the maintenance of a long-term therapeutic effect. Similarly, the management of potency by multiple MSC injections within a short interval and the dosage of MSC infusions need to be further explored.
Because of the complicated pathogenesis of diabetes mellitus, intrinsic dysfunction of the bone marrow SC niche ultimately results in MSC failure [23]. Some strategies for reducing the functional inability of BM-MSCs need to be recognized. HbA 1c reduction and insulin requirements require close clinical follow-up to improve the effi cacy of MSC treatment in type II diabetes [23]. Similarly, ED studies using a diabetic animal model need to observe changing insulin resistance when using MSC therapies. While available data from animal and human studies are encouraging, MSC therapy may signify a new paradigm for glycemic control in type-2 diabetes.
The main biocomponent of the secretome is the exosome, which is a naturally occurring membrane nanoparticle of 30-120 nm in diameter that mediates intercellular communication by delivering biomolecules into recipient cells [69,70]. Exosomes carry many molecules, including miRNAs, proteins, and lipids as a composite cargo, as well as the exosome cargo, which is transferable to different cell types. These recipient cells undergo expressional and functional changes with exosome uptake [71,72]. The role of exosomes in diabetic ED needs further study.
The nanosized exosomes derived from MSCs may become a valuable therapeutic strategy in regenerative therapies compared with transplanted exogenous MSCs. There are many advantages of nanosized exosomes compared with exogenous MSCs. Exosomes are more natural to preserve and transfer, have lower immunogenicity, and are safer for therapeutic administration [73]. Exosomes derived from BMSCs may become a treatment for diabetes-induced ED. MSCs with hypoxic preconditioning may provide additional benefi t in diabetes-induced ED, due to increased angiogenesis and neuroprotection [74].
Erythropoietin [75] is a potent cytokine capable of reducing apoptosis of Schwann cells. However, the expression of EPO in MSC is limited, though overexpression of EPO in MSC signifi cantly improves neuroprotective actions. EPO-MSCs have the potential to reduce apoptosis of diabetes-triggered Schwann cells. Thus, suppression is likely due to the reduction of oxidative stress and apoptosis-related protein factors. Studies have revealed that the placenta (P)-derived MSCs have potent paracrine and differentiation potential effects in diabetic nude rats [76]. P-MSCs that survived three weeks accelerated the recovery of ischemic damage by increased generation of arterioles, the formation of capillaries, and the secretion of various proangiogenic factors [76].
MSCs combined with pioglitazone, or exendin-4 demonstrated substantial benefi t compared with MSCs alone in regards to cardioprotective effects [77]. Recently, Jeon et al. [78] showed that stromal cell-derived factor-(SDF)-1-expressing engineered MSCs improved erectile function in STZ-induced diabetic ED rats [23]. A recent phase-I clinical study proved the safety, tolerability, and effi ciency of intracavernous autologous BM-MSC injections to treat ED in diabetic patients [79].

ADSCs
Adipose tissue is also a possible source for SCs, as ADSCs have self-renewal and multipotency characteristics similar to BMSCs [20,21]. The main advantages of ADSCs are that they are accessible to culture and easily collected from patients by a minimally invasive procedure, such as liposuction. The successful transplantation of allogeneic and xenogeneic ADSC illustrates their low immunogenicity [80,81]. Growing evidence suggests the success of ADSC in several ED models [82]. Intracavernosal unmodifi ed ADSCs have been shown to restore erectile function in numerous rat ED models [42,83].
The preservation of neuronal and endothelial cells of CC in rat ED models has been observed after the intracavernous administration of cultured ADSCs [42,46]. Rats with diabetic ED treated with autologous ADSCs displayed improvement of erectile function, as well as reduced apoptosis of cavernosal tissues, but few labeled ADSCs were identifi ed [46]. The therapeutic benefi t of ADSCs appears to be an indirect mechanism, whereby ADSCs improve the extracellular environment and local tissue function via the direct transformation of ADSCs into local cell types [46]. Intracavernosal injection of ADSC to a VEGF-treated group of ED in a rodent diabetic model demonstrated improved erectile function linked to an amplifi ed expression of smooth muscle, endothelial, and pericyte markers [84]. The potential of ADSCs to regenerate and repair various tissues deserves more focus [85]. At eight weeks, erectile function was restored by increased endothelial and smooth muscle cells, nNOS-positive nerve fi bers, and eNOS phosphorylation in diabetic mice [86].
This benefi t witnessed in animal models [45,87] suggests that SC therapy may recover erectile function in humans [23].
Furthermore, the overexpression of adrenomedullin by ADSC enhanced erectile function in diabetic rats, likely by amplifi ed VE-cadherin and eNOS expressions in diabetic rats [88].
Various forms of fi brosis involve the TGF1-Smad signaling pathway. Hepatocyte growth factor (HGF) is known to inhibit the TGF1-Smad signaling pathway and attenuate renal fi brosis in diabetic rats [89,90]. Similarly, penile fi brosis occurs as a pathological response to diabetes. Erectile function was improved by ADSC monotherapy in streptozotocin-induced diabetic rats, and the benefi t was augmented when combined with HGF, resulting in a higher number of endothelial and smooth muscle cells and a lower cell apoptotic index in the CC [91].
In other experiments, a streptozotocin-diabetic rat, transplantation with pigment epithelium-derived factor (PEDF)-transfected ADSCs successfully improved ICP/MAP ratios as compared with untreated ADSC [92]. Overexpression of PEDF resulted in higher survival rates and decreased apoptosis of ADSC [92]. ADSC transplantation restored erectile function in a diabetic rat model by attenuating the harmful effects of hyperglycemia. Thus, the therapeutic potential of ADSC for treating ED, as well as the additional benefi ts of PEDF overexpression, is an exciting development [92]. At the early stages of elevated glucose levels in type-2 diabetic rats,  [95]. ADSC-derived exosomes also induced a benefi cial effect on erectile function in a type-2 diabetic rat model [96]. Exosomes were isolated from the supernatants of cultured ADSC by ultracentrifugation. ADSCderived exosomes, similar to ADSC, were capable of rescuing CC endothelial and smooth muscle cells by inhibiting apoptosis and thus promoting the recovery of erectile function in a type-2 diabetic rat model (using a high-fat diet and low-dose streptozotocin administered by intraperitoneal injection) [64].
ADSCs-based Microtissues (MT) in STZ-induced diabetic rats with ED induced expression of Nerve Growth Factor (NGF), VEGF, and tumor necrosis factor-stimulated gene-6 [97]. Also, MT treatment improved ICP, nNOS levels, and endothelial and smooth muscle contents and reduced local infl ammation in the CC of diabetic rats. MTs combined with intracavernosal ADSC enhanced erectile function and histopathological changes in streptozotocin-induced diabetic rats [97]. Very recently, the injection of ADSCs into the tunica albuginea during the active phase of Peyronie's disease prevents the development of fi brosis [98].
ADSCs and platelet-rich plasma co-transplantation is an attractive option in therapies using autologous cells.
However, transplantation of ADSCs is often exposed to hostile environments in which local oxidative stress, hypoxia, and infl ammation induce early cell loss. Reduced survival of transplanted ADSCs will dramatically reduce their therapeutic effects. Of note, a current study in a rat model of type 2 diabetes showed that hypoxia-preconditioning promoted ADSC-based repair of diabetes-induced ED by augmenting angiogenesis and neuroprotection [99].

