Effects of Pulsed Electric Field Pretreatment on Black Tea Processing and Its Impact on Cold-Brewed Tea.

This study applied pulsed electric fields (PEFs) to accelerate the withering and drying processes during cold-brewed black tea production. PEF pretreatment was administered at 1.0, 1.5, and 2.0 kV/cm electric field strengths, combined with varying withering times from 8 to 12 hr. During the 12-hour withering process, the redness value (a*) and total color change (∆E) of PEF-treated leaves significantly increased (p < 0.05). Furthermore, the homogenous redness of tea leaves during fermentation depended on the PEF strength applied. In addition, PEF pretreatment remarkably reduced the drying time, up to a 50% reduction at a 2.0 kV/cm field strength. Additionally, the 2.0 kV/cm PEF-pretreated black tea exhibited a notable 42% increase in theaflavin (TF) content and a 54% increase in thearubigin (TR) content. Sensory evaluation scores were highest for black tea that received PEF pretreatment at 2.0 kV/cm. These findings highlight the significant potential of PEFs in enhancing the efficiency of withering and drying processes while positively impacting the physicochemical and sensory properties of cold-brewed black tea.

The Effects of Jet-Milling and Pulsed Electric Fields on the Preservation of Spinach Juice Lutein Contents during Storage.

This study aimed to investigate the effects of jet-milling on the lutein extraction contents of spinach powder (SP), as well as the effects of pulsed electric field (PEF), as a non-thermal pasteurization technology, on the preservation of spinach juice (SJ) lutein contents. SP particles were divided into SP-coarse (Dv50 = 315.2 µm), SP-fine (Dv50 = 125.20 µm), and SP-superfine (Dv50 = 5.59 µm) fractions, and SP-superfine was added to SJ due to its having the highest contents of lutein extract. PEFs and thermal treatment were applied to evaluate the effects of preserving the lutein content of PEF during storage (25 days). The juice was then designated as untreated (no pasteurization), PEF-1,2 (SJ treated with PEF 20 kV/cm 110 kJ/L, 150 kJ/L), or Thermal-1,2 (SJ treated with 90 °C, 10 min and 121 °C, 15 min). The sizes and surface shapes of the superfine SP particles were more homogeneous and smoother than those of the other samples. SJ made with SP-superfine and treated with PEF had the highest lutein content and antioxidant activities among the group during storage. A complex of jet-milling and PEF could have great potential as a method to improve the lutein contents of lutein-enriched juice in the food industry.

IFITM3-mediated activation of TRAF6/MAPK/AP-1 pathways induces acquired TKI resistance in clear cell renal cell carcinoma.

PURPOSE: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. MATERIALS AND METHODS: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. RESULTS: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. CONCLUSIONS: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

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.

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.

Synaptophysin, CD117, and GATA3 as a Diagnostic Immunohistochemical Panel for Small Cell Neuroendocrine Carcinoma of the Urinary Tract.

Although SCNEC is based on its characteristic histology, immunohistochemistry (IHC) is commonly employed to confirm neuroendocrine differentiation (NED). The challenge here is that SCNEC may yield negative results for traditional neuroendocrine markers. To establish an IHC panel for NED, 17 neuronal, basal, and luminal markers were examined on a tissue microarray construct generated from 47 cases of 34 patients with SCNEC as a discovery cohort. A decision tree algorithm was employed to analyze the extent and intensity of immunoreactivity and to develop a diagnostic model. An external cohort of eight cases and transmission electron microscopy (TEM) were used to validate the model. Among the 17 markers, the decision tree diagnostic model selected 3 markers to classify NED with 98.4% accuracy in classification. The extent of synaptophysin (>5%) was selected as the initial parameter, the extent of CD117 (>20%) as the second, and then the intensity of GATA3 (≤1.5, negative or weak immunoreactivity) as the third for NED. The importance of each variable was 0.758, 0.213, and 0.029, respectively. The model was validated by the TEM and using the external cohort. The decision tree model using synaptophysin, CD117, and GATA3 may help confirm NED of traditional marker-negative SCNEC.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Valproic acid enforces the priming effect of sphingosine-1 phosphate on human mesenchymal stem cells.

Engraftment and homing of mesenchymal stem cells (MSCs) are modulated by priming factors including the bioactive lipid sphingosine-1-phosphate (S1P), by stimulating CXCR4 receptor signaling cascades. However, limited in vivo efficacy and the remaining priming molecules prior to administration of MSCs can provoke concerns regarding the efficiency and safety of MSC priming. Here, we showed that valproic acid (VPA), a histone deacetylase inhibitor, enforced the priming effect of S1P at a low dosage for human umbilical cord-derived MSCs (UC-MSCs). A DNA-methylation inhibitor, 5-azacytidine (5-Aza), and VPA increased the expression of CXCR4 in UC-MSCs. In particular, UC-MSCs primed with a suboptimal dose (50 nM) of S1P in combination with 0.5 mM VPA (VPA+S1P priming), but not 1 µM 5-Aza, significantly improved the migration activity in response to stromal cell-derived factor 1 (SDF-1) concomitant with the activation of both MAPKp42/44 and AKT signaling cascades. Both epigenetic regulatory compounds had little influence on cell surface marker phenotypes and the multi-potency of UC-MSCs. In contrast, VPA+S1P priming of UC-MSCs potentiated the proliferation, colony forming unit-fibroblast, and anti-inflammatory activities, which were severely inhibited in the case of 5-Aza treatment. Accordingly, the VPA+S1P-primed UC-MSCs exhibited upregulation of a subset of genes related to stem cell migration and anti-inflammation response. Thus, the present study demonstrated that VPA enables MSC priming with S1P at a low dosage by enhancing their migration and other therapeutic beneficial activities. This priming strategy for MSCs may provide a more efficient and safe application of MSCs for treating a variety of intractable disorders.