Assessment of the Therapeutic Effectiveness of Glutathione-Enhanced Mesenchymal Stem Cells in Rat Models of Chronic Bladder Ischemia-Induced Overactive Bladder and Detrusor Underactivity.

Overactive bladder (OAB) and detrusor underactivity (DUA) are representative voiding dysfunctions with a chronic nature and limited treatment modalities, and are ideal targets for stem cell therapy. In the present study, we investigated the therapeutic efficacy of human mesenchymal stem cells (MSCs) with a high antioxidant capacity generated by the Primed Fresh OCT4 (PFO) procedure in chronic bladder ischemia (CBI)-induced OAB and DUA rat models. Sixteen-week-old male Sprague-Dawley rats were divided into three groups (sham, OAB or DUA, and stem cell groups; n=10, respectively). CBI was induced by bilateral iliac arterial injury (OAB, 10 times; DUA, 30 times) followed by a 1.25% cholesterol diet for 8 weeks. Seven weeks after injury, rats in the stem cell and other groups were injected with 1×106 PFO-MSCs and phosphate buffer, respectively. One week later, bladder function was analyzed by awake cystometry and bladders were harvested for histological analysis. CBI with a high-fat diet resulted in atrophy of smooth muscle and increased collagen deposits correlating with reduced detrusor contractility in both rat models. Arterial injury 10 and 30 times induced OAB (increased number of non-voiding contractions and shortened micturition interval) and DUA (prolonged micturition interval and increased residual volume), respectively. Injection of PFO-MSCs with the enhanced glutathione dynamics reversed both functional and histological changes; it restored the contractility, micturition interval, residual volume, and muscle layer, with reduced fibrosis. CBI followed by a high-fat diet with varying degrees of arterial injury induced OAB and DUA in rats. In addition, PFO-MSCs alleviated functional and histological changes in both rat models.

Therapeutic effects of axitinib, an anti-angiogenic tyrosine kinase inhibitor, on interstitial cystitis.

To investigate the therapeutic effects of axitinib, a tyrosine kinase inhibitor, in an interstitial cystitis (IC) rat model. IC patients with or without Hunner lesion and non-IC controls were enrolled (n = 5/group). Bladder tissues were stained with vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), platelet-derived growth factor (PDGF), and PDGF receptor B (PDGFR-B). The IC group showed extensive VEGFR-2 and PDGFR-B staining compared with controls. Next, ten-week-old female Sprague Dawley rats were divided into three groups (n = 10/group): sham, hydrochloride (HCl), and axitinib groups. One week after HCl instillation (day 0), the axitinib group received oral axitinib (1 mg/kg) for five consecutive days and pain was evaluated daily. Bladder function, histology and genetics were evaluated on day 7. The pain threshold significantly improved 3 days after axitinib administration. Axitinib decreased non-voiding contraction and increased the micturition interval and micturition volume and alleviated urothelial denudation, angiogenesis, mast cell infiltration, and fibrosis. HCl instillation increased the expression of tyrosine kinase receptors, including VEGFR-2 and PDGFR-B; axitinib administration inhibited their expression. Oral administration of axitinib improved pain, voiding profiles, and urothelial integrity by inhibiting angiogenesis in IC rat model. Axitinib may have potential therapeutic efficacy in IC patients.

Safety of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells for Treating Interstitial Cystitis: A Phase I Study.

There are still no definite treatment modalities for interstitial cystitis (IC). Meanwhile, stem cell therapy is rising as potential alternative for various chronic diseases. This study aimed to investigate the safety of the clinical-grade mesenchymal stem cells (MSCs) derived from human embryonic stem cells (hESCs), code name MR-MC-01 (SNU42-MMSCs), in IC patients. Three female IC patients with (1) symptom duration >6 months, (2) visual pain analog scale (VAS) ≥4, and (3) one or two Hunner lesions <2 cm in-office cystoscopy within 1 month were included. Under general anesthesia, participants received cystoscopic submucosal injection of SNU42-MMSCs (2.0 × 107/5 mL) at the center or margin of Hunner lesions and other parts of the bladder wall except trigone with each injection volume of 1 mL. Follow-up was 1, 3, 6, 9, and 12 months postoperatively. Patients underwent scheduled follow-ups, and symptoms were evaluated with validated questionnaires at each visit. No SNU42-MMSCs-related adverse events including immune reaction and abnormalities on laboratory tests and image examinations were reported up to 12-month follow-up. VAS pain was temporarily improved in all subjects. No de novo Hunner lesions were observed and one lesion of the first subject was not identifiable on 12-month cystoscopy. This study reports the first clinical application of transurethral hESC-derived MSC injection in three patients with IC. hESC-based therapeutics was safe and proved to have potential therapeutic efficacy in IC patients. Stem cell therapy could be a potential therapeutic option for treating IC.

