Use of Human Pluripotent Stem Cell Derived‐Cardiomyocytes to Study Drug‐Induced Cardiotoxicity

Agnes Maillet1, Kim Peng Tan1, Liam R. Brunham2

1 Translational Laboratory in Genetic Medicine, Agency for Science Technology and Research, 2 Department of Medicine, Centre for Heart Lung Innovation, University of British Columbia, Vancouver
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 22.5
DOI:  10.1002/cptx.30
Online Posting Date:  August, 2017
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Abstract

Drug‐induced cardiotoxicity is the one of the most common causes of drug withdrawal from market. A major barrier in managing the risk of drug‐induced cardiotoxicity has been the lack of relevant models to study cardiac safety. Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have great potential in drug discovery and cardiotoxcity screens as they display many characteristics of the human myocardium and offer unlimited supply. This unit describes how to use pluripotent stem cells derived cardiomyocytes to study drug‐induced cardiotoxicty using doxorubicin as an example. We present a workflow that explains procedure for editing hPSC using the CRISPR/Cas9 system and for differentiation of hPSC into cardiomyocytes. We also report protocols to study drug effect on ROS production, intracellular calcium concentration, formation of DNA double strand breaks, gene expression and electrophysiological properties of hPSC‐CMs. © 2017 by John Wiley & Sons, Inc.

Keywords: genome‐editing; differentiation; cardiomyocytes; cardiotoxicity; functional tests

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Generation and Maintenance of hPSC‐ and Edited hPSC‐Derived Cardiomyocytes
  • Basic Protocol 2: Characterization of hPSC‐Derived Cardiomyocytes
  • Basic Protocol 3: Characterization of Dic in hPSC‐Derived Cardiomyocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Generation and Maintenance of hPSC‐ and Edited hPSC‐Derived Cardiomyocytes

  Materials
  • Matrigel (Corning Matrigel hESC‐Qualified Matrix, cat. no. 354277)
  • Ice
  • DMEM/F12 (Thermo Fisher Scientific, cat. no. 11965‐092)
  • Human pluripotent stem cells (hPSC)
  • mTesRTM1 Basal Medium (STEMCELL Technologies, cat. no. 05851)
  • mTesRTM1 5× Supplement (STEMCELL Technologies, cat. no. 05852)
  • 0.02% EDTA (Sigma, cat. no. E8008)
  • RPMI Medium 1640 (Thermo Fisher Scientific, cat. no. 11875‐119)
  • Y27632 (Tocris, cat. no. 1254)
  • CRIPSR/Cas9 construct targeting gene of interest (with puromycin selection cassette)
  • Puromycin
  • QIAamp DNA Blood Mini Kit (Qiagen, cat. no. 51104)
  • Q5 High‐fidelity DNA polymerase and buffer (New England BioLabs, cat. no. M0491L)
  • 10× NEBuffer 2.1 (New England BioLabs, cat. no. B7202S)
  • Nuclease‐free water (Promega, cat. no. P1197)
  • T7E1 enzyme (New England BioLabs, cat. no. M0302L)
  • Proteinase K (Qiagen, cat. no. 19133)
  • Agarose gel system
  • 10 mM dNTP Mix (Promega, cat. no.U1515)
  • QIAquickPCR Purification kit (Qiagen, cat. no. 28106) containing:
  • EB buffer
  • One shot TOP10 chemically competent E. coli (Thermo Fisher, cat. no. C404006)
  • Selective agar plates (LB ampicillin)
  • Ampicillin
  • LB medium
  • QIAprep Miniprep Kit (Qiagen, cat. no. 27106)
  • CHIR99021 (Tocris, cat. no. 4423)
  • B‐27 supplement without insulin (Thermo Fisher Scientific, cat. no. 0050129SA)
  • IWP2 (Tocris, cat. no. 3533)
  • B‐27 supplement (Thermo Fisher Scientific, cat. no. 17504‐044)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Sterile tissue culture plates (6‐, 12‐ and 96‐well)
  • Centrifuge
  • Water bath
  • Neon Transfection System (Thermo Fisher Scientific, cat. no. MPK5000)
  • 1.5‐ml microtubes
  • Hemacytometer
  • Heat block
  • Thermal cycler

