Hwan Yeul Yu

Mesenchymal Stem Cell Therapy Alleviates Interstitial Cystitis by Activating Wnt Signaling Pathway

Interstitial cystitis (IC) is a syndrome characterized by urinary urgency, frequency, pelvic pain, and nocturia in the absence of bacterial infection or identifiable pathology. IC is a devastating disease that certainly decreases quality of life. However, the causes of IC remain unknown and no effective treatments or cures have been developed. This study evaluated the therapeutic potency of using human umbilical cord-blood-derived mesenchymal stem cells (UCB-MSCs) to treat IC in a rat model and to investigate its responsible molecular mechanism. IC was induced in 10-week-old female Sprague-Dawley rats via the instillation of 0.1 M HCl or phosphate-buffered saline (PBS; sham). After 1 week, human UCB-MSC (IC+MSC) or PBS (IC) was directly injected into the submucosal layer of the bladder. A single injection of human UCB-MSCs significantly attenuated the irregular and decreased voiding interval in the IC group. Accordingly, denudation of the epithelium and increased inflammatory responses, mast cell infiltration, neurofilament production, and angiogenesis observed in the IC bladders were prevented in the IC+MSC group. The injected UCB-MSCs successfully engrafted to the stromal and epithelial tissues and activated Wnt signaling cascade. Interference with Wnt and epidermal growth factor receptor activity by small molecules abrogated the benefits of MSC therapy. This is the first report that provides an experimental evidence of the therapeutic effects and molecular mechanisms of MSC therapy to IC using an orthodox rat animal model. Our findings not only provide the basis for clinical trials of MSC therapy to IC but also advance our understanding of IC pathophysiology.

Mesenchymal stem cells protect against the tissue fibrosis of ketamine-induced cystitis in rat bladder

Abuse of the hallucinogenic drug ketamine promotes the development of lower urinary tract symptoms that resemble interstitial cystitis. The pathophysiology of ketamine-induced cystitis (KC) is largely unknown and effective therapies are lacking. Here, using a KC rat model, we show the therapeutic effects of human umbilical cord-blood (UCB)-derived mesenchymal stem cells (MSCs). Daily injection of ketamine to Sprague-Dawley rats for 2-weeks resulted in defective bladder function, indicated by irregular voiding frequency, increased maximum contraction pressure, and decreased intercontraction intervals and bladder capacity. KC bladders were characterized by severe mast-cell infiltration, tissue fibrosis, apoptosis, upregulation of transforming growth factor-β signaling related genes, and phosphorylation of Smad2 and Smad3 proteins. A single administration of MSCs (1 × 106) into bladder tissue not only significantly ameliorated the aforementioned bladder voiding parameters, but also reversed the characteristic histological and gene-expression alterations of KC bladder. Treatment with the antifibrotic compound N-acetylcysteine also alleviated the symptoms and pathological characteristics of KC bladder, indicating that the antifibrotic capacity of MSC therapy underlies its benefits. Thus, this study for the first-time shows that MSC therapy might help to cure KC by protecting against tissue fibrosis in a KC animal model and provides a foundation for clinical trials of MSC therapy.

Histopathological characteristics of interstitial cystitis/bladder pain syndrome without Hunner lesion

To assess the distinct histopathological characteristics and their clinical significance between non‐Hunner‐type and Hunner‐type interstitial cystitis (IC)/bladder pain syndrome (BPS).

The fibrosis of ketamine, a noncompetitive N-methyl-d-aspartic acid receptor antagonist dose-dependent change in a ketamine-induced cystitis rat model

Abstract Ketamine abusers have greatly increased in number worldwide during recent years. The consumption of ketamine has increased, as have the number of published accounts of devastating urological sequelae. However, the mechanism of ketamine-associated urinary tract dysfunction remains unclear. This study was to evaluate the ketamine dose-dependency of ketamine-induced cystitis (KC) in a rat model. A total of 42 Sprague-Dawley rats (female, 10-week-old) were used. Each of the 7 KC rat models were induced by 1, 5, 10, 25 and 50 mg/kg ketamine intravenous injection for two weeks. For the sham group (n = 7), a phosphate-buffered saline (PBS) vehicle was used rather than ketamine hydrochloride. The cystometric parameters, histological examinations, staining for Masson’s trichome, cytokeratin, toluidine blue and quantitative PCR were measured at two weeks following the intervention. The voiding interval gradually decreased depending upon the ketamine dose of 1, 5, 10, 25, or 50 mg/kg, respectively, and was decreased compared with Sham. Bladder capacity was decreased as ketamine dose increased. In particular, the increase of fibrosis and submucosal apoptosis were found according to the increase of the ketamine dose. The bladder apoptosis in the KC rat model makes the fibrotic bladder change, and led us to hypothesize that fibrosis could contribute to the lower urinary-tract symptoms. We suggest that according to the pathophysiology evidence, fibrosis induced by apoptosis plays a key role in KC.

