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Add-Mix-Read Assays for Assessing Cell Health Using an iPSC-Derived Cardiomyocyte Cell...

Add-Mix-Read Assays for Assessing Cell Health Using an iPSC-Derived Cardiomyocyte Cell Model

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Abstract

Early screening for off-target effects has become an integral component of the drug discovery process, because proactive testing reduces high attrition of lead compounds during medicinal chemistry efforts and clinical trials. Such screening requires cell models that accurately predict relevant in vivo human responses and drug efficacy. Additionally assays to assess cell health must be easily miniaturized and amenable to ultra high-throughput environments while providing a complete picture of cell health. Homogeneous (add-mix-read) assay reagents are user-friendly alternatives to traditional methods of monitoring cell viability. Reagents have been developed to monitor various aspects of cell health, including viability, cytotoxicity and apoptosis. The assay reagents can be used individually, or in some cases multiplexed, to generate a multi-parameter readout of cell health from cells within a single assay well. Here we present data generated with add-mix-read cell health assays using an human induced pluriopotent stemcell (iPSC)-derived cardiomyocyte cell model to provide a more complete picture of cell health.

Andrew Niles1, Chad Zimprich1, Tracy Worzella1, and Blake Anson2 1Promega Corporation 2Cellular Dynamics International Publication Date: 2012

Introduction

Induced pluriopotent stem cells (iPSCs) are an emerging research tool with applications in a variety of research areas, including toxicity screening. Lethal cardiotoxic side effects have resulted in the removal of therapies from the market for safety reasons. (1) Consequently, screening for off-target effects, including cardiotoxicity, is a vital part of the drug development process.

To demonstrate the applicability of iPSC-derived cardiomyocytes as an off-target model for toxicity screening, we conducted parallel experiments using a target human K562 cancer cell line model (chronic myelogenous leukemia) alongside iPSC-derived cardiomyocytes (iCell® Cardiomyocytes; Cellular Dynamics International). Select drugs known to target cancerous cells were tested, and the toxicity profiles of the two cell types were compared.

To assess overall cell health and cytotoxicity, we used the ApoTox-Glo™ Triplex Assay, a combination of chemistries used to measure viability, as indicated by an intact cell membrane; cytotoxicity, as indicated by a compromised cell membrane; and apoptosis, as indicated by caspase activation.

Assay Principle

The ApoTox-Glo™ Triplex Assay combines three assay chemistries to easily assess viability, cytotoxicity and apoptosis events in the same cell-based assay well. First, viability and cytotoxicity are determined by measuring two differential protease biomarkers simultaneously with the addition of a single nonlytic reagent containing two peptide substrates. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where it is cleaved to generate a fluorescent signal proportional to the number of living cells. This live-cell protease activity marker becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium. A second, cell-impermeant, fluorogenic peptide substrate (bis-AAF-R110 Substrate) is used simultaneously to measure dead-cell protease activity that has been released from cells that have lost membrane integrity. This results in ratiometric, inversely correlated measures of cell viability and cytotoxicity. The ratio of viable cells to dead cells is independent of cell number and, therefore, can be used to normalize data. A second reagent containing luminogenic DEVD-peptide substrate for caspase-3/7 and Ultra-Glo™ Recombinant Thermostable Luciferase is added. Caspase-3/7 cleavage of the substrate releases luciferin, which is a substrate for luciferase and generates light. The light output, measured with a luminometer, correlates with caspase-3/7 activation as a key biomarker of apoptosis.

6601MBFigure 1. The ApoTox-Glo™ Triplex Assay combines three assay chemistries to easy assess viability, cytotoxicity and apoptosis in the same cell-based assay well.
Overview of the ApoTox-Glo Triplex Assay protocol. Figure 2. Overview of the ApoTox-Glo™ Assay Protocol. The ApoTox-Glo™ Assay can be performed in 1.5ml tubes or in assay plates. Reagents are added and mixed to ensure homogeneity. Signal is read with a single-tube or multi-mode plate reader.

Cellular Material

iCell® Cardiomyocytes from Cellular Dynamics International are derived from human iPSCs (2) . These cardiomyocytes have been developed to serve as a parallel cardiac model to aid in drug discovery by improving predictability of drug efficacy and toxicity. These cells are terminally differentiated, fully functional human cardiomyocytes and therefore provide results that more accurately predict the relevant in vivo cardiac response (2) (3) .