Basic Protocol 2: Characterization of hPSC‐Derived Cardiomyocytes

  Materials
  • hPSC‐CMs in 12‐well culture plates (see protocol 1)
  • 1× Dulbecco's phosphate‐buffered saline with Ca2+ and Mg2+ (PBS; Thermo Fisher Scientific)
  • TrypLE Select (Thermo Fisher Scientific, cat. no. 12604‐013)
  • RMPI medium 1640 (Thermo Fisher Scientific, Cat# 11875‐119)
  • Superscript II Reverse Transcription Kit (Thermo Fisher Scientific, cat. no. 18064071)
  • SYBR Select Master Mix (Thermo Fisher Scientific, cat. no. 4472920)
  • PCR master mix (see Table 22.5.4)
  • Cardiomyocytes maintenance medium (RPMI/B‐27 insulin)
  • Fetal bovine serum (FBS)
  • 10 μM Y27632 (Tocris, cat. no. 1254)
  • 0.1% Gelatin (STEMCELL Technologies, cat. no. 07903)
  • Formaldehyde (FA)
  • Washing solution: 1× PBS + 0.3% TritonX‐100
  • Blocking solution (see recipe)
  • Antibody diluent (see recipe)
  • Anti‐cTnT (Abcam, cat. no. ab10214): 1:400
  • Anti‐sarcomeric alpha actinin (Abcam, cat. no. ab9465): 1:200
  • Goat anti‐Mouse IgG2b Secondary Antibody, Alexa Fluor 647 conjugate (Thermo Scientific, cat.no. A‐21242)
  • Vectashield with DAPI (Vector Laboratories, cat. no. H‐1200)
  • Nail polish
  • Wash buffer I (5% FBS/PBS)
  • Zombie Violet (BioLegend, cat. no. 423113)
  • Permeabilization solution: 0.2% Tween/PBS
  • Wash buffer II (5% FBS/PBS + 0.1% Tween)
  • Anti‐Mlc2a (Synaptic Systems, cat. no. 311 011): 1:200
  • Alexa Fluor 647 Mouse IgG2b Isotype control (Biolegend, cat. no. 400330)
  • StepOnePlus (Applied Biosystem)
Table 2.5.4   MaterialsComposition of the Mix for the Real‐time PCR

Reagents Amount per well
cDNA (10 ng) 1 μl
Sybr Green Mix 10 μl
Forward primer (10 μM) 1 μl
Reverse primer (10 μM) 1 μl
dH 2O 6 μl

  • 37°C incubator
  • 15‐ml conical tubes
  • Centrifuge
  • 96‐well microplate
  • Thermal cycler
  • 1000‐μl pipettes
  • Hemacytometer
  • Tissue culture plates (12‐well)
  • Coverslips
  • Parafilm
  • 15‐cm petri dishes
  • Microscope slides
  • Epifluorescence microscope
  • 100‐μM cell strainer
  • Flow cytometry BD FACSCanto II
  • 5‐ml round‐bottom tubes