Future perspectives
The effects and safety issues for diabetes-associated ED treatment need further delineation to improve the quality of life for affl icted men.
Metabolic disorders are commonly observed after the pathological effects of diabetes have occurred [105]. Further discovery of the pathophysiological mechanisms will come. SPIONs effectively incorporated into ADSCs had no adverse effects on SC properties. ADSCs and platelet-rich plasma co-transplantation is another novel approach to cell therapy in regenerative medicine.
Platelet-rich plasma can enhance the properties of ADSCs and also needs further investigation [108]. Conversely, the processes related to culturing and isolating ADSCs have boundaries; these comprise the high cost of amenities and staff, the underlying threat of contamination with undefi ned proteins and foreign serum, and changes in functional characteristics due to repeated culturing procedures [109,110].
Soluble factors released from MSCs may benefi t MSC effects [111]. High levels of cellular senescence, apoptosis, and altered differentiation capacity in ADSC isolated from type-2 diabetics, have been observed [112]. Thus, the addition of adjuncts that increase differentiation and proliferation is needed to fortify ADSCs. focused on type-1 diabetes, which is an autoimmune condition characterized by a complete loss of insulin secretion, leading to hyperglycemia. Therefore, the development of autologous MSC therapies depends on a better understanding of the extrinsic host milieu on MSC function.
Advancements in technology and experimental techniques have provided an insight into how aging affects the properties of MSCs. Given that human life expectancy is expected to increase, the topic of cell aging and therapeutic applications continues to be an area of interest.
The more recent evidence suggests a developmental affi liation between pericytes and MSCs based on cell markers and differentiation potential [115,116]. As a novel stem cell source, pericytes are generally considered to be the origin of MSCs. Pericytes have crucial roles in blood vessel function/ stability, angiogenesis, endothelial cell proliferation/ differentiation, wound healing, blood-brain barrier function, and hematopoietic stem cell maintenance [117]. All of these properties make pericytes preferred cells in the fi eld of tissue engineering. Similar to other types of stem cells, pericytes act as a repair system in response to injury by maintaining the structural integrity of blood vessels [118]. Pericytes have recently been recognized for their central role in blood vessel formation. Pericytes are multipotent cells that are heterogeneous in their origin, function, morphology, and surface markers. In situ, pericytes are recognized by their localization to the abluminal side of the blood vessel wall and closely associated with endothelial cells, in combination with the expression of markers such as CD146, neural glial 2, platelet derived growth factor receptor , -smooth muscle actin, nestin and/or leptin receptor [116]. Similar to other types of stem cells, pericytes act as a repair system in response to injury by maintaining the structural integrity of blood vessels The role of pericytes is not restricted to the formation and development of the vasculature: they have been shown to possess stem cell-like characteristics and may differentiate into cell types from different lineages. While this assumption relies mainly on indirect evidence, the data supports the possibility that a precursor of the MSC is natively associated with the blood vessel wall and belongs to a subset of perivascular cells. Addressing this aspect may help improve the novelty of the subject in diabetes.

Conclusion
Though few stem cell-based studies have been directed toward type-2 diabetes, MSC-based therapies may provide better multifaceted metabolic corrections and concurrently offer long-term benefi ts to diabetic patients ( Figure 1). MSC therapy in diabetic men with ED appears very close to addressing the effectiveness and safety of regenerative technology ( Figure  1) [119]. MSC seems to be safe and effective in the shorter term and may provide genomic or epigenetic changes in the longer term.
It is useful for future MSC clinical trials to include histology confi rmation and more extensive multicenter trials with various study protocols to compare treatment templates, including dose, duration, and a number of MSC injections [120].
Adult MSC has the advantage of avoiding the ethical issues of ESCs, and besides, published literature shows a very low probability of malignant transformation and tumor formation [121]. Diabetic patients need to be counseled and treated for many problems [122] hopefully, and regenerative effects will soon be brought into clinical practice.