-
[show abstract]
[hide abstract]
ABSTRACT: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) at different stages (approximate days 30, 60, and 90) were used to determine the appropriate stage for functional and morphological assessment of drug effects in vitro. The hiPS-CMs had spontaneous beating activity, and β-adrenergic function was comparable in all stages of differentiation. Microelectrode array analyses using ion channel blockers indicated that the electrophysiological properties of these ion channels were comparable at all differentiation stages. Ultrastructural analysis using electron microscopy showed that myofibrillar structures at days 60 and 90 were similarly distributed and more mature than that at day 30. Analysis of motion vectors in contracting cells showed that the velocity of contraction was the highest at day 90 and was the most mature among the three stages. Gene expression analysis demonstrated that expression of some genes related to myofilament and sarcoplasmic reticulum increased with maturation of morphological and contractile properties. In conclusion, day 30 cardiomyocytes are useful for basic screening such as the assessment of electrophysiological properties, and days 60 and 90 are the appropriate differentiation stage for morphological assays. For the assay of contractile function associated with subcellular components such as sarcoplasmic reticulum, day 90 cardiomyocytes are the most suitable.
Journal of Biomolecular Screening 06/2012; 17(9):1192-203. · 2.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cardiomyocytes derived from embryonic stem cells (ES-CMs) and induced pluripotent stem cells (iPS-CMs) are useful for toxicity and pharmacology screening. In the present study, we found that cardiomyocyte-rich beating cell clusters (CCs) emerged from murine embryonic stem cell (mESC)-derived beating EBs and from human-induced pluripotent stem cell (hiPSC)-derived beating EBs dissociated by gentle pipetting with a thin glass pipette. The percentage of cardiac troponin T (cTnT)-positive cells in the beating CCs obtained from mESC-derived and hiPSC-derived beating EBs was higher (81.5% and 91.6%, respectively) than in beating-undissociated EBs (13.7% and 67.1%, respectively). For mESCs, the yield of cTnT-positive cells from beating CCs was estimated to be 1.6 times higher than that of beating EBs. The bromodeoxyuridine labeling index of mouse ES-CMs and human iPS-CMs in beating CCs was 1.5- and 3.2-fold, respectively, greater than those in beating EBs. To investigate the utility of the cells in toxicity assessment, we showed that doxorubicin, a cardiotoxic drug, induced myofilament disruption in cardiomyocytes isolated by this method. This simple method enables preparation of mouse ES-CMs and human iPS-CMs with better proliferative activity than beating EBs not dissociated by pipetting, and the cardiomyocytes are useful for drug-induced myocardial toxicity testing.
Journal of Biomolecular Screening 01/2012; 17(5):683-91. · 2.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although embryonic stem cell (ESC)-derived cardiomyocytes may be a powerful tool in drug discovery, their potential has not yet been fully explored. Nor has a detailed comparison with adult heart tissue been performed. We have developed a method for efficient production of cardiomyocyte-rich embryoid bodies (EBs) from murine ESCs. Analysis of global gene expression profiles showed that EBs on day 7 and/or 21 of differentiation (d7CMs and d21CMs, respectively) were similar to adult heart tissue for genes categorized as regulators of muscle contraction or voltage-gated ion channel activity, although d21CMs were more mature than d7CMs for contractile components related to morphological structures. Calcium and sodium channel blockers altered Ca2+ transients, and isoproterenol, a beta-adrenergic compound, increased the rate of beating in d7CMs and d21CMs. Our gene analytic system therefore enabled us to identify genes that are expressed in the physiological pathways associated with ion channels and structural components in d7CMs and d21CMs. We conclude that EBs might be of use for the basic screening of drugs that might affect contractile function through ion channels.
Journal of Biomolecular Screening 03/2009; 14(3):239-45. · 2.05 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To verify the concept that cell-free organ/tissue-specific mRNAs leaking from drug-damaged organs/tissues into peripheral blood could be toxicological biomarkers for identification of the target organs of drug toxicity, we attempted to detect liver-specific mRNAs in peripheral blood from rats with chemical-induced hepatotoxicity. We selected alpha(1)-microglobulin/bikunin precursor (Ambp) and albumin mRNAs as tentative liver-specific biomarkers and successfully detected them by reverse transcription (RT)-PCR in peripheral blood 24 h after D-galactosamine HCl (D-gal) or acetaminophen administration. Moreover, albumin mRNA was detected 2 h after D-gal administration, although plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were still unchanged. On the other hand, in peripheral blood from rat with bupivacaine HCl-induced skeletal muscle damage, neither Ambp nor albumin mRNA was detectable while plasma creatine kinase, ALT, and AST levels prominently increased 2 or 12 h after dosing. Furthermore, Ambp mRNA was also detectable in filtered plasma from rats with liver damage, indicating that cell-free Ambp mRNA can be present in peripheral blood. In conclusion, cell-free, liver-specific Ambp, and albumin mRNAs were detectable in peripheral blood from rats with chemical-induced liver damage. It is believed that the detection of cell-free organ/tissue-specific mRNA in peripheral blood is a promising approach in the survey of toxicological biomarkers.
Toxicological Sciences 10/2008; 106(2):538-45. · 4.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously showed that a toxicogenomics analysis of drug-induced phospholipidosis enabled the identification of 12 specific gene markers and the establishment of an in vitro real-time PCR screening assay for the assessment of the phospholipidosis-inducing potential of compounds. The purpose of this study was to transfer our PCR-based assay into a 96-well microplate-based multiple mRNAs measuring assay (ArrayPlate assay) in order to increase throughput. Specifically, we determined the expression of the 12 marker genes using real-time PCR and ArrayPlate in human hepatoma HepG2 cells that were treated for 24h with each of amiodarone and 80 proprietary compounds. The following three performance criteria were satisfied in the ArrayPlate analysis: 1. Sensitivity-the expression of mRNA for all target genes was detected at quantifiable levels. 2. Repeatability-signal intensities and fold change values of each marker gene were highly repeatable. 3. Correlation-fold change values and their average values, which were used as indices of phospholipidosis induction potential, showed apparent correlation between the ArrayPlate and real-time PCR assays. Thus, the in vitro screening assay for compound-induced phospholipidosis should be transferable from a PCR-based assay to the higher-throughput ArrayPlate-based method.
Toxicology in Vitro 01/2007; 20(8):1506-13. · 2.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Some cationic amphiphilic compounds are known to have the potential to induce phospholipidosis. This study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. In electron microscopy, HepG2 cells developed lamellar myelin-like bodies in their lysosomes, the characteristic change of phospholipidosis, after treatment with these compounds for 72 h. DNA microarray analysis performed 6 and 24 h after treatment showed alterations in gene expression reflecting the inhibition of lysosomal phospholipase activity and lysosomal enzyme transport, and the induction of phospholipid and cholesterol biosynthesis. Seventeen genes that showed a similar expression profile following treatment were selected as candidate markers. Real-time PCR analysis confirmed that 12 gene markers showed significant concordance with lamellar myelin-like body formation. Furthermore, the average fold change values of these markers correlated well with the magnitude of this pathological change. In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.
Toxicological Sciences 03/2005; 83(2):282-92. · 4.65 Impact Factor