Basic Protocol 3: Characterization of Dic in hPSC‐Derived Cardiomyocytes

  Materials
  • hPSC‐CMs in Matrigel‐coated 12‐well culture plates
  • 1× Dulbecco's phosphate‐buffered saline with Ca2+ and Mg2+ (PBS; Thermo Fisher Scientific)
  • RMPI Media 1640 (Thermo Fisher Scientific, Cat# 11875‐119)
  • Y27632 (Tocris, Cat# 1254)
  • Cardiomyocytes maintenance medium (RPMI/B‐27 insulin)
  • Fetal bovine serum (FBS)
  • 0.1% Gelatin (STEMCELL Technologies, cat. no. 07903)
  • Doxorubicin (Sigma‐Aldrich, cat. no. D1515)
  • Washing solution: 1× PBS + 0.3% TritonX‐100
  • Blocking solution (see recipe)
  • Antibody anti‐ϒH2Ax (Abcam, cat. no. ab10214)
  • Antibody diluent (see recipe)
  • Goat anti‐Mouse IgG2b Secondary Antibody, Alexa Fluor 647 conjugate (Thermo Scientific, cat. no. A‐21242)
  • Vectashield with DAPI (Vector Laboratories, Cat# H‐1200)Prolong Gold
  • Nail polish
  • MitoSOX Red (ThermoFisher, cat. no. M36008)
  • H2DCFDA (ThermoFisher, cat. no. C6827)
  • Fluo‐4, AM (ThermoFisher, cat. no. F14201)
  • Fibronectin (Sigma‐Aldrich, cat. no. F1141)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich, cat. no. D2650)
  • Trizol (ThermoFisher, cat. no. 15596026)
  • TruSeq RNA sample preparation kit (Illumina)
  • 100‐μM cell strainer
  • Coverslips
  • 37°C incubator
  • Parafilm
  • 15‐cm petri dishes
  • Microscope slides
  • Epifluorescence microscope
  • CellProfiler software
  • 1000‐μl pipette
  • 15‐ml conical tubes
  • Centrifuge
  • Gelatin‐coated glass‐bottom culture plates (12‐well)
  • Olympus FV1000 inverted confocal microscope
  • Maestro 12‐well plate (Axion)
  • Maestro MEA system
  • 2100 Bioanalyzer (Agilent)
  • Hiseq 2500 sequencer
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Figures

