Protein‐free: A hydrogel containing phenylboronate was optimized so as to undergo rapid glucose‐dependent changes in the state of hydration under physiological aqueous conditions. A localized ...dehydration of the gel surface to form a “skin layer” enabled control of the release of insulin from the gel. This dehydration is induced by fluctuations in the glucose concentration in the range between normo‐ and hyperglycemia.
Maintenance of islet function after in vitro culture is crucial for both transplantation and research. Here we evaluated the effects of encapsulation in alginate fiber on the function of human islets ...which were distributed by the Alberta Islet Distribution Program. Encapsulated human islets from 15 deceased donors were cultured under 5.5 or 25 mM glucose conditions in vitro. The amounts of C-peptide and glucagon secreted from encapsulated islets into the culture media were measured periodically, and immunohistochemical studies were performed. Encapsulated islets maintained C-peptide and glucagon secretion for more than 75 days in 5 cases; in two cases, their secretion was also successfully detected even on day 180. α- and β-cell composition and β-cell survival in islets were unaltered in the fiber after 75 or 180 days of culture. The encapsulated islets cultured with 5.5 mM glucose, but not those with 25 mM glucose, exhibited glucose responsiveness of C-peptide secretion until day 180. We demonstrate that alginate encapsulation enabled human islets to maintain their viability and glucose responsiveness of C-peptide secretion after long-term in vitro culture, potentially for more than for 180 days.
Abstract : Objectives : Personal lifestyle, including diet, exercise, and sleep, might have an impact on work engagement, though previous studies have not focused on these relationships. The aim of ...this study was to examine whether dietary intake of fish, regular exercise, sufficient sleep, abstinence from alcohol, and abstinence from tobacco were positively associated with work engagement. Methods : We recruited adults aged 40-74 years who attended the health checkups with a particular focus on the metabolic syndrome in central Tokyo. In December 2015, 797 people responded to a questionnaire and 592 (74.3%) who had regular jobs were selected for this study. Work engagement was assessed on the 9-item Utrecht Work Engagement Scale (UWES-9). Bivariate and multivariate regression analyses were performed to examine the relationships between lifestyle and UWES-9. Results : Dietary intake of fish, regular exercise, sufficient sleep, and abstinence from tobacco were significantly correlated with the total UWES-9 score, even after adjusting for age, sex, and depressive and anxiety symptoms. The results suggested a doseresponse relationship between dietary fish intake and work engagement. Conclusions : Dietary fish intake, regular exercise, sufficient sleep, and abstinence from tobacco might be lifestyle factors that can serve as resources for work engagement. These findings could be useful in motivating employees to make lifestyle improvements and convincing employers and managers that lifestyle is important not only for health but also for productivity.
Dexamethasone (Dex) regulates aqueous humor outflow by inducing reorganization of the cytoskeleton and extracellular matrix (ECM) production. Rho kinase (ROCK) has an important role in this process, ...but the upstream pathway leading to its activation remains elusive. The purpose of the study was to determine the role of autotaxin (ATX), an enzyme involved in the generation of lysophosphatidic acid (LPA), in the Dex-induced fibrotic response and ECM production in human trabecular meshwork (HTM) cells.
The expression of ATX in specimens from glaucoma patients was investigated by immunohistochemistry. Regulation of ATX expression and the changes in actin cytoskeleton, ECM production, myosin light chain (MLC) and cofilin phosphorylation, ATX secretion, and lysophospholipase D (lysoPLD) activity induced by Dex treatment in HTM cells were determined by immunofluorescence, real-time quantitative PCR, immunoblot, and the two-site immunoenzymetric and lysoPLD assays.
Significant ATX expression was found in conventional outflow pathway specimens from glaucoma patients. Dex treatment induced increases in ATX mRNA levels, protein expression, and secretion in HTM cells in association with reorganization of cytoskeleton and ECM accumulation. Significant suppression of these aforementioned changes was observed after ATX/LPA-receptor/ROCK inhibition as well as suppression of fibrotic changes and MLC and cofilin phosphorylation in HTM cells.
