Therapeutic Efficacy of Human Embryonic Stem Cell-Derived Multipotent Stem/Stromal Cells in Diabetic Detrusor Underactivity: A Preclinical Study

Mesenchymal stem/stromal cell (MSC) therapy is a promising approach for treatment of as yet incurable detrusor underactivity (DUA), which is characterized by decreased detrusor contraction strength and/or duration, leading to prolonged bladder emptying. In the present study, we demonstrated the therapeutic potential of human embryonic stem cell (ESC)-derived multipotent MSCs (M-MSCs) in a diabetic rat model of DUA. Diabetes mellitus (DM) was induced by intraperitoneal injection of streptozotocin (STZ) (50 mg/kg) into 8-week-old female Sprague-Dawley rats. Three weeks later, various doses of M-MSCs (0.25, 0.5, and 1 × 106 cells) or an equivalent volume of PBS were injected into the outer layer of the bladder. Awake cystometry, organ bath, histological, and gene expression analyses were evaluated 1 week (short-term) or 2 and 4 weeks (long-term) after M-MSC transplantation. STZ-induced diabetic rats developed DUA, including phenotypes with significantly longer micturition intervals, increased residual urine amounts and bladder capacity, decreased micturition pressure on awake cystometry, and contractile responses to various stimuli in organ bath studies. Muscle degeneration, mast cell infiltration, fibrosis, and apoptosis were present in the bladders of DM animals. A single local transplantation of M-MSCs ameliorated DUA bladder pathology, including functional changes and histological evaluation, and caused few adverse outcomes. Immunostaining and gene expression analysis revealed that the transplanted M-MSCs supported myogenic restoration primarily by engrafting into bladder tissue via pericytes, and subsequently exerting paracrine effects to prevent apoptotic cell death in bladder tissue. The therapeutic efficacy of M-MSCs was superior to that of human umbilical cord-derived MSCs at the early time point (1 week). However, the difference in efficacy between M-MSCs and human umbilical cord-derived MSCs was statistically insignificant at the later time points (2 and 4 weeks). Collectively, the present study provides the first evidence for improved therapeutic efficacy of a human ESC derivative in a preclinical model of DM-associated DUA.


Animal Model and Study Design
Female Sprague-Dawley rats (8 weeks old) were used in this study. streptozotocin (STZ) (Sigma Chemical Company, St. Louis, MO, USA) was used to induce type I diabetes. After fasting overnight, rats were injected intraperitoneally with STZ (50 mg/kg) dissolved in 0.1 M citrate acid buffer (pH 4.5) solution. For the non-diabetic control group, rats were injected with an equivalent volume of citrate buffer solution. Blood glucose level was measured 72 h later using samples obtained by tail prick. Rats with blood glucose higher than 200 mg/dL (16.7 mmoL/L) were selected as diabetic rats and used for subsequent studies. Three weeks after induction of diabetes, diabetic rats were anesthetized with 0.2 mL tiletamine (Zoletil1; Virbac Laboratories, Carros, France) for stem cell transplantation. To evaluate the therapeutic efficacy of M-MSCs, the rats were distributed into five groups: non-diabetic control (sham), untreated diabetic (DM), and diabetic rats that received a single administration of the indicated dosage of human ESC-derived M-MSCs (0.25, 0.5, and 1 × 10 6 cells; DM groups + 250 K, 500 K, 1000 K) resuspended in 200 µL phosphate buffered saline (PBS). For non-diabetic and untreated DM groups, PBS was injected instead of stem cells. PBS and M-MSCs were directly injected into the outer layer (serosa) of the anterior wall of the bladder as previously described [24,26]. Human UC-MSCs were transplanted using the same procedure. The therapeutic effect of stem cell transplantation was examined using awake cystometry and organ bath evaluation, as well as histological and gene expression analysis 1 week (short-term) or 2 and 4 weeks (long-term) after stem cell injection.