Videos

Literature Cited

Literature Cited
  Albini, A., Pennesi, G., Donatelli, F., Cammarota, R., De Flora, S., & Noonan, D. M. (2010). Cardiotoxicity of anticancer drugs: The need for cardio‐oncology and cardio‐oncological prevention. Journal of the National Cancer Institute, 102, 14–25. doi: 10.1093/jnci/djp440.
  Babiarz, J. E., Ravon, M., Sridhar, S., Ravindran, P., Swanson, B., Bitter, H., … Kolaja, K. L. (2012). Determination of the human cardiomyocyte mRNA and miRNA differentiation network by fine‐scale profiling. Stem Cells and Development, 21, 1956–1965. doi: 10.1089/scd.2011.0357.
  Braam, S. R., Tertoolen, L., van de Stolpe, A., Meyer, T., Passier, R., & Mummery, C. L. (2010). Prediction of drug‐induced cardiotoxicity using human embryonic stem cell‐derived cardiomyocytes. Stem Cell Research, 4, 107–116. doi: 10.1016/j.scr.2009.11.004.
  Burridge, P. W., Matsa, E., Shukla, P., Lin, Z. C., Churko, J. M., Ebert, A. D., … Wu, J. C. (2014). Chemically defined generation of human cardiomyocytes. Nature Methods, 11, 855–860. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/24930130 doi: 10.1038/nmeth.2999.
  Clements, M., & Millar, V. (2015). Bridging functional and structural cardiotoxicity assays using human stem cell‐ derived cardiomyocytes for a more comprehensive risk assessment. GE Healthcare, 44, 10012.
  Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., … Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, N.Y.), 339, 819–823. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23287718 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC3795411 doi: 10.1126/science.1231143.
  Dahlmann, J., Kensah, G., Kempf, H., Skvorc, D., Gawol, A., Elliott, D. A., … Gruh, I. (2013). The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells. Biomaterials, 34, 2463–2471. doi: 10.1016/j.biomaterials.2012.12.024.
  Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR‐Cas9. Science, 346, 1258096–1258096. Available at: Retrieved from http://www.sciencemag.org/content/346/6213/1258096.long. doi: 10.1126/science.1258096.
  Eschenhagen, T., Mummery, C., & Knollmann, B. C. (2015). Modelling sarcomeric cardiomyopathies in the dish: From human heart samples to iPSC cardiomyocytes. Cardiovascular Research, 105, 424–438. doi: 10.1093/cvr/cvv017.
  Force, T., & Kolaja, K. L. (2011). Cardiotoxicity of kinase inhibitors: The prediction and translation of preclinical models to clinical outcomes. Nature reviews. Drug discovery, 10, 111–126. Available at: Retrieved from https://doi.org/10.1038/nrd3252. doi: 10.1038/nrd3252.
  Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013). ZFN, TALEN, and CRISPR/Cas‐based methods for genome engineering. Trends in Biotechnology, 31, 397–405. doi: 10.1016/j.tibtech.2013.04.004.
  Groarke, J. D., & Nohria, A. (2015). Editorial: Anthracycline cardiotoxicity a new paradigm for an old classic. Circulation, 131, 1946–1949. doi: 10.1161/CIRCULATIONAHA.115.016704.
  Ichikawa, Y., Ghanefar, M., Bayeva, M., Wu, R., Khechaduri, A., Naga Prasad, S. V., … Ardehali, H. (2014). Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. Journal of Clinical Investigation, 124, 617–630. doi: 10.1172/JCI72931.
  Jones, R. L., Swanton, C., & Ewer, M. S. (2006). Anthracycline cardiotoxicity. Expert Opinion on Drug Safety, 5, 791–809. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17044806. doi: 10.1517/14740338.5.6.791.
  Kim, S.‐Y., Kim, S.‐J., Kim, B.‐J., Rah, S.‐Y., Chung, S. M., Im, M.‐J., & Kim, U.‐H. (2006). Doxorubicin‐induced reactive oxygen species generation and intracellular Ca2+ increase are reciprocally modulated in rat cardiomyocytes. Experimental & Molecular Medicine, 38, 535–545. doi: 10.1038/emm.2006.63.
  Kuznetsov, A. V., Margreiter, R., Amberger, A., Saks, V., & Grimm, M. (2011). Changes in mitochondrial redox state, membrane potential and calcium precede mitochondrial dysfunction in doxorubicin‐induced cell death. Biochimica et Biophysica Acta ‐ Molecular Cell Research, 1813, 1144–1152. doi: 10.1016/j.bbamcr.2011.03.002.
  Lian, X., Zhang, J., Azarin, S. M., Zhu, K., Hazeltine, L. B., Bao, X., … Palecek, S. P. (2013). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β‐catenin signaling under fully defined conditions. Nature Protocols, 8, 162–175. Available at: Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3612968&tool=pmcentrez&rendertype=abstract. doi: 10.1038/nprot.2012.150.
  Ma, J., Guo, L., Fiene, S. J., Anson, B. D., Thomson, J. A., Kamp, T. J., … January, C. T. (2011). High purity human‐induced pluripotent stem cell‐derived cardiomyocytes: Electrophysiological properties of action potentials and ionic currents. American Journal of Physiology. Heart and Circulatory Physiology, 301, H2O06–2017. Available at: Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid‐4116414&tool‐pmcentrez&rendertype‐abstract http://ajpheart.physiology.org/content/301/5/H2O06. doi: 10.1152/ajpheart.00694.2011.