Intravital imaging and single cell transcriptomic analysis for engraftment of mesenchymal stem cells in an animal model of interstitial cystitis/bladder pain syndrome.

Mesenchymal stem cell (MSC) therapy is a promising treatment for various intractable disorders including interstitial cystitis/bladder pain syndrome (IC/BPS). However, an analysis of fundamental characteristics driving in vivo behaviors of transplanted cells has not been performed, causing debates about rational use and efficacy of MSC therapy. Here, we implemented two-photon intravital imaging and single cell transcriptome analysis to evaluate the in vivo behaviors of engrafted multipotent MSCs (M-MSCs) derived from human embryonic stem cells (hESCs) in an acute IC/BPS animal model. Two-photon imaging analysis was performed to visualize the dynamic association between engrafted M-MSCs and bladder vasculature within live animals until 28 days after transplantation, demonstrating the progressive integration of transplanted M-MSCs into a perivascular-like structure. Single cell transcriptome analysis was performed in highly purified engrafted cells after a dual MACS-FACS sorting procedure and revealed expression changes in various pathways relating to pericyte cell adhesion and cellular stress. Particularly, FOS and cyclin dependent kinase-1 (CDK1) played a key role in modulating the migration, engraftment, and anti-inflammatory functions of M-MSCs, which determined their in vivo therapeutic potency. Collectively, this approach provides an overview of engrafted M-MSC behavior in vivo, which will advance our understanding of MSC therapeutic applications, efficacy, and safety.

A Preclinical Study of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells for Treating Detrusor Underactivity by Chronic Bladder Ischemia.

BACKGROUND: The therapeutic effects of human embryonic stem cell-derived multipotent mesenchymal stem cells (M-MSCs) were evaluated for detrusor underactivity (DUA) in a rat model with atherosclerosis-induced chronic bladder ischemia (CBI) and associated mechanisms. METHODS: Sixteen-week-old male Sprague-Dawley rats were divided into five groups (n = 10). The DUA groups underwent 30 bilateral repetitions of endothelial injury to the iliac arteries to induce CBI, while the sham control group underwent a sham operation. All rats used in this study received a 1.25% cholesterol diet for 8 weeks. M-MSCs at a density of 2.5, 5.0, or 10.0 × 105 cells (250 K, 500 K, or 1000 K; K = a thousand) were injected directly into the bladder 7 weeks post-injury, while the sham and DUA group were treated only with vehicle (phosphate buffer solution). One week after M-MSC injection, awake cystometry was performed on the rats. Then, the bladders were harvested, studied in an organ bath, and prepared for histological and gene expression analyses. RESULTS: CBI by iliac artery injury reproduced voiding defects characteristic of DUA with decreased micturition pressure, increased micturition interval, and a larger residual volume. The pathological DUA properties were improved by M-MSC treatment in a dose-dependent manner, with the 1000 K group producing the best efficacy. Histological analysis revealed that M-MSC therapy reduced CBI-induced injuries including bladder fibrosis, muscular loss, and apoptosis. Transplanted M-MSCs mainly engrafted as vimentin and NG2 positive pericytes rather than myocytes, leading to increased angiogenesis in the CBI bladder. Transcriptomes of the CBI-injured bladders were characterized by the complement system, inflammatory, and ion transport-related pathways, which were restored by M-MSC therapy. CONCLUSIONS: Single injection of M-MSCs directly into the bladder of a CBI-induced DUA rat model improved voiding profiles and repaired the bladder muscle atrophy in a dose-dependent manner.