The results of this study, including the robust induction of ATX by Dex treatment, in association with fibrotic changes and ECM production in HTM cells, collectively suggest a potential role for ATX-LPA pathway in the regulation of aqueous humor outflow and IOP in glaucomatous eyes.
Commercially available enzyme-linked immunosorbent assay (ELISA) kits are often used to monitor vascular endothelial growth factor (VEGF) levels in exudative age-related macular degeneration. To test ...their accuracy, this study performed measurements using the ELISA kits in the presence of anti-VEGF drugs.
The concentrations of bevacizumab, pegaptanib, or ranibizumab at 28 days and aflibercept at 28 and 56 days after an injection were estimated based on previous pharmacokinetic studies. Vascular endothelial growth factor concentrations were measured with two widely used VEGF ELISA kits in the presence of anti-VEGF drugs or control mouse immunoglobulin G (IgG). The monocyte chemotactic protein-1 (MCP-1) ELISA kit was used as a non-VEGF ELISA control kit.
The concentrations of aflibercept, bevacizumab, pegaptanib, and ranibizumab were estimated at 0.14 to 7.2, 4.9, 8.6, and 0.11 to 1.1 μg/mL, respectively. ELISA underestimated the VEGF concentration 2- to 100-fold lower in the presence of an anti-VEGF drug, except for pegaptanib, at all VEGF concentrations tested (7.8-1500 pg/mL). Vascular endothelial growth factor at 1000 pg/mL was measured as 92, 150, and 170 pg/mL in the presence of aflibercept (7.2 μg/mL), bevacizumab (4.9 μg/mL), and ranibizumab (1.1 μg/mL), respectively (all P < 0.0001), and the measured VEGF concentration decreased proportionately by 90% to 92% with aflibercept, 85% to 94% with bevacizumab, and 83% to 99% with ranibizumab. The control mouse IgG did not interfere with the measurement of VEGF. Ranibizumab did not affect the measurements with MCP-1 ELISA.
Investigators should exercise caution when interpreting measurements of VEGF ELISA in patients being treated with an anti-VEGF drug.
Diabetes mellitus is caused by breakdown of blood glucose homeostasis, which is maintained by an exquisite balance between insulin and glucagon produced respectively by pancreatic beta cells and ...alpha cells. However, little is known about the mechanism of inducing glucagon secretion from human alpha cells. Many methods for generating pancreatic beta cells from human pluripotent stem cells (hPSCs) have been reported, but only two papers have reported generation of pancreatic alpha cells from hPSCs. Because NKX6.1 has been suggested as a very important gene for determining cell fate between pancreatic beta and alpha cells, we searched for the factors affecting expression of NKX6.1 in our beta cell differentiation protocols. We found that BMP antagonism and activation of retinoic acid signaling at stage 2 (from definitive endoderm to primitive gut tube) effectively suppressed NKX6.1 expression at later stages. Using two different hPSCs lines, treatment with BMP signaling inhibitor (LDN193189) and retinoic acid agonist (EC23) at Stage 2 reduced NKX6.1 expression and allowed differentiation of almost all cells into pancreatic alpha cells in vivo after transplantation under a kidney capsule. Our study demonstrated that the cell fate of pancreatic cells can be controlled by adjusting the expression level of NKX6.1 with proper timing of BMP antagonism and activation of retinoic acid signaling during the pancreatic differentiation process. Our method is useful for efficient induction of pancreatic alpha cells from hPSCs.
To complement islet transplantation for type1 diabetic patients, cell-based therapy using pluripotent stem cells such as ES cells and iPS cells is promising. Many papers have already reported the ...induction of pancreatic β cells from these cell types, but a suspension culture system has not usually been employed. The aim of this study is to establish a suspension culture method for inducing functional islet-like cells from human iPS cells.
We used 30 ml spinner type culture vessels for human iPS cells throughout the differentiation process. Differentiated cells were analyzed by immunostaining and C-peptide secretion. Cell transplantation experiments were performed with STZ-induced diabetic NOD/SCID mice. Blood human C-peptide and glucagon levels were measured serially in mice, and grafts were analyzed histologically.
