Glutathione dynamics is a potential predictive and therapeutic trait for neoadjuvant chemotherapy response in bladder cancer.

Radical cystectomy with preoperative cisplatin-based neoadjuvant chemotherapy (NAC) is the standard care for muscle-invasive bladder cancers (MIBCs). However, the complete response rate to this modality remains relatively low, and current clinicopathologic and molecular classifications are inadequate to predict NAC response in patients with MIBC. Here, we demonstrate that dysregulation of the glutathione (GSH) pathway is fundamental for MIBC NAC resistance. Comprehensive analysis of the multicohort transcriptomes reveals that GSH metabolism and immune-response genes are enriched in NAC-resistant and NAC-sensitive MIBCs, respectively. A machine-learning-based tumor/stroma classifier is applied for high-throughput digitalized immunohistochemistry analysis, finding that GSH dynamics proteins, including glutaminase-1, are associated with NAC resistance. GSH dynamics is activated in cisplatin-resistant MIBC cells, and combination treatment with a GSH dynamics modulator and cisplatin significantly suppresses tumor growth in an orthotopic xenograft animal model. Collectively, these findings demonstrate the predictive and therapeutic values of GSH dynamics in determining the NAC response in MIBCs.

Activating transcription factor-2 supports the antioxidant capacity and ability of human mesenchymal stem cells to prevent asthmatic airway inflammation.

Glutathione (GSH), an abundant nonprotein thiol antioxidant, participates in several biological processes and determines the functionality of stem cells. A detailed understanding of the molecular network mediating GSH dynamics is still lacking. Here, we show that activating transcription factor-2 (ATF2), a cAMP-response element binding protein (CREB), plays a crucial role in maintaining the level and activity of GSH in human mesenchymal stem cells (MSCs) by crosstalking with nuclear factor erythroid-2 like-2 (NRF2), a well-known master regulator of cellular redox homeostasis. Priming with ascorbic acid 2-glucoside (AA2G), a stable vitamin C derivative, increased the expression and activity of ATF2 in MSCs derived from human embryonic stem cells and umbilical cord. Subsequently, activated ATF2 crosstalked with the CREB1-NRF2 pathway to preserve the GSH dynamics of MSCs through the induction of genes involved in GSH synthesis (GCLC and GCLM) and redox cycling (GSR and PRDX1). Accordingly, shRNA-mediated silencing of ATF2 significantly impaired the self-renewal, migratory, proangiogenic, and anti-inflammatory capacities of MSCs, and these defects were rescued by supplementation of the cells with GSH. In addition, silencing ATF2 attenuated the ability of MSCs to alleviate airway inflammatory responses in an ovalbumin-induced mouse model of allergic asthma. Consistently, activation of ATF2 by overexpression or the AA2G-based priming procedure enhanced the core functions of MSCs, improving the in vivo therapeutic efficacy of MSCs for treating asthma. Collectively, our findings suggest that ATF2 is a novel modulator of GSH dynamics that determines the core functionality and therapeutic potency of MSCs used to treat allergic asthma.

Intrinsic Phase Stability and Inherent Bandgap of Formamidinium Lead Triiodide Perovskite Single Crystals.

Understanding the intrinsic phase stability and inherent band gap of formamidinium lead triiodide (FAPbI3 ) perovskites is crucial to further improve the performance of perovskite solar cells (PSCs). Herein, we explored the α- to δ-phase transition and band gap of FAPbI3 single crystals grown by an inverse temperature solubility method. We found that the residual γ-butyrolactone solvents in the inner empty space of the FAPbI3 single crystal accelerate the phase transition at kinetics. By adopting 2-methoxyethanol as the solvent, over 2000 h of stable α-FAPbI3 crystals could be acquired. This proves that although FAPbI3 is regarded as unstable at thermodynamics, it could own excellent kinetic stability without any doping or additives because of the slow solid to solid phase transition instead of the fast phase transition assisted by the solvents. Furthermore, we revealed that the bulk FAPbI3 single crystal with a size above 100 µm can have an inherent band gap of 1.41 eV. Thus, our work provides key scientific guidance for high-performance FAPbI3 -based PSCs.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer.