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.

Glutathione dynamics determine the therapeutic efficacy of mesenchymal stem cells for graft-versus-host disease via CREB1-NRF2 pathway.

Glutathione (GSH), the most abundant nonprotein thiol functioning as an antioxidant, plays critical roles in maintaining the core functions of mesenchymal stem cells (MSCs), which are used as a cellular immunotherapy for graft-versus-host disease (GVHD). However, the role of GSH dynamics in MSCs remains elusive. Genome-wide gene expression profiling and high-throughput live-cell imaging assays revealed that CREB1 enforced the GSH-recovering capacity (GRC) of MSCs through NRF2 by directly up-regulating NRF2 target genes responsible for GSH synthesis and redox cycling. MSCs with enhanced GSH levels and GRC mediated by CREB1-NRF2 have improved self-renewal, migratory, anti-inflammatory, and T cell suppression capacities. Administration of MSCs overexpressing CREB1-NRF2 target genes alleviated GVHD in a humanized mouse model, resulting in improved survival, decreased weight loss, and reduced histopathologic damages in GVHD target organs. Collectively, these findings demonstrate the molecular and functional importance of the CREB1-NRF2 pathway in maintaining MSC GSH dynamics, determining therapeutic outcomes for GVHD treatment.

Ascorbic Acid 2-Glucoside Stably Promotes the Primitiveness of Embryonic and Mesenchymal Stem Cells Through Ten-Eleven Translocation- and cAMP-Responsive Element-Binding Protein-1-Dependent Mechanisms.

Aims: The naive or primitive states of stem cells (SCs) residing in specific niches are unstable and difficult to preserve in vitro. Vitamin C (VitC), in addition to suppressing oxygen radicals, exerts pleiotropic effects to preserve the core functions of SCs. However, this compound is labile and readily oxidized, resulting in cellular toxicity and preventing its reliable application in this context. We found that a VitC derivative, ascorbic acid 2-glucoside (AA2G), stably maintains the naive pluripotency of murine embryonic SCs (mESCs) and the primitiveness of human mesenchymal SCs (hMSCs) without cellular toxicity. Results: The beneficial effects of AA2G and related molecular mechanisms were evaluated in mESCs, induced pluripotent-SCs (iPSCs), and hMSCs. AA2G was stable in aqueous solution and barely induced cellular toxicity in cultured SCs, unlike VitC. AA2G supplementation recapitulated the well-known effects of VitC, including induction of ten-eleven translocation-dependent DNA demethylation in mESCs and suppression of p53 during generation of murine iPSCs. Furthermore, supplementation of hMSCs with AA2G improved therapeutic outcomes in an asthma mouse model by promoting their self-renewal, engraftment, and anti-inflammatory properties. Particularly, activation of the cAMP-responsive element-binding protein-1 (CREB1) pathway contributed to the ability of AA2G to maintain naive pluripotency of mESCs and functionality of hMSCs. Innovation and Conclusion: Given its long-lasting effects and low cellular toxicity, AA2G supplementation is useful to support the naive pluripotency of mESCs and the primitiveness of hMSCs, affecting their developmental potency and therapeutic efficacy. Furthermore, we demonstrate the significance of the CREB1 pathway in the mechanism of action of AA2G.

Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis.

Molecular programs involved in embryogenesis are frequently upregulated in oncogenic dedifferentiation and metastasis. However, their precise roles and regulatory mechanisms remain elusive. Here, we showed that CDK1 phosphorylation of TFCP2L1, a pluripotency-associated transcription factor, orchestrated pluripotency and cell-cycling in embryonic stem cells (ESCs) and was aberrantly activated in aggressive bladder cancers (BCs). In murine ESCs, the protein interactome and transcription targets of Tfcp2l1 indicated its involvement in cell cycle regulation. Tfcp2l1 was phosphorylated at Thr177 by Cdk1, which affected ESC cell cycle progression, pluripotency, and differentiation. The CDK1-TFCP2L1 pathway was activated in human BC cells, stimulating their proliferation, self-renewal, and invasion. Lack of TFCP2L1 phosphorylation impaired the tumorigenic potency of BC cells in a xenograft model. In patients with BC, high co-expression of TFCP2L1 and CDK1 was associated with unfavorable clinical characteristics including tumor grade, lymphovascular and muscularis propria invasion, and distant metastasis and was an independent prognostic factor for cancer-specific survival. These findings demonstrate the molecular and clinical significance of CDK1-mediated TFCP2L1 phosphorylation in stem cell pluripotency and in the tumorigenic stemness features associated with BC progression.

