24 research outputs found
Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias
Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted
Bivalent predicates in Saami languages from an areal perspective
Non peer reviewe
Chronic Cardiotoxicity Assays Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs)
Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are increasingly used in cardiac safety assessment, disease modeling and regenerative medicine. A vast majority of cardiotoxicity studies in the past have tested acute effects of compounds and drugs; however, these studies lack information on the morphological or physiological responses that may occur after prolonged exposure to a cardiotoxic compound. In this review, we focus on recent advances in chronic cardiotoxicity assays using hiPSC-CMs. We summarize recently published literature on hiPSC-CMs assays applied to chronic cardiotoxicity induced by anticancer agents, as well as non-cancer classes of drugs, including antibiotics, anti-hepatitis C virus (HCV) and antidiabetic drugs. We then review publications on the implementation of hiPSC-CMs-based assays to investigate the effects of non-pharmaceutical cardiotoxicants, such as environmental chemicals or chronic alcohol consumption. We also highlight studies demonstrating the chronic effects of smoking and implementation of hiPSC-CMs to perform genomic screens and metabolomics-based biomarker assay development. The acceptance and wide implementation of hiPSC-CMs-based assays for chronic cardiotoxicity assessment will require multi-site standardization of assay protocols, chronic cardiac maturity marker reproducibility, time points optimization, minimal cellular variation (commercial vs. lab reprogrammed), stringent and matched controls and close clinical setting resemblance. A comprehensive investigation of long-term repeated exposure-induced effects on both the structure and function of cardiomyocytes can provide mechanistic insights and recapitulate drug and environmental cardiotoxicity
Human cardiomyocytes are more susceptible to irreversible electroporation by pulsed electric field than human esophageal cells
Abstract Pulse electric fieldābased (PEF) ablation is a technique whereby short highāintensity electric fields inducing irreversible electroporation (IRE) are applied to various tissues. Here, we implemented a standardized in vitro model to compare the effects of biphasic symmetrical pulses (100 pulses, 1ā10Ā Ī¼s phase duration (d), 10ā1000āHz pulse repetition rate (f)) using two different human cellular models: humanāinduced pluripotent stem cellāderived cardiomyocytes (hiPSCāCMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEFāinduced irreversibly electroporated cell monolayer areas and the corresponding electric field thresholds (EFTs) for both cardiac and esophageal cultures. Our results suggest marked cell type specificity with EFT estimated to be 2ā2.5 times lower in hiPSCāCMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85ākV/cm for the treatment of 100 pulses with dĀ =Ā 5Ā Ī¼s, fĀ =Ā 10Ā Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with dĀ =Ā 10Ā Ī¼s, fĀ =Ā 10Ā Hz). PEF treatment can result in increased temperature around the stimulating electrodes and lead to unanticipated thermal tissue damage that is proportional to the peak temperature rise and to the duration of the PEFāinduced elevated temperatures. In our study, temperature increases ranged from less than 1Ā°C to as high as 30Ā°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed āT returned to 50% of its maximum recorded temperature in tens of seconds
Differences in PGE<sub>2</sub> Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1Ī² and TRIF/IRF3
<div><p>Prostaglandin E2 (PGE<sub>2</sub>) is induced <i>in vivo</i> by bacterial products including TLR agonists. To determine whether PGE<sub>2</sub> is induced directly or via IL-1Ī², human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE<sub>2</sub> in macrophages exclusively via IL-1Ī²-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE<sub>2</sub> production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30ā60%. Recombinant human IL-1Ī² augmented <i>COX-2</i> and <i>mPGES-1</i> mRNA and PGE<sub>2</sub> production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down <i>TRIF</i>, <i>TRAM</i>, or <i>IRF3</i> genes by siRNA inhibited IL-1Ī²-induced <i>COX-2</i> and <i>mPGES-1</i> mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE<sub>2</sub> production by exogenous IL-1Ī². Our data showed that TLR2 agonists induce PGE<sub>2</sub> in monocytes independently from IL-1Ī². In the case of TLR4, IL-1Ī² augments PGE<sub>2</sub> production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.</p></div
ZVAD reduced PGE<sub>2</sub> production and <i>COX-2</i>/<i>mPGES-1</i> mRNA expression in LPS-activated monocytes but not macrophages.
