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 field‐based (PEF) ablation is a technique whereby short high‐intensity 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, 1–10 μs phase duration (d), 10–1000 Hz pulse repetition rate (f)) using two different human cellular models: human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) and human esophageal smooth muscle cells (hESMCs) cultured in monolayer format. We report the PEF‐induced 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 2–2.5 times lower in hiPSC‐CMs than in hESMCs when subjected to identical PEF treatments (e.g., 0.90 vs 1.85 kV/cm for the treatment of 100 pulses with d = 5 μs, f = 10 Hz, and 0.65 vs 1.67 kV/cm for the treatment of 100 pulses with d = 10 μs, f = 10 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 PEF‐induced elevated temperatures. In our study, temperature increases ranged from less than 1°C to as high as 30°C, however, all temperature changes were transient and quickly returned to baseline and the highest observed ∆T returned to 50% of its maximum recorded temperature in tens of seconds

Differences in PGE₂ Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

<div><p>Prostaglandin E2 (PGE₂) is induced <i>in vivo</i> by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, 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 PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented <i>COX-2</i> and <i>mPGES-1</i> mRNA and PGE₂ 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-1β-induced <i>COX-2</i> and <i>mPGES-1</i> mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ 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

IRF3 is phosphorylated in monocytes but not in macrophages following activation with LPS.

<p>Monocytes (lanes 1–6) and differentiated macrophages (lanes 7–10) 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-1β alone at 100 ng/ml (lanes 3, 9), or with LPS + IL1β (lanes 4, 10); or with Pam3CSK4 + IL-1β (lane 6). Following incubation, cell lysates were prepared and resolved in SDS-PAGE. Phospho-IRF3, total IRF3, and β-actin were detected after Western Blotting. The experiment was performed 3 times with similar results.</p

LPS induced PGE₂ but not IL-1β secretion in macrophages from caspase-1 KO mice.

<p>Macrophages were isolated from the peritoneal cavity of wild type (WT, solid lines) or caspase-1 KO mice (broken lines) and were incubated <i>in vitro</i> with LPS for 24 h. Cell culture supernatants were assayed for PGE₂ (A) and for IL-1β (B). The data are shown as means ± STDEV for triplicate wells. The experiment was performed three times with similar results.</p

ZVAD reduced PGE₂ 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 A–D) or in the presence of ZVAD (A–D, broken lines). After overnight incubation, cell culture supernatants were assayed for IL-1β (A, C) and for PGE₂ (B, D). The data is shown as mean ± STDEV for PGE₂ and IL-1β 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_t 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>β-actin</i> primers performed in the same samples and are shown as mean ΔC_t±STDEV calculated for triplicate wells. Asterisks denote statistical significance of the differences in the means of ΔC_t values between LPS-treated cells cultured with or without ZVAD. ΔC_t 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), ***p≤0.001; ns, not significant (p≥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-1β IL-1R signaling to IL-6 vs. PGE₂ 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 PGE₂. (B–C) 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 (ΔMFI) was calculated by subtracting the isotope control MFI from the experimental values. The IL-1R1 ΔMFI for untreated and LPS-activated cells was 13.2±6.4 and 73.2±8.1 for monocytes (B) and 13.0±3.3 and 5.2±5.5 for macrophages (C), respectively, n = 3. Histograms show results from one representative experiment, each measuring at least 10,000 events. (D–E) Monocytes were incubated with human rIL-1β 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 PGE₂ (D) or for IL-6 (E). The data in A, D, and E are shown as means ± STDEV for triplicate wells in the PGE₂ and IL-6 and assay. This is representative of three experiments with similar results, ***p≤0.001, **p≤0.01; ns, not significant.</p