Current and Future Directions of Stem Cell Therapy for Bladder Dysfunction.

Stem cells are capable of self-renewal and differentiation into a range of cell types and promote the release of chemokines and progenitor cells necessary for tissue regeneration. Mesenchymal stem cells are multipotent progenitor cells with enhanced proliferation and differentiation capabilities and less tumorigenicity than conventional adult stem cells; these cells are also easier to acquire. Bladder dysfunction is often chronic in nature with limited treatment modalities due to its undetermined pathophysiology. Most treatments focus on symptom alleviation rather than pathognomonic changes repair. The potential of stem cell therapy for bladder dysfunction has been reported in preclinical models for stress urinary incontinence, overactive bladder, detrusor underactivity, and interstitial cystitis/bladder pain syndrome. Despite these findings, however, stem cell therapy is not yet available for clinical use. Only one pilot study on detrusor underactivity and a handful of clinical trials on stress urinary incontinence have reported the effects of stem cell treatment. This limitation may be due to stem cell function loss following ex vivo expansion, poor in vivo engraftment or survival after transplantation, or a lack of understanding of the precise mechanisms of action underlying therapeutic outcomes and in vivo behavior of stem cells administered to target organs. Efficacy comparisons with existing treatment modalities are also needed for the successful clinical application of stem cell therapies. This review describes the current status of stem cell research on treating bladder dysfunction and suggests future directions to facilitate clinical applications of this promising treatment modality, particularly for bladder dysfunction.

Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels.

We tried to establish a reliable detrusor underactivity (DUA) rat model and to investigate pathophysiology of chronic bladder ischemia (CBI) on voiding behavior and bladder function. Adult male rats were divided into five groups. The arterial injury (AI) groups (AI-10, AI-20, AI-30) underwent vascular endothelial damage (VED) of bilateral iliac arteries (with 10, 20, and 30 bilateral repetitions of injury, respectively) and received a 1.25% cholesterol diet. The sham group underwent sham operation and received the same diet. Controls received a regular diet. After 8 weeks, all rats underwent unanesthetized cystometrogram. Bladder tissues were processed for organ bath investigation, immunohistochemistry staining, and genome-wide gene expression analysis. Awake cystometry analysis showed that frequency of voiding contractions and micturition pressure were lower in the AI-30 group than in sham group (p < 0.01). Contractile responses to various stimuli were lower in AI-20 and AI-30 groups (both p < 0.001). In the AI-20 and AI-30 groups, atherosclerotic occlusion in the iliac arteries, tissue inflammation, fibrosis, denervation, and apoptosis of bladder muscle were prominent compared to the sham. Mechanistically, the expression of purinergic receptor P2X-1 was reduced in the AI-30 group, and the genome-wide gene expression analysis revealed that genes related to IL-17 and HIF-1 signaling pathways including INF-γ receptor-1 and C-X-C motif chemokine ligand-2 were upregulated in the CBI-induced DUA rat model. A rat model of progressive VED successfully induced DUA. Abnormal tissue inflammation, fibrosis, denervation, and bladder muscle tissue apoptosis may be involved in CBI-induced DUA pathophysiology.

N-acetylcysteine prevents bladder tissue fibrosis in a lipopolysaccharide-induced cystitis rat model.