<p>Monocytes (A, B, E, F) and differentiated macrophages (C, D, G, H) were cultured with LPS at indicated concentrations in the absence (solid lines in AāD) or in the presence of ZVAD (AāD, broken lines). After overnight incubation, cell culture supernatants were assayed for IL-1Ī² (A, C) and for PGE<sub>2</sub> (B, D). The data is shown as mean Ā± STDEV for PGE<sub>2</sub> and IL-1Ī² protein concentration calculated for triplicate wells. Monocytes (E, F) and macrophages (G, H) were incubated alone (open bars) or with 20 ng/ml of LPS in the absence (solid bars) or in the presence of ZVAD (striped bars) and were assayed for <i>COX-2</i> mRNA and for <i>mPGES-1</i> mRNA expression at 6 and 12 h post treatment by qPCR in triplicate wells. The C<sub>t</sub> values for <i>COX-2</i> and <i>mPGES-1</i> mRNA expression in monocytes (E) and macrophages (G) were normalized using qPCR reactions with <i>Ī²-actin</i> primers performed in the same samples and are shown as mean ĪC<sub>t</sub>Ā±STDEV calculated for triplicate wells. Asterisks denote statistical significance of the differences in the means of ĪC<sub>t</sub> values between LPS-treated cells cultured with or without ZVAD. ĪC<sub>t</sub> values were used to calculate <i>COX-2</i> and <i>mPGES-1</i> mRNA fold increases over time 0 in monocytes (F) and macrophages (H), ***pā¤0.001; ns, not significant (pā„0.05). The experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098517#pone-0098517-g001" target="_blank">Figure 1</a> was performed 3 times with cells obtained from different donors. The data is shown for one representative donor.</p
Different contributions of rIL-1Ī² IL-1R signaling to IL-6 vs. PGE<sub>2</sub> production in human monocytes and macrophages.
<p>(A) Monocytes and differentiated macrophages were left untreated (solid bars) or were incubated with LPS alone at 20 ng/ml or with LPS in the presence of the IL-1R inhibitor Kineret (open and striped bars, respectively). Cell culture supernatants were assayed for PGE<sub>2</sub>. (BāC) The expression level of IL-1R1 was determined by FACS analysis on monocytes (B) and macrophages (C) untreated (dashed lines) or following activation with LPS at 20 ng/ml overnight (solid lines); filled histograms in panels B and C show staining with IgG-PE isotype control antibody. The Delta Mean Fluorescence Intensity (ĪMFI) was calculated by subtracting the isotope control MFI from the experimental values. The IL-1R1 ĪMFI for untreated and LPS-activated cells was 13.2Ā±6.4 and 73.2Ā±8.1 for monocytes (B) and 13.0Ā±3.3 and 5.2Ā±5.5 for macrophages (C), respectively, nā=ā3. Histograms show results from one representative experiment, each measuring at least 10,000 events. (DāE) Monocytes were incubated with human rIL-1Ī² at 0, 10 or 100 ng/ml in the absence or presence of Kineret for 18 h (open and solid bars, respectively). Cell culture supernatants were assayed for PGE<sub>2</sub> (D) or for IL-6 (E). The data in A, D, and E are shown as means Ā± STDEV for triplicate wells in the PGE<sub>2</sub> and IL-6 and assay. This is representative of three experiments with similar results, ***pā¤0.001, **pā¤0.01; ns, not significant.</p
Caspase-1 and IL-1Ī² do not play a role in TLR-2 ligand-induced PGE<sub>2</sub> production in monocytes.
<p>Monocytes were treated with Pam3CSK4 (A, B) or with FSL-1 (C, D) with or without ZVAD for 18 h (broken and solid lines respectively). Cell culture supernatants were assayed for IL-1Ī² (A, C) and for PGE<sub>2</sub> (B, D). (E) IL-R1 surface expression was determined by FACS analysis on untreated monocytes (dashed line) or on monocytes following activation with Pam3CSK4 or with FSL-1 at 500 ng/ml overnight (solid and dotted lines, respectively); filled histogram shows staining of untreated monocytes with isotype control. IL-1R1 ĪMFI for untreated monocytes and for monocytes activated with Pam3CSK4 or with FSL-1 was 14.2Ā±5.1, 55.5Ā±10.5, and 47.3Ā±8.4, respectively, nā=ā3. Histograms show results from one representative experiment, each measuring at least 10,000 events. (F) TLR2 agonist does not prime monocytes for rIL-1Ī² response. Monocytes were primed with Pam3CSK4 for 1 h at indicated concentrations (open bars) or were primed with Pam3CSK4 followed by addition of rIL-1Ī² at 100 ng/ml for 18 h (solid bars). Cell culture supernatants were collected and assayed for PGE<sub>2</sub> protein. The data are shown as mean Ā± STDEV for PGE<sub>2</sub> and IL-1Ī² protein concentration calculated from triplicate wells in the PGE<sub>2</sub> assay and in IL-1Ī² ELISA, respectively. This is representative of three experiments; ns, not significant.</p
IRF3 is phosphorylated in monocytes but not in macrophages following activation with LPS.
<p>Monocytes (lanes 1ā6) and differentiated macrophages (lanes 7ā10) were left untreated (lanes 1 and 7) or were treated for 1 h with: LPS alone at 1 ng/ml (lanes 2, 8); Pam3CSK4 alone at 50 ng/ml (lane 5); with IL-1Ī² alone at 100 ng/ml (lanes 3, 9), or with LPS + IL1Ī² (lanes 4, 10); or with Pam3CSK4 + IL-1Ī² (lane 6). Following incubation, cell lysates were prepared and resolved in SDS-PAGE. Phospho-IRF3, total IRF3, and Ī²-actin were detected after Western Blotting. The experiment was performed 3 times with similar results.</p