  Maddah, M., Heidmann, J. D., Mandegar, M. A., Walker, C. D., Bolouki, S., Conklin, B. R., & Loewke, K. E. (2015). A non‐invasive platform for functional characterization of stem‐cell‐derived cardiomyocytes with applications in cardiotoxicity testing. Stem Cell Reports, 4, 621–631. doi: 10.1016/j.stemcr.2015.02.007.
  Maillet, A., Tan, K., Chai, X., Sadananda, S. N., Mehta, A., Ooi, J., … Brunham, L. R. (2016). Modeling doxorubicin‐induced cardiotoxicity in human pluripotent stem cell derived‐cardiomyocytes. Scientific Reports, 6, 25333. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/27142468. doi: 10.1038/srep25333.
  Mohr, J. C., Zhang, J., Azarin, S. M., Soerens, A. G., de Pablo, J. J., Thomson, J. A., … Kamp, T. J. (2010). The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials, 31, 1885–1893. doi: 10.1016/j.biomaterials.2009.11.033.
  Mummery, C. L., Zhang, J., Ng, E. S., Elliott, D. A., Elefanty, A. G., & Kamp, T. J. (2012). Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: A methods overview. Circulation Research, 111, 344–358. doi: 10.1161/CIRCRESAHA.110.227512.
  Ng, E. S., Davis, R. P., Azzola, L., Stanley, E. G., & Elefanty, A. G. (2005). Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood, 106, 1601–1603. doi: 10.1182/blood‐2005‐03‐0987.
  Passier, R., Oostwaard, D. W., Snapper, J., Kloots, J., Hassink, R. J., Kuijk, E., … Mummery, C. (2005). Increased cardiomyocyte differentiation from human embryonic stem cells in serum‐free cultures. Stem Cells, 23, 772–780. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15917473. doi: 10.1634/stemcells.2004‐0184.
  Pfeffer, B., Tziros, C., & Katz, R. J. (2009). Current concepts of anthracycline cardiotoxicity: Pathogenesis, diagnosis and prevention. British Journal of Cardiology, 16, 85–89.
  Preda, M. B., Burlacu, A., & Simionescu, M. (2013). Defined‐size embryoid bodies formed in the presence of serum replacement increases the efficiency of the cardiac differentiation of mouse embryonic stem cells. Tissue and Cell, 45, 54–60. doi: 10.1016/j.tice.2012.09.005.
  Puppala, D., Collis, L. P., Sun, S. Z., Bonato, V., Chen, X., Anson, B., … Engle, S. J. (2013). Comparative gene expression profiling in human‐induced pluripotent stem cell‐derived cardiocytes and human and cynomolgus heart tissue. Toxicological Sciences, 131, 292–301. doi: 10.1093/toxsci/kfs282.
  Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR‐Cas9 system. Nature Protocols, 8, 2281–2308. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/24157548 http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid‐3969860&tool‐pmcentrez&rendertype‐abstract http://www.ncbi.nlm.nih.gov/pubmed/24157548 http://www.nature doi: 10.1038/nprot.2013.143.
  Sharma, A., Wu, J. C., & Wu, S. M. (2013). Induced pluripotent stem cell‐derived cardiomyocytes for cardiovascular disease modeling and drug screening. Stem Cell Research & Therapy, 4, 150. Available at: Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=4056681&tool=pmcentrez&rendertype=abstract. doi: 10.1186/scrt380.
  Šimůnek, T., Štěrba, M., Popelová, O., Adamcová, M., Hrdina, R., & Gerši, V. (2009). Anthracycline‐induced cardiotoxicity: Overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacological Reports, 61, 154–171. doi: 10.1016/S1734‐1140(09)70018‐0.
  Singal, P. K., Li, T., Kumar, D., Danelisen, I., & Iliskovic, N. (2000). Adriamycin‐induced heart failure: Mechanism and modulation. Molecular and Cellular Biochemistry, 207, 77–86. doi: 10.1023/A:1007094214460.
  Volkova, M., Russell, R., & Russell, R. III (2011). Anthracycline cardiotoxicity: Prevalence, pathogenesis and treatment. Current Cardiology Reviews, 7, 214–220. Available at: Retrieved from http://www.scopus.com/inward/record.url?eid‐2‐s2.0‐84861325625&partnerID‐40&md5‐ea09081347a23cf723531289e4a1e15b http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322439/pdf/CCR‐7‐214.pdf. doi: 10.2174/157340311799960645.
  Xu, X. Q., Graichen, R., Soo, S. Y., Balakrishnan, T., Rahmat, S. N. B., Sieh, S., … Davidson, B. P. (2008). Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. Differentiation; Research in Biological Diversity, 76, 958–970. Available at: Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18557764. doi: 10.1111/j.1432‐0436.2008.00284.x.,
  Zhang, S., Liu, X., Bawa‐Khalfe, T., Lu, L.‐S., Lyu, Y. L., Liu, L. F., & Yeh, E. T. H. (2012). Identification of the molecular basis of doxorubicin‐induced cardiotoxicity. Nature Medicine, 18, 1639–1642. doi: 10.1038/nm.2919.
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