Therapeutic options for non-Hunner type interstitial cystitis (IC), which is histologically characterized by fibrosis and mast cell infiltration, are limited. We developed a rat model that replicates chronic inflammation and fibrosis and evaluated the therapeutic effect of N-acetylcysteine (NAC), a well-known anti-fibrotic agent, on the model. Intravesical instillation of lipopolysaccharide (LPS, 750 µg) after protamine sulfate (10 mg) was conducted twice per week for five consecutive weeks. One week after final instillation, 200 mg/kg NAC (n = 10, IC + NAC group) or phosphate-buffered saline (n = 10, IC group) was daily injected intraperitoneally once daily for 5 days. LPS instillation induced bladder fibrosis, mast cell infiltration, and apoptotic tissue damage. Functionally, LPS insult led to irregular micturition, decreased inter-contraction intervals, and decreased micturition volume. NAC significantly improved most of the voiding parameters and reversed histological damages including fibrosis. NAC inhibited the induction and nuclear localization of phospho-Smad2 protein in bladder tissues and the upregulation of genes related to fibrosis, such as Tgfb2, Tgfb3, Smad2, Smad3, Cxcl10, and Card10. This is the first study to demonstrate the beneficial effects on NAC in restoring voiding function, relieving tissue fibrosis and related bladder injuries, in the LPS-induced cystitis rat model.

Diagnosis in a Preclinical Model of Bladder Pain Syndrome Using a Au/ZnO Nanorod-based SERS Substrate.

To evaluate the feasibility of ZnO nanorod-based surface enhanced Raman scattering (SERS) diagnostics for disease models, particularly for interstitial cystitis/bladder pain syndrome (IC/BPS), ZnO-based SERS sensing chips were developed and applied to an animal disease model. ZnO nanorods were grown to form nano-sized porous structures and coated with gold to facilitate size-selective biomarker detection. Raman spectra were acquired on a surface enhanced Raman substrate from the urine in a rat model of IC/BPS and analyzed using a statistical analysis method called principal component analysis (PCA). The nanorods grown after the ZnO seed deposition were 30 to 50 nm in diameter and 500 to 600 nm in length. A volume of gold corresponding to a thin film thickness of 100 nm was deposited on the grown nanorod structure. Raman spectroscopic signals were measured in the scattered region for nanometer biomarker detection to indicate IC/BPS. The Raman peaks for the control group and IC/BPS group are observed at 641, 683, 723, 873, 1002, 1030, and 1355 cm-1, which corresponded to various bonding types and compounds. The PCA results are plotted in 2D and 3D. The Raman signals and statistical analyses obtained from the nano-sized biomarkers of intractable inflammatory diseases demonstrate the possibility of an early diagnosis.

Synergistic Effects of N-Acetylcysteine and Mesenchymal Stem Cell in a Lipopolysaccharide-Induced Interstitial Cystitis Rat Model.

The purpose of this study was to reduce the amount of stem cells used in treating preclinical interstitial cystitis (IC model) by investigating the synergistic effects of multipotent mesenchymal stem cells (M-MSCs; human embryonic stem cell-derived) and N-acetylcysteine (NAC). Eight-week-old female Sprague-Dawley rats were divided into seven groups, i.e., sham (n = 10), lipopolysaccharide/protamine sulfate (LPS/PS; n = 10), LPS/PS + NAC (n = 10), LPS/PS with 25K MSC (n = 10), LPS/PS with 50K MSC (n = 10) LPS/PS + 25K MSC + NAC (n = 10), and LPS/PS + 50K MSC + NAC (n = 10). To induce the IC rat model, protamine sulfate (10 mg, 45 min) and LPS (750 µg, 30 min) were instilled once a week for five consecutive weeks via a transurethral PE-50 catheter. Phosphate-buffered saline (PBS) was used in the sham group. One week after the final instillation, M-MSCs with two suboptimal dosages (i.e., 2.5 or 5.0 × 104 cells) were directly transplanted into the outer-layer of the bladder. Simultaneously, 200 mg/kg of NAC or PBS was intraperitoneally injected daily for five days. The therapeutic outcome was evaluated one week after M-MSC or PBS injection by awake cystometry and histological analysis. Functionally, LPS/PS insult led to irregular micturition, decreased intercontraction intervals, and decreased micturition volume. Both monotherapy and combination therapy significantly increased contraction intervals, increased urination volume, and reduced the residual volume, thereby improving the urination parameters compared to those of the LPS group. In particular, a combination of NAC dramatically reduced the amount of M-MSCs used for significant restoration in histological damage, including inflammation and apoptosis. Both M-MSCs and NAC-based therapy had a beneficial effect on improving voiding dysfunction, regenerating denudated urothelium, and relieving tissue inflammation in the LPS-induced IC/BPS rat model. The combination of M-MSC and NAC was superior to MSC or NAC monotherapy, with therapeutic efficacy that was comparable to that of previously optimized cell dosage (1000K) without compromised therapeutic efficacy.