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

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.

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.

Transglutaminase 2 mediates hypoxia-induced selective mRNA translation via polyamination of 4EBPs.

Hypoxia selectively enhances mRNA translation despite suppressed mammalian target of rapamycin complex 1 activity, contributing to gene expression reprogramming that promotes metastasis and survival of cancer cells. Little is known about how this paradoxical control of translation occurs. Here, we report a new pathway that links hypoxia to selective mRNA translation. Transglutaminase 2 (TG2) is a hypoxia-inducible factor 1-inducible enzyme that alters the activity of substrate proteins by polyamination or crosslinking. Under hypoxic conditions, TG2 polyaminated eukaryotic translation initiation factor 4E (eIF4E)-bound eukaryotic translation initiation factor 4E-binding proteins (4EBPs) at conserved glutamine residues. 4EBP1 polyamination enhances binding affinity for Raptor, thereby increasing phosphorylation of 4EBP1 and cap-dependent translation. Proteomic analyses of newly synthesized proteins in hypoxic cells revealed that TG2 activity preferentially enhanced the translation of a subset of mRNA containing G/C-rich 5'UTRs but not upstream ORF or terminal oligopyrimidine motifs. These results indicate that TG2 is a critical regulator in hypoxia-induced selective mRNA translation and provide a promising molecular target for the treatment of cancers.

Estimation of spatially continuous daytime particulate matter concentrations under all sky conditions through the synergistic use of satellite-based AOD and numerical models.

Satellite-derived aerosol optical depth (AOD) products are one of main predictors to estimate ground-level particulate matter (PM10 and PM2.5) concentrations. Since AOD products, however, are only provided under high-quality conditions, missing values usually exist in areas such as clouds, cloud shadows, and bright surfaces. In this study, spatially continuous AOD and subsequent PM10 and PM2.5 concentrations were estimated over East Asia using satellite- and model-based data and auxiliary data in a Random Forest (RF) approach. Data collected from the Geostationary Ocean Color Imager (GOCI; 8 times per day) in 2016 were used to develop AOD and PM models. Three schemes (i.e. G1, A1, and A2) were proposed for AOD modeling according to target AOD data (GOCI AOD and AERONET AOD) and the existence of satellite-derived AOD. The A2 scheme showed the best performance (validation R2 of 0.74 and prediction R2 of 0.73 when GOCI AOD did not exist) and the resultant AOD was used to estimate spatially continuous PM concentrations. The PM models with location information produced successful estimation results with R2 of 0.88 and 0.90, and rRMSE of 26.9 and 27.2% for PM10 and PM2.5, respectively. The spatial distribution maps of PM well captured the seasonal and spatial characteristics of PM reported in the literature, which implies the proposed approaches can be adopted for an operational estimation of spatially continuous AOD and PMs under all sky conditions.

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.

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.

Efficient, stable solar cells by using inherent bandgap of α-phase formamidinium lead iodide.

In general, mixed cations and anions containing formamidinium (FA), methylammonium (MA), caesium, iodine, and bromine ions are used to stabilize the black α-phase of the FA-based lead triiodide (FAPbI3) in perovskite solar cells. However, additives such as MA, caesium, and bromine widen its bandgap and reduce the thermal stability. We stabilized the α-FAPbI3 phase by doping with methylenediammonium dichloride (MDACl2) and achieved a certified short-circuit current density of between 26.1 and 26.7 milliamperes per square centimeter. With certified power conversion efficiencies (PCEs) of 23.7%, more than 90% of the initial efficiency was maintained after 600 hours of operation with maximum power point tracking under full sunlight illumination in ambient conditions including ultraviolet light. Unencapsulated devices retained more than 90% of their initial PCE even after annealing for 20 hours at 150°C in air and exhibited superior thermal and humidity stability over a control device in which FAPbI3 was stabilized by MAPbBr3.

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.

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.