ABSTRACT
The alignment of each cell in human myocardium is considered critical for the efficient movement of cardiac tissue. We investigated 96-well microstripe-patterned plates to align human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), which resemble fetal myocardium. The aligned CMs (ACMs) cultured on the microstripe-patterned plates exhibited pathology, motor function, gene expression, and drug response that more closely resembled those of adult cells than did unaligned CMs cultured on a flat plate (FCMs). We used these ACMs to evaluate drug side effects and efficacy, and to determine whether these were similar to adult-like responses. When CMs from patients with hypertrophic cardiomyopathy (HCMs) were seeded and cultured on the microstripe-patterned plates or layered on top of the ACMs, both sets of HCMs showed increased heart rate and synchronized contractions, indicating improved cardiac function. It is suggested that the ACMs could be used for drug screening as cells representative of adult-like CMs and be transplanted in the form of a cell sheet for regenerative treatment of heart failure.
Subject(s)
Cell Differentiation , Induced Pluripotent Stem Cells , Myocytes, Cardiac , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Cardiomyopathy, Hypertrophic , Cells, Cultured , Myocardium/cytology , Myocardium/metabolism , Tissue Engineering/methods , Cell Culture TechniquesABSTRACT
Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.
Subject(s)
Antioxidants/administration & dosage , Mitochondria/physiology , Mitochondrial Diseases/prevention & control , Oxidative Stress/physiology , Animals , Cell Line, Tumor , Cell Respiration , Cell Survival , Dietary Supplements , Flow Cytometry , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Humans , Membrane Potential, Mitochondrial , Microscopy, Confocal , Microscopy, Fluorescence , Mitochondria/pathology , Mitochondrial Diseases/chemically induced , Mitochondrial Diseases/diet therapy , Mitochondrial Diseases/pathology , Oxidation-Reduction , Rats , Superoxides/metabolism , Time Factors , Xanthophylls/administration & dosageABSTRACT
BACKGROUND: Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. METHODS: We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. RESULTS: We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. CONCLUSIONS: The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.
Subject(s)
Fluoroimmunoassay/methods , Interferon-gamma/analysis , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay , Humans , Intercellular Signaling Peptides and Proteins/immunology , Sensitivity and SpecificityABSTRACT
An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.