Longitudinal intravital imaging of transplanted mesenchymal stem cells elucidates their functional integration and therapeutic potency in an animal model of interstitial cystitis/bladder pain syndrome.

Rationale: Mesenchymal stem cell (MSC) therapy may be a novel approach to improve interstitial cystitis/bladder pain syndrome (IC/BPS), an intractable disease characterized by severe pelvic pain and urinary frequency. Unfortunately, the properties of transplanted stem cells have not been directly analyzed in vivo, which hampers elucidation of the therapeutic mechanisms of these cells and optimization of transplantation protocols. Here, we monitored the behaviors of multipotent stem cells (M-MSCs) derived from human embryonic stem cells (hESCs) in real time using a novel combination of in vivo confocal endoscopic and microscopic imaging and demonstrated their improved therapeutic potency in a chronic IC/BPS animal model. Methods: Ten-week-old female Sprague-Dawley rats were instilled with 10 mg of protamine sulfate followed by 750 µg of lipopolysaccharide weekly for 5 weeks. The sham group was instilled with phosphate-buffered saline (PBS). Thereafter, the indicated dose (0.1, 0.25, 0.5, and 1×106 cells) of M-MSCs or PBS was injected once into the outer layer of the bladder. The distribution, perivascular integration, and therapeutic effects of M-MSCs were monitored by in vivo endoscopic and confocal microscopic imaging, awake cystometry, and histological and gene expression analyses. Results: A novel combination of longitudinal intravital confocal fluorescence imaging and microcystoscopy in living animals, together with immunofluorescence analysis of bladder tissues, demonstrated that transplanted M-MSCs engrafted following differentiation into multiple cell types and gradually integrated into a perivascular-like structure until 30 days after transplantation. The beneficial effects of transplanted M-MSCs on bladder voiding function and the pathological characteristics of the bladder were efficient and long-lasting due to the stable engraftment of these cells. Conclusion: This longitudinal bioimaging study of transplanted hESC-derived M-MSCs in living animals reveals their long-term functional integration, which underlies the improved therapeutic effects of these cells on IC/BPS.

Downregulation of WNT11 is associated with bladder tissue fibrosis in patients with interstitial cystitis/bladder pain syndrome without Hunner lesion.

This study assessed the functional role of WNT genes and the association between WNT signalling cascades and fibrosis in interstitial cystitis/bladder pain syndrome (IC/BPS) patients. Twenty-five patients (3 males, 22 females; mean age 59.7 ± 10.9 years), included 7 non-Hunner-type IC (NHIC), 18 Hunner-type IC (HIC), and 5 non-IC (control) groups. The expression of sonic hedgehog, WNT gene family, and genes previously reported as biomarkers for IC/BPS were examined using RT-PCR in biopsy specimens from the mucosa and submucosa layer of the bladder. WNT2B, WNT5A, WNT10A, and WNT11 functions in the urothelium were evaluated by silencing in an HBlEpC cell line. Pelvic Pain and Urgency/Frequency Patient Symptom Scale scores, O'Leary-Sant Symptom and Problem Index scores, and Visual Analogue Scores did not differ between the NHIC and HIC groups. However, HIC patients had significantly shorter symptom duration (30.9 vs 70.8 months, p = 0.046), higher daily urinary frequency (16.1 versus 8.5 times, p = 0.006), and smaller bladder capacity (208.6 versus 361.4 ml, p = 0.006) than NHIC patients. Overall WNT gene expression was lower in NHIC than HIC patients. Bladder epithelial tissues from HIC patients were characterised by the downregulation of WNT11. Silencing of WNT11, WNT2B, WNT5A, and WNT10A in HBlEpCs resulted in fibrotic changes, indicated by fibrotic morphology, increased fibrosis-related gene expression, and nuclear localisation of phosphorylated SMAD2, and increased vimentin and fibronectin levels. Downregulation of WNT11 results in fibrotic changes of bladder epithelial cells and is associated with the pathogenesis and differential diagnosis of NHIC. Decreased expression of WNT11 is a potential biomarker for predicting NHIC.