Evaluation of Bladder Function and Tissue Preparation
Cystometric evaluation was performed in conscious unrestrained animals in metabolic cages. Three days prior to the cystometrogram, intravesical pressure (IVP) and intra-abdominal pressure (IAP) were measured. The urethra was approached using a PE-50 catheter (Clay Adams, Parsippany, NJ, USA) connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) and a microinjection pump (PHD22/2000 pump; Harvard Apparatus). Voiding volumes were recorded by means of a fluid collector connected to a force displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus) as normal saline was infused into the bladder at a rate of 0.4 mL/min. IVP, IAP, and voiding volume were recorded continuously using Acq Knowledge 3.8.1 software and an MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) at a sampling rate of 50 Hz. The mean values from three reproducible voiding cycles of each individual animal were used for the analysis. Non-voiding contractions (NVCs) were considered to have occurred when the IVP increments exceeded 15 cm H 2 O from baseline in the absence of expelled urine. Bladder pressure (BP) was defined as the lowest bladder pressure during filling; micturition pressure was defined as the maximum BP during the micturition cycle; micturition volume (MV) was defined as the volume of expelled urine; and residual volume (RV) was defined as the urine volume remaining after voiding. Consistent with prior studies, the micturition interval (MI) was defined as the interval between micturition contraction cycle, representing the times between resting to basal pressure in the preceding micturition cycle and rising from basal pressure in the following cycle. Bladder capacity (BC) was defined as MV + RV. Bladder voiding efficiency (BVE) was defined as 100 × MV/BC. The mean values from three reproducible micturition cycles were evaluated in five individual animals per group. After evaluation of voiding function, rat bladders were harvested. Half of each bladder was cryopreserved in liquid nitrogen for RNA isolation. The remaining half was fixed in 4% buffered formalin and embedded in paraffin for histological examination or immunohistochemical staining.

Gene Expression Analyses
Total RNA was isolated using a RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). Reverse transcription was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). The indicated transcripts were quantified by real-time quantitative PCR (RQ-PCR) using a PikoReal Real-Time PCR System (Thermo Scientific, Foster City, CA, USA) and iQ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA), as described previously [30,31]. The threshold cycle (Ct) of the indicated genes was subtracted from that of Gapdh, an endogenous control gene, and the expression level of the target genes was quantified as 100% × 2 −∆Ct(target-Gapdh) , representing the percentage of Gapdh. Duplicate RQ-PCR assays were performed using five separate animals per group and the mean values from each individual animal were used for the analysis.

Organ Bath Study
Bladders (n = 5 animals/group) were cut into two strips with the mucosa along the longitudinal axis. The strips were mounted in an organ bath system (Danish Myo Technology, Aarhus, Denmark) containing 15 mL Krebs buffer. Bladder strips were subjected to a resting tension of 1 g and allowed to stabilize for at least 60 min. Contractions were recorded as changes in bladder strip tension from baseline in response to 80 mM KCl, a concentration gradient of carbachol (3-100 mM), electrical field stimulation (EFS; 1,2,4,8,16, and 32 Hz), and 1 mM ATP. All tissue responses (g) were normalized to tissue weight (g tissue) for the analysis (g/g tissue). Drug concentrations are expressed as final concentration in the organ bath.

Statistical Analysis
Data are expressed as means ± standard error of the mean (SEM), and were analyzed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA). Statistical significance was assessed using a one-way or two-way ANOVA followed by Bonferroni post-hoc tests. A p-value < 0.05 was considered statistically significant. Source data with the exact experimental values, number of replicates, and statistical test results can be found in the Supplementary Table.
We next examined the overall contractile response in an organ bath study. Consistent with the awake cystometry results, bladder strips from the DM group exhibited significant defects in the contractile responses to 80 mM KCl, 1 mM ATP; a defective frequency response to EFS; and an impaired concentration response curve to carbachol (Cch) relative to non-diabetic animals. M-MSC therapy significantly restored defects in contractile responses to these stimuli ( Figure 1c).

Long-Term Therapeutic Effects of M-MSC Transplantation
In our previous study of an IC/BPS rat model, the therapeutic effects of a single M-MSC administration on bladder voiding function were sustained for 2-4 weeks following transplantation [26]. Therefore, we examined the long-term therapeutic effects of M-MSCs in the diabetic DUA model by measuring bladder voiding parameters using awake cystometry 2 and 4 weeks after a single administration of 1 × 10 6 M-MSCs. The DM group presented a gradually elongated MI, larger RV, and decreased micturition. In contrast to the short-term (1-week) follow-up, M-MSC transplantation did not substantially restore DUA voiding phenotypes in either the 2-or 4-week long-term follow-ups (Figure 2a,b). In the long-term follow-ups, minimal effects were found in the BVE in all tested groups (Figure 2b).