The immortalized K562 cell line was isolated from a female patient with CML from a pleural effusion nine days before her death (4) . These cells, which have a Philadelphia chromosome translocation, have been extensively characterized and serve as a model system for the development of cancer therapies targeting CML (5) .

For all experiments iCell® Cardiomyocytes were plated and maintained as previously described by the manufacturer (6) . K562 cells were plated and treated in high glucose RPMI 1640 supplemented with 10% fetal bovine serum.

Toxicity Profiles of iCell® Cardiomyocytes and K562 Cells in Response to Treatment with FDA-approved Anti-Leukemia Drugs

The tyrosine kinase inhibitor, imatinib, targets the mutant bcr-abl kinase found in the leukocytes of patients with CML (7) . In this experiment, cells were prepared as described above. A serial titration of imatinib was applied to each cell line and incubated for 24 hours at 37°C, 5% CO2. After treatment, the ApoTox-Glo™ Assay was performed as per manufacturer instructions  (8) .

At imatinib concentrations greater than 1µM, cytotoxicity is noted in both cell lines (Figure 1).  The compound causes a profound decrease in cancer cell viability with a commensurate increase in caspase-3/7 activity (Figure 1).  This concentration-dependent, apoptotic profile is consistent with the reported on-target activity of the drug. Conversely, cardiomyocyte toxicity suggests a troubling off-target effect of imatinib treatment which occurs without caspase activation. This in vitro cytotoxic result would warrant further study to gauge potential clinical risk. The apparent high-dose diminution of caspase and cytoxicity biomarker signals observed after 24 hours of imatinib treatment with K562 cells, is consistent with early onset activation kinetics and consequent time-dependent biomarker decay.  A full time course of exposure (4, 12, 24 hours) is suggested to fully characterize the kinetics of the cytotoxic event. Bortezomib has been shown to direct anti-tumor activity in hematopoietic malignancies by specifically inhibiting part of a complex of proteins containing the 28S proteasome. On target efficacy is thought to occur by interference with the misfolded protein recycling function of the proteasome, which allows cancerous cells to maintain replicative potential (9) .

10420MA_600pxFigure 3. Comparison of toxicity profiles for iCell® Cardiomyocytes (Panel A) and K562 (Panel B) cells treated with imatinib. Note that the viability falls off in both cell lines at imatinib concentrations exceeding 1µM. The mechanism of cell death in K562 cells appears to be via apoptosis as indicated by the increase in caspase-3/7 activity that precisely accompanies the increase in cytotoxicity.

Note that the viability falls off in both cell lines at imatinib concentrations exceeding 1µM. The mechanism of cell death in K562 cells appears to be via apoptosis as indicated by the increase in caspase-3/7 activity that precisely accompanies the increase in cytotoxicity.

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Bortezomib shows potent specificity to K562 cells and dose dependent effects on cell health, with a decrease in viability and corresponding increase in cytotoxicity (Figure 2). The increase in caspase-3/7 signal indicates the mechanism is due to apoptosis induction. A decrease in cytotoxicity signal at higher concentrations of bortezomib in the K562 model is likely a result of activation kinetics and time-dependent biomarker decay (Figure 2). The cardiomyocytes do not show apparent off-target effects on cell health. It should be noted however that bortezomib and other proteasome inhibitors are know to partially inhibit the viability assay protease at concentrations greater than 1µM, which is more than 50-fold greater than the EC50 for K562 cells.

10421MA_600pxFigure 4. Comparison of toxicity profiles for iCell® Cardiomyocytes (Panel A) and K562 cells (Panel B) treated with bortezomib.

The decrease in cell viability is specific for K562 cells and is accompanied by induction of caspase-3/7 activity, indicating the mechanism of cell death is apoptosis.

The decrease in cell viability is specific for K562 cells and is accompanied by induction of caspase-3/7 activity, indicating the mechanism of cell death is apoptosis.

/~/media/images/resources/figures/10400-10499/10421ma_600px.jpg?la=en

Summary

Identifying compounds with undesirable off-target effects early in the drug discovery process requires two things: cellular models that generate results highly predictive of the human in vivo response and cell health assays that are easily performed in a highly miniaturized format. iCell® Cardiomyocytes serve as a cardiac model to aid in toxicity and efficacy screens as terminally differentiated, fully functional, normal human cardiomyocytes. The ApoTox-Glo™ Assay provides a multiplexed assay that measures several parameters of cell health in the same well, allowing researchers to gain a more complete picture of the effects treatments are having on their cells. Together with an appropriate cellular target (K562 cell line) and human test system (iCell® Cardiomyocytes), the homogeneous, add-mix-read, ApoTox-Glo™ assay reagent enabled a rapid and robust assessment of compound efficacy, off-target safety margin, and toxicity-based mechanism of action in a single experiment.