Small hypoxia-primed mesenchymal stem cells attenuate graft-versus-host disease.

Mesenchymal stem cells (MSCs) are of particular interest for the treatment of immune-related diseases due to their immunosuppressive capacity. Here, we show that Small MSCs primed with Hypoxia and Calcium ions (SHC-MSCs) exhibit enhanced stemness and immunomodulatory functions for treating allogeneic conflicts. Compared with naïve cultured human umbilical cord blood-derived MSCs, SHC-MSCs were resistant to passage-dependent senescence mediated via the monocyte chemoattractant protein-1 and p53/p21 cascade and secreted large amounts of pro-angiogenic and immunomodulatory factors, resulting in suppression of T-cell proliferation. SHC-MSCs showed DNA demethylation in pluripotency, germline, and imprinted genes similarly to very small embryonic-like stem cells, suggesting a potential mutual relationship. Genome-wide DNA methylome and transcriptome analyses indicated that genes related to immune modulation, cell adhesion, and the cell cycle were up-regulated in SHC-MSCs. Particularly, polo-like kinase-1 (PLK1), zinc-finger protein-143, dehydrogenase/reductase-3, and friend-of-GATA2 play a key role in the beneficial effects of SHC-MSCs. Administration of SHC-MSCs or PLK1-overexpressing MSCs significantly ameliorated symptoms of graft-versus-host disease (GVHD) in a humanized mouse model, resulting in significantly improved survival, less weight loss, and reduced histopathologic injuries in GVHD target organs compared with naïve MSC-infused mice. Collectively, our findings suggest that SHC-MSCs can improve the clinical treatment of allogeneic conflicts, including GVHD.

The Therapeutic Effect of Human Embryonic Stem Cell-Derived Multipotent Mesenchymal Stem Cells on Chemical-Induced Cystitis in Rats.

PURPOSE: To evaluate the therapeutic effect of human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (M-MSCs) on ketamine-induced cystitis (KC) in rats. METHODS: To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS) was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×106 cells) of M-MSCs (KC+M-MSC group) or PBS vehicle (KC group) were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×105 cells) to that of an identical dose of adult bone marrow (BM)-derived MSCs. RESULTS: Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×105) of cells, at which point BM-derived MSCs did not substantially improve bladder function. CONCLUSIONS: This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic damage.

Improved efficacy and in vivo cellular properties of human embryonic stem cell derivative in a preclinical model of bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease characterized by severe pelvic pain and urinary frequency. Mesenchymal stem cell (MSC) therapy is a promising approach to treat incurable IC/BPS. Here, we show greater therapeutic efficacy of human embryonic stem cell (hESC)-derived multipotent stem cells (M-MSCs) than adult bone-marrow (BM)-derived counterparts for treating IC/BPS and also monitor long-term safety and in vivo properties of transplanted M-MSCs in living animals. Controlled hESC differentiation and isolation procedures resulted in pure M-MSCs displaying typical MSC behavior. In a hydrochloric-acid instillation-induced IC/BPS animal model, a single local injection of M-MSCs ameliorated bladder symptoms of IC/BPS with superior efficacy compared to BM-derived MSCs in ameliorating bladder voiding function and histological injuries including urothelium denudation, mast-cell infiltration, tissue fibrosis, apoptosis, and visceral hypersensitivity. Little adverse outcomes such as abnormal growth, tumorigenesis, or immune-mediated transplant rejection were observed over 12-months post-injection. Intravital confocal fluorescence imaging tracked the persistence of the transplanted cells over 6-months in living animals. The infused M-MSCs differentiated into multiple cell types and gradually integrated into vascular-like structures. The present study provides the first evidence for improved therapeutic efficacy, long-term safety, and in vivo distribution and cellular properties of hESC derivatives in preclinical models of IC/BPS.