Long-Term Therapeutic Effects of M-MSC Transplantation
In our previous study of an IC/BPS rat model, the therapeutic effects of a single M-MSC administration on bladder voiding function were sustained for 2-4 weeks following transplantation [26]. Therefore, we examined the long-term therapeutic effects of M-MSCs in the diabetic DUA model by measuring bladder voiding parameters using awake cystometry 2 and 4 weeks after a single administration of 1 × 10 6 M-MSCs. The DM group presented a gradually elongated MI, larger RV, and decreased micturition. In contrast to the short-term (1-week) follow-up, M-MSC transplantation did not substantially restore DUA voiding phenotypes in either the 2-or 4-week long-term followups (Figure 2a,b). In the long-term follow-ups, minimal effects were found in the BVE in all tested groups (Figure 2b).

Determining Optimal Cell Dosage for M-MSC Therapy
Considering the risk of neoplasia in stem cell therapy, in addition to the time and cost required for isolation and expansion of stem cells, determining the critical cell dosage for therapeutic efficacy is of significant clinical relevance. Therefore, we reduced the transplanted cell numbers to 2.5 × 10 5 (250 K) and 5.0 × 10 5 (500 K), and examined the therapeutic efficacies of these dosages in diabetic DUA over the short-term follow-up period. Awake cystometry revealed that the significant beneficial effects of M-MSCs on bladder voiding functions were also conferred by administration of as few as 2.5 × 10 5 M-MSCs (Figure 3a,b), although lower dosages of M-MSCs had decreased therapeutic effects relative to the 1 × 10 6 M-MSC group. Therapeutic efficacy did not significantly differ between the 250 K and 500 K groups.
To further compare the dose-dependent effects of M-MSC transplantation, we evaluated the severity of pathologies present in diabetic DUA patients. Histological examinations revealed that severe muscle atrophy, tissue fibrosis, and infiltration of toluidine blue-stained mast cells were present in DM bladders (Figure 4a

Determining Optimal Cell Dosage for M-MSC Therapy
Considering the risk of neoplasia in stem cell therapy, in addition to the time and cost required for isolation and expansion of stem cells, determining the critical cell dosage for therapeutic efficacy is of significant clinical relevance. Therefore, we reduced the transplanted cell numbers to 2.5 × 10 5 (250 K) and 5.0 × 10 5 (500 K), and examined the therapeutic efficacies of these dosages in diabetic DUA over the short-term follow-up period. Awake cystometry revealed that the significant beneficial effects of M-MSCs on bladder voiding functions were also conferred by administration of as few as 2.5 × 10 5 M-MSCs (Figure 3a,b), although lower dosages of M-MSCs had decreased therapeutic effects relative to the 1 × 10 6 M-MSC group. Therapeutic efficacy did not significantly differ between the 250 K and 500 K groups. confer significant therapeutic effects. These histological findings, together with the awake cystometry results, demonstrated that a single administration of 5.0 × 10 5 M-MSCs into the bladder was the optimal cell dosage for treatment of diabetic DUA.   Three representative areas per slide were randomly selected from five individual animals per group. Data were normalized against sham-operated rats and are presented as mean ± SEM (n = 5, *** p < 0.001, relative to DM group; # p < 0.05 and ### p < 0.001 relative to 500 K group, one-way ANOVA with Bonferroni post-hoc comparison). The exact experimental and statistical values can be found in the Supplementary

Therapeutic Efficacy of M-MSCs Relative to Umbilical Cord-Derived Stem Cells
We next compared the therapeutic efficacy of M-MSCs by transplanting either 5.0 × 10 5 M-MSCs or 5.0 × 10 5 human UC-MSCs into DM animals, followed by short-term (1-week) follow-up. Awake cystometry revealed that administration of UC-MSCs also decreased MI, increased micturition pressure, and decreased post-void RV in DM animals (Figure 5a,b). However, animals that received UC-MSC transplantation exhibited more frequent NVCs relative to animals that received M-MSC therapy, suggesting that M-MSCs offered superior stabilization of intravesical pressure (Figure 5a,b). Therefore, transplantation of M-MSCs conferred more potent and stable therapeutic outcomes than UC-MSCs.

Therapeutic Efficacy of M-MSCs Relative to Umbilical Cord-Derived Stem Cells
We next compared the therapeutic efficacy of M-MSCs by transplanting either 5.0 × 10 5 M-MSCs or 5.0 × 10 5 human UC-MSCs into DM animals, followed by short-term (1-week) follow-up. Awake cystometry revealed that administration of UC-MSCs also decreased MI, increased micturition pressure, and decreased post-void RV in DM animals (Figure 5a,b). However, animals that received UC-MSC transplantation exhibited more frequent NVCs relative to animals that received M-MSC therapy, suggesting that M-MSCs offered superior stabilization of intravesical pressure (Figure 5a,b). Therefore, transplantation of M-MSCs conferred more potent and stable therapeutic outcomes than UC-MSCs.