We obtained spherical pancreatic beta-like cells from human iPS cells and detected verifiable amounts of C-peptide secretion in vitro. We demonstrated reversal of hyperglycemia in diabetic model mice after transplantation of these cells, maintaining non-fasting blood glucose levels along with the human glycemic set point. We confirmed the secretion of human insulin and glucagon dependent on the blood glucose level in vivo. Immunohistological analysis revealed that grafted cells became α, β and δ cells in vivo.
These results suggest that differentiated cells derived from human iPS cells grown in suspension culture mature and function like pancreatic islets in vivo.
•Functional islet-like cells were induced from human iPS cells by suspension culture.•They ameliorated hyperglycemia in diabetic mice and secreted human insulin and glucagon.•Grafted cells became α, β and δ cells in vivo.
To determine if model-based iterative reconstruction (MBIR) can improve visualization of the Adamkiewicz artery on multi-detector row computed tomographic (CT) images compared with adaptive ...statistical iterative reconstruction (ASIR) and filtered back projection (FBP).
This retrospective study was approved by the institutional review board, and written informed consent for the CT examination was obtained. Thirty-three patients underwent contrast material-enhanced 64-section multi-detector row CT for assessment of aortic aneurysm or dissection. Helical data were reconstructed by using FBP, ASIR, and MBIR. The signal-to-noise ratio of the aorta and contrast-to-noise ratio of the anterior spinal artery relative to the spinal cord were measured on multiplanar reformatted images. Visualization of the Adamkiewicz artery and its continuity with the intercostal or lumbar artery were evaluated by using a four-point scale. All image analyses were performed by two blinded, independent observers. The one-way analysis of variance and the Wilcoxon signed-rank test were used for statistical analysis.
MBIR showed significantly better signal-to-noise and contrast-to-noise ratios than did ASIR and FBP (P < .05 for all comparisons) with good interobserver agreement (intraclass correlation coefficient of 0.93 for signal-to-noise ratio and 0.75 for contrast-to-noise ratio). The visualization score of the Adamkiewicz artery was also significantly better when MBIR was used (3.4 ± 0.8 and 3.6 ± 0.7 for observers A and B, respectively) than when ASIR (2.7 ± 1.1 and 3.0 ± 1.0, respectively) or FBP (2.5 ± 1.2 and 3.1 ± 0.9, respectively) was used.
Use of the MBIR algorithm led to improved multi-detector row CT visualization of the Adamkiewicz artery when compared with the use of ASIR and FBP.
Maturity-onset diabetes of the young (MODY) is a heterozygous monogenic diabetes; more than 14 disease genes have been identified. However, the pathogenesis of MODY is not fully understood because ...the patients' pancreatic beta cells are inaccessible. To elucidate the pathology of MODY, we established MODY3 patient-derived iPS (MODY3-iPS) cells using non-integrating Sendai virus (SeV) vector and examined the mutant mRNA and protein of HNF1A (Hepatocyte Nuclear factor 1A) after pancreatic lineage differentiation. Our patient had a cytosine insertion in the HNF1A gene (P291fsinsC) causing frameshift and making a premature termination codon (PTC). We confirmed these MODY3-iPS cells possessed the characteristics of pluripotent stem cells. After we differentiated them into pancreatic beta cells, transcripts of HNF1A gene were cloned and sequenced. We found that P291fsinsC mutant transcripts were much less frequent than wild ones, but they increased after adding cycloheximide (CHX) to the medium. These results suggested that mutant mRNA was destroyed by nonsense-mediated mRNA decay (NMD). Moreover, we were not able to detect any band of mutant proteins in pancreatic lineage cells which were differentiated from MODY3-iPSCs by western blot (WB) analysis. A scarcity of the truncated form of mutant protein may indicate that MODY3 might be caused by a haplo-insufficiency effect rather than a dominant negative manner.
Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because ...numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.
PDF