References

  1. Hishigaski, H. and Kuhara, S. (2001) hERGAPDbase: A database documenting hERG channel inhibitory potentials and APD-prolongation activities of chemical compounds Database Bar017.
  2. Ma, J. et al. (2011) High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents. Am. J. Physiol. 301, H2006-H2017.
  3. Guo, L. et al. (2011) Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes. Tox. Sci. 123, 281–9.
  4. Lozzio, C.B. and Lozzio, B.B. (1975) Human chronic myelogenous leukemia cell-line with positive Philedelphia chromosome. Blood 45, 321–44.
  5. Lozzio, C.B. and Lozzio, B.B. (1979) Properties and usefulness of the original K-562 human myelogenous leukemia cell line. Leukemia Research 3, 363–70.
  6. Cellular Dynamics International (2011) iCell® Cardiomyocytes User's Guide
  7. Hunter, T. (2007) Treatment for chronic myelogenous leukemia: the long road to imatinib J. Clin. Invest. 117, 2036–43.
  8. ApoTox-GloTriplex Assay Protocol TM322, Promega Corporation
  9. Moore, B.S., Eustáquio, A.S., McGlinchey, R.P. (2008) Advances in and applications of proteasome inhibitors. Curr. Opin. Chem. Biol. 12, 434–40.

How to Cite This Article

Niles, A., Zimprich, C., Worzella, T., and Anson, B. Add-mix-read assays for assessing cell health using an iPS cardiomyocyte cell model. [Internet] 2012. [cited: year, month, date]. Available from: http://www.promega.lu/resources/pubhub/add-mix-read-assays-for-assessing-cell-health-using-an-ips-cardiomyocyte-cell-model/

Niles, A., Zimprich, C., Worzella, T., and Anson, B. Add-mix-read assays for assessing cell health using an iPS cardiomyocyte cell model. Promega Corporation Web site. http://www.promega.lu/resources/pubhub/add-mix-read-assays-for-assessing-cell-health-using-an-ips-cardiomyocyte-cell-model/ Updated 2012. Accessed Month Day, Year.

ApoTox-Glo is a trademark of Promega Corporation.

iCell is a registered trademark of Cellular Dynamics International.

Figures

6601MBFigure 1. The ApoTox-Glo™ Triplex Assay combines three assay chemistries to easy assess viability, cytotoxicity and apoptosis in the same cell-based assay well.
Overview of the ApoTox-Glo Triplex Assay protocol. Figure 2. Overview of the ApoTox-Glo™ Assay Protocol. The ApoTox-Glo™ Assay can be performed in 1.5ml tubes or in assay plates. Reagents are added and mixed to ensure homogeneity. Signal is read with a single-tube or multi-mode plate reader.
10420MA_600pxFigure 3. Comparison of toxicity profiles for iCell® Cardiomyocytes (Panel A) and K562 (Panel B) cells treated with imatinib. Note that the viability falls off in both cell lines at imatinib concentrations exceeding 1µM. The mechanism of cell death in K562 cells appears to be via apoptosis as indicated by the increase in caspase-3/7 activity that precisely accompanies the increase in cytotoxicity.

Note that the viability falls off in both cell lines at imatinib concentrations exceeding 1µM. The mechanism of cell death in K562 cells appears to be via apoptosis as indicated by the increase in caspase-3/7 activity that precisely accompanies the increase in cytotoxicity.

/~/media/images/resources/figures/10400-10499/10420ma_600px.jpg?la=en
10421MA_600pxFigure 4. Comparison of toxicity profiles for iCell® Cardiomyocytes (Panel A) and K562 cells (Panel B) treated with bortezomib.

The decrease in cell viability is specific for K562 cells and is accompanied by induction of caspase-3/7 activity, indicating the mechanism of cell death is apoptosis.

The decrease in cell viability is specific for K562 cells and is accompanied by induction of caspase-3/7 activity, indicating the mechanism of cell death is apoptosis.

/~/media/images/resources/figures/10400-10499/10421ma_600px.jpg?la=en

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