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.

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.

MP38-01 DEVELOPMENT OF ATHEROSCLEROSIS-INDUCED CHRONIC PELVIC ISCHEMIA RAT MODEL FOR REPRODUCING THE DETRUSOR UNDERACTIVITY

INTRODUCTION AND OBJECTIVES: To our best knowledge, animal models reproducing detrusor underactivity (DUA) are scarce. Previous studies suggested that atherosclerosis, a common agingassociated disorder, has a role in the pathogenesis of lower urinary tract dysfunction, such as DUA. We tried to develop a rat model of atherosclerosis-induced chronic bladder ischemia and investigate the effect of chronic bladder ischemia on voiding behavior and bladder function. METHODS: Adult male rats were divided into four groups. The arterial injury (AI) group underwent endothelial injury of the iliac arteries (AI-10, 10 times of injury at each iliac artery; AI-30, 30 times) and received a 2% cholesterol diet. The sham group underwent sham operation and received a 2% cholesterol diet. The control group received a regular diet. After 8 weeks, a metabolic cage study and cystometry were performed without anesthesia. Histological examination of the iliac arteries and the bladder was performed. The bladder was also processed for organ bath study. RESULTS: The metabolic cage study showed that in the AI-30 group, micturition interval, voided volume, and residual volume were significantly increased. Cystometry showed that the frequency of reflex bladder contractions and micturition pressure were significantly lower in the AI-30 group. Histological study showed that in the AI group alone, atherosclerotic occlusion occurred in the iliac arteries as well as in the downstream bladder microvessels. Contractile responses of bladder strips to various stimuli in AI-30 group were significantly less than in sham group (Figure). CONCLUSIONS: Pelvic arterial occlusive disease plus vascular endothelial dysfunction may cause progressive vascular damage resulting in bladder dysfunction that develops the detrusor underactivity

PD33-02 MESENCHYMAL STEM CELLS IMPROVED BLADDER FUNCTION AND HISTOPATHOLOGICAL FEATURES IN PROTAMINE SULFATE-LIPOPOLYSACCHARIDE INDUCED INTERSTITIAL CYSTITISIN RAT BLADDER

INTRODUCTION AND OBJECTIVES: To evaluate the therapeutic effect of mesenchymal stem cells (MSCs)inthe chronic interstitial cystitis (IC) rat model. METHODS: To make IC rat model female 8-week-old SpragueDawley ratswere given protamine sulfate (PS, 10mg) and lipopolysaccharide (LPS, 750ug) weekly for 5 weeks. One week after final administration of PS/LPS, rat were divided into five groups: sham (n1⁄410); PBS (n1⁄410, IC); 0.25 million of MSCs (n1⁄410, ICþMSCs); 0.5 million of MSCs (n1⁄410, ICþMSCs); 1 million of MSCs (n1⁄410, ICþMSCs) were injected into the submucosal layer of the anterior wall and dome of the bladder.The therapeutic effect of MSCs was examined by awakecystometry,histological and gene expression analysis 1 week after MSCs injection. RESULTS: IC rat groupexhibited irregular voiding frequency, decreased inter-contraction intervals, micturition volume and increased residual volume. A single injection of MSCs significantly improved most of voiding parameters by increased the inter-contraction interval, increased micturition volume and decreased residual volume. The bladder of IC group rats were characterized with severe denudated urothelium with inflammation, increased mastcell infiltration and paralleled with down-regulation of Wnt-8a, Wnt-8b and Wnt-11. MSCs therapy significantly upregulatedShh and Wnt family genes (Smo, Wnt5a, Wnt8a, Wnt8b, Wnt10a, and Wnt11) as well as their downstream growth factors (Igf1, Igf2) which were characteristically downregulated in bladders of the LPS-IC group. A single transplantation of 1X106 MSCs (LPS-IC þ MSCs group) significantly ameliorated thesedefective voiding parameters.Of note, a single injection of MSCs significantly not only improved the bladder voiding parameters but also reversed the histological and gene expression alternations characteristic for IC bladder. CONCLUSIONS: We demonstrate that MSC therapy have therapeutic effect to repair voiding function and regenerate denudated urothelium and stabilize mast cell infiltration in the most proper IC rat model.

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.

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.