Cellular Properties of Transplanted M-MSCs
Because M-MSC transplantation significantly restored muscle degeneration, we determined whether engrafted M-MSCs could promote regeneration of the detrusor muscle in DM animals 1 week after GFP + M-MSC transplantation. Co-immunostaining of GFP + cells with two muscle-specific proteins, α-SMA and desmin, revealed that the majority of the engrafted M-MSCs did not robustly express muscle markers, although some GFP + /α-SMA + double positive cells were detected proximal to the serosa where M-MSCs were injected (Figure 6a). However, cells with strong GFP signals were frequently detected proximal to the muscular bundle.
In our previous studies of IC/BPS, the majority of transplanted M-MSCs were integrated into pericytes, expressing the pericyte markers vimentin or NG2 [24,26]. Therefore, we further characterized the GFP + transplanted cells by co-staining with the pericyte markers vimentin and NG2. The majority of GFP + cells located between the serosa and muscularis were strongly labeled with both vimentin and NG2 (Figure 6b). In particular, the engrafted GFP + cells proximal to the muscular bundle strongly expressed the pericyte-specific NG2 protein, indicating that implanted M-MSCs differentiated into stromal and perivascular cells, rather than directly into myocytes. Consistent with

Cellular Properties of Transplanted M-MSCs
Because M-MSC transplantation significantly restored muscle degeneration, we determined whether engrafted M-MSCs could promote regeneration of the detrusor muscle in DM animals 1 week after GFP + M-MSC transplantation. Co-immunostaining of GFP + cells with two muscle-specific proteins, α-SMA and desmin, revealed that the majority of the engrafted M-MSCs did not robustly express muscle markers, although some GFP + /α-SMA + double positive cells were detected proximal to the serosa where M-MSCs were injected (Figure 6a). However, cells with strong GFP signals were frequently detected proximal to the muscular bundle.  In our previous studies of IC/BPS, the majority of transplanted M-MSCs were integrated into pericytes, expressing the pericyte markers vimentin or NG2 [24,26]. Therefore, we further characterized the GFP + transplanted cells by co-staining with the pericyte markers vimentin and NG2. The majority of GFP + cells located between the serosa and muscularis were strongly labeled with both vimentin and NG2 (Figure 6b). In particular, the engrafted GFP + cells proximal to the muscular bundle strongly expressed the pericyte-specific NG2 protein, indicating that implanted M-MSCs differentiated into stromal and perivascular cells, rather than directly into myocytes. Consistent with these results, staining for human specific Ki-67, a cell proliferation marker, was negative in muscle fibers and was only positive in pericytes (Figure 6c). The GFP + cells located in the endothelium slightly overlapped with CD31 + endothelial cells (Figure 6b).

Paracrine Effects of M-MSCs for Suppressing Apoptosis in Diabetic DUA
As the engrafted M-MSCs mainly integrated into pericytes rather than directly into myocytes, we examined whether engrafted M-MSCs could support a microenvironment favorable for restoration of detrusor muscle injury. In the bladders of DM animals, TUNEL + apoptotic cells were significantly increased, while injection of as few as 2.5 × 10 5 M-MSCs ameliorated apoptosis in both the mucosal and muscular layers (Figure 7a,b).
restoration of detrusor muscle injury. In the bladders of DM animals, TUNEL + apoptotic cells were significantly increased, while injection of as few as 2.5 × 10 5 M-MSCs ameliorated apoptosis in both the mucosal and muscular layers (Figure 7a,b).

Discussion
In the present study, the STZ-induced diabetic DUA rat model was used to evaluate the therapeutic effects of M-MSCs derived from human ESCs in DM-related DUA. Additionally, we demonstrated that M-MSCs integrated into pericytes and could exert paracrine effects, not only to restore detrusor muscle damage, but also to prevent apoptosis in diabetic bladders.
To our knowledge, this is the first study to demonstrate the therapeutic efficacy of M-MSCs in a diabetic DUA model. In a previous study by Zhang et al., autologous adipose tissue-derived MSCs (ADSCs, 3.0 × 10 6 cells) were injected via tail vein or directly into bladders. ADSCs significantly

Discussion
In the present study, the STZ-induced diabetic DUA rat model was used to evaluate the therapeutic effects of M-MSCs derived from human ESCs in DM-related DUA. Additionally, we demonstrated that M-MSCs integrated into pericytes and could exert paracrine effects, not only to restore detrusor muscle damage, but also to prevent apoptosis in diabetic bladders.
To our knowledge, this is the first study to demonstrate the therapeutic efficacy of M-MSCs in a diabetic DUA model. In a previous study by Zhang et al., autologous adipose tissue-derived MSCs (ADSCs, 3.0 × 10 6 cells) were injected via tail vein or directly into bladders. ADSCs significantly ameliorated diabetic bladder dysfunction, and the route of stem cell injection did not affect the therapeutic efficacy of ADSCs [36]. In addition, Zhang et al. suggested that defocused low-energy shock waves recruited endogenous stem cells by increasing secretion of NGF and VEGF [37]. In the present preclinical study, we used a lower dose of M-MSCs derived from human ESCs, and the injection route was via direct bladder transplantation, according to our previously developed protocols in the IC/BPS rat model [24,26].
The advantage of ESCs as an alternative source of MSCs is that ESCs can be indefinitely maintained due to their self-renewal capacity, and at the same time can differentiate into any cell type due to their pluripotency [38,39]. Despite these therapeutic advantages, safety concerns such as the risk of tumorigenesis remain a major obstacle to the therapeutic application of hESC-derived cells [40]. However, the recent successful clinical treatment of ocular disorders with hESC-based therapeutics partly diminished this concern [41,42]. Consistently, our previous 1 year study using a chronic IC/BPS model failed to identify adverse outcomes, including tumorigenesis, abnormal growth, immune reactions, or differentiation of transplanted M-MSCs into unwanted cell types [24,26].
Although the present preclinical diabetic DUA study did not identify any adverse reactions, the safety issues surrounding ESC-based therapies must be thoroughly investigated before this modality can be applied in clinical settings [40]. In addition, the potential for rejection via antigenicity and immunological processes should be carefully considered. T-cells and major histocompatibility complex class 1 (MHC-1) play pivotal roles in the rejection of hESCs and their derivatives [43]. However, prior studies identified that the immune-stimulatory capacity of hESCs and their derivatives is very low [44]. In addition, MSCs derived from iPSCs haven been considered as a potential means to circumvent immune rejection.
Prior studies have demonstrated that iPSC-derived MSCs have therapeutic effects in a wide range of intractable disorders in multiple animal models [23,45,46]. Hyperglycemia can induce cellular damage through oxidative stress and other mechanisms. Excess mitochondrial glucose oxidation produces electrons that generate reactive oxygen species [47]. In vivo experimental studies in animal models identified that iPSC-derived MSCs caused functional changes in detrusor smooth muscle, neuronal impairment, and urothelial dysfunction [48]. Further, the severity of diabetic cystopathy depends primarily on disease duration and management.
DUA or UAB result from various pathological processes that can be classified as idiopathic, neurogenic, or myogenic [49]. Idiopathic DUA is defined as being "free of evident neuropathy, functional or anatomical bladder obstruction and shows low or no detrusor pressure combined with low maximum flow and large post-void residual, or urinary retention" [50]. Neurogenic DUA occurs due to any alteration of neural control mechanisms [51]. Myogenic DUA can result from alterations in ion storage and/or exchange, energy generation, or degenerative changes to bladder smooth muscle [52].
Notably, the pathogenesis of DM-associated bladder DUA is multifactorial with changes in the physiology of detrusor smooth muscle cells, innervation or function of the neuronal component, or urothelial dysfunction [53][54][55]. Detrusor contraction is mediated by acetylcholine released by the pelvic nerve acting on muscarinic receptors. Many neurotransmitters such as adenosine triphosphate, nitric oxide, and prostanoids are released from urothelial cells and these mediators can be increased in response to urothelial injury or inflammation, resulting in a variety of abnormalities in DM-induced bladder dysfunction [55]. Therefore, changes in the innervating neurons and the physiology of urothelium are associated with diabetic bladder dysfunction.
The pathophysiology of DUA in our diabetic DUA rat model is presumed to be myogenic. DUA pathology developed 21 days after induction of DM, with increased smooth muscle atrophy and apoptosis leading to decreased detrusor contractility, while little differences were observed in the neurofilaments and urothelium. The significant decrease in detrusor volume in our diabetic model might result in over-expression of receptors associated with detrusor contraction to maintain contraction (Figure 8b-d); however, alteration in the function of these receptors should be evaluated further. In addition, the Ngf transcript level was downregulated in our diabetic model but was not restored to a significant level in one week after M-MSC transplantation (Figure 8a). Reduced NGF production in the bladder or a deficiency in NGF transport to the bladder afferent pathway are observed in patients and animal models with diabetic cystopathy. In their study on rats with STZ-induced DM, Katsumi Sasaki et al. found a significant time dependent decrease in NGF levels in the bladder and in L6-S1 dorsal root ganglia (DRG) that was associated with voiding dysfunction, attributable to defects in δ and C-fiber bladder afferents [56]. Therefore, reduced NGF production in the bladder and/or impaired transport of NGF to L6 to S1 DRG, which contains bladder afferent neurons, could be an important mechanism underlying the induction of diabetic bladder dysfunction [54][55][56]. Therefore, limited recovery of nerves or Ngf expression in present study might be related to the limited long-term efficacy of M-MSC therapy.
According to the histological results, our diabetic DUA rat model was characterized by increased infiltration of mast cells in the bladder, which was significantly prevented by M-MSC transplantation (Figure 4a,c). A recent study employing a mouse ex vivo bladder afferent nerve recording, retrograde tracing of bladder-innervating DRG neurons, and in vivo spinal cord signaling assays revealed that histamine, an inflammatory mast cell mediator, enhances the sensitivity of bladder afferents to distension via interaction with histamine H1 receptor and transient receptor potential vanilloid-1 [53]. Sustained hyperglycemia resulted in oxidative stress and inflammation, which increased the mast cells and histamine production. However, as DM leads to damage in the peripheral neurons of the bladder, the effect of increased mechanosensitivity by the inflammatory mast cell mediator might have been neutralized. Taken together, further studies such as investigating the changes in peripheral nerves including DRG and urothelial physiology or inflammation are required to elucidate whether the mode of action for M-MSC therapy in diabetic cystopathy could include myogenic regeneration as well as denervation secondary to autonomic neuropathy or urothelium restoration.
Acontractile bladder is defined as occurring when "the detrusor cannot contract during urodynamic studies resulting in prolonged bladder emptying within a normal time span," and is generally grouped with DUA under the clinical diagnosis of UAB [57]. Awake cystometry on long-term follow-up of DM animals (2 and 4 weeks following transplantation) revealed severely decreased contractility of the detrusor, which resembled detrusor acontractility or detrusor areflexia. A longer duration of STZ-induced DM could have caused more severe bladder damage that was not responsive to transplantation of M-MSCs. Implementation of stem cell therapy should be considered prior to the development of advanced disease, which is less likely to be responsive to any therapeutic modality.
Previously, we demonstrated that transplanted M-MSCs replenish denuded urothelium in the early stages of disease and progressively contribute to perivascular cells in IC/BPS rat models [24,26]. The precise mechanism for restoration of bladder function in the present diabetic DUA model is unclear, but histological changes suggested that M-MSCs could have ameliorated smooth muscle apoptosis and differentiated into pericytes in vessel-like structures proximal to the muscular layer. The paracrine effects of M-MSCs integrated into the vasculature could promote regeneration of damaged myocytes, restoring detrusor contractility. However, the in-depth mechanisms of action for M-MSC therapy should be further investigated in crucial future studies.
The primary limitation of the present study is that only one diabetic animal model was used. Diabetes can be generally classified as type 1 diabetes (the inability to produce enough insulin due to loss of insulin-producing β-cells) and type 2 diabetes (non-response of peripheral tissues to insulin leading to impaired uptake of glucose from the bloodstream). STZ is toxic to pancreatic β-cells and induces type 1 diabetes. However, most DM patients have type 2 diabetes, which has multifactorial etiologies, including genetics, environment, diet, and autoimmune pathologies [58].
However, because hyperglycemia is the primary causative factor of DM complications, animal models of both type 1 and type 2 diabetes are extensively used for investigation of DM complications.

Conclusions
Effective therapeutic strategies for DUA are limited. Ttransplantation of M-MSCs into the bladders of DM animals alleviated DUA by integrating into pericytes, providing a favorable microenvironment for myogenic restoration in the DM rat model with detrusor muscle atrophy. In addition, M-MSCs more effectively restored bladder function than currently available MSC therapy. However, duration of the therapeutic effect of M-MSC transplantation was short-term, limited to under 2 weeks. Therefore, further investigations are required to further improve the functionality and therapeutic potential of MSC therapy and to successfully translate this preclinical study into clinical applications.