Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes (hiPSC‐CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways

Liang Guo1, Sandy Eldridge1, Mike Furniss2, Jodie Mussio2, Myrtle Davis3

1 These authors contributed equally to this work, 2 Laboratory of Investigative Toxicology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 3 Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150035
Online Posting Date:  September, 2015
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Abstract

There is a need to develop mechanism‐based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC‐CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC‐CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. © 2015 by John Wiley & Sons, Inc.

Keywords: human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs); cell signaling pathways; protein expression; nanofluidic proteomic immunoassay (NIA); real‐time impedance and field potential–based cell assay (RTCA); CardioECR system; multiparameter imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation, Maintenance, and Characterization of Human Induced Pluripotent Stem Cell–Derived Cardiomyocyte Cultures
  • Basic Protocol 2: Multiparameter Biochemical and Imaging Assays to Evaluate Cell Viability, Mitochondrial Membrane Potential, Caspase Activation, ATP, LDH, and Cardiac Troponin
  • Basic Protocol 3: Real‐Time Monitoring of Cardiomyocyte Function by Impedance‐Based Contractility and Electrical Activity by Field Potential Measurement
  • Basic Protocol 4: Analysis of ErbB, AKT, AND Erk1/2 Protein Expression and Pathway Modulation in Human Induced Pluripotent Stem Cell–Derived Cardiomyoctyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation, Maintenance, and Characterization of Human Induced Pluripotent Stem Cell–Derived Cardiomyocyte Cultures

  Materials
  • Gelatin (Sigma, cat. no. G1890)
  • 1 mg/ml fibronectin (Sigma, cat. no. F1141‐1 mg) stock solution
  • Dulbecco's phosphate‐buffered saline with Ca2+/Mg2+ (DPBS; Lonza, cat. no. 17‐513) and without Ca2+/Mg2+ (CMF‐DPBS; Lonza, cat. no. 17‐512)
  • Human induced pluripotent stem cell–derived cardiomyocytes (iCell Cardiomyocytes, Cellular Dynamics International) and plating/thawing and maintenance media (Cellular Dynamics International)
  • 70% ethanol
  • 32% or 40% paraformaldehyde (Electron Microscopy Sciences, cat. no. 15714 or 15715)
  • 0.1% (v/v) Triton X‐100 (Sigma, cat. no. T8787) CMF‐DPBS
  • Odyssey Blocking Buffer (LI‐COR, cat. no. 927‐40003)
  • Primary antibodies:
    • Rabbit anti–cardiac troponin I (anti‐cTnI; Abcam, cat. no. ab52862)
    • Mouse anti‐myomesin (clone B4; University of Iowa Developmental Studies Hybridoma Bank)
  • Secondary antibodies:
    • Anti‐rabbit antibody conjugated with FITC (Life Technologies, cat. no. A11008)
    • Anti‐mouse antibody conjugated with FITC (Life Technologies, cat. no. A11029)
  • 2.5 μg/ml Hoechst 33258 (Enzo Life Sciences, cat. no. ENZ‐52402) in CMF‐DPBS
  • Water bath with adjustable temperature setting (37° to 60°C)
  • 250‐ml polystyrene bottles
  • 0.22‐μm bottle‐top filtration units
  • Sterile tissue culture plates (6‐, 12‐, or 24‐well), E‐plate Cardio 96‐well plates (ACEA Biosciences, cat. no. 6417035001) and E‐plate CardioECR 48‐well plates (ACEA Biosciences, cat. no. 300600950), or Acoustic uClear 96‐well plates (Greiner, cat. no. 655090)
  • Floating microcentrifuge tube rack
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Automated cell counter (Vi‐CELL XR Cell Viability Analyzer, Beckman Coulter) or hemacytometer with 0.4% trypan blue solution (Sigma, cat. no.T8154)
  • Inverted phase‐contrast microscope
  • 50 ml reagent transfer reservoirs for multichannel pipettors
  • 100‐μl to 1‐ml single‐channel pipettor
  • 300‐μl 8‐channel pipettor
  • 100‐μl to 1 ml‐low‐adhesion pipet tips
  • Tilting plate holder (Thomas Scientific, cat. no. 1226C51)
  • Nikon ECLIPSE Ti fluorescence microscope equipped with Nikon DS‐Ri1 digital camera (Nikon Instrument)
  • IN Cell Analyzer 2000 (GE Healthcare)

Basic Protocol 2: Multiparameter Biochemical and Imaging Assays to Evaluate Cell Viability, Mitochondrial Membrane Potential, Caspase Activation, ATP, LDH, and Cardiac Troponin

  Materials
  • Hoechst 33258 (Enzo Life Sciences, cat. no. ENZ‐52402)
  • Human induced pluripotent stem cell–derived cardiomyocytes (iCell Cardiomyocytes, Cellular Dynamics International) and plating/thawing and maintenance media (Cellular Dynamics International); live or fixed (see protocol 1)
  • Cell Meter JC‐10 assay kit (AAT Bioquest, cat. no. 22800)
  • Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP; Tocris, cat. no. 0452)
  • CellTiter‐Glo Luminescent Cell Viability Assay (ATP) kit (Promega, cat. no. G7570)
  • CytoTox 96 Non‐Radioactive Cytotoxicity Assay Lactate Dehydrogenase (LDH) kit (Promega, cat. no. G1780)
  • Caspase‐Glo 3/7 Assay kit (Promega, cat. no. G8090)
  • Serum‐free DMEM: DMEM (no glucose; Life Technologies, cat. no. 11966‐025) supplemented with 1 mM sodium pyruvate (Life Technologies, cat. no. 11360‐070) and 10 mM D‐(+)‐galactose (Sigma‐Aldrich, cat. no. G5388‐100 G)
  • Trastuzumab (Genentech, Inc.) or Neuregulin‐1β (NRG) [Recombinant human neuregulin‐1β (NRG1‐β1/HRG1‐β1 EGF domain), R&D Systems, cat. no. 396‐HB‐050]
  • Doxorubicin‐HCl (Enzo Life Sciences, cat. no. BML‐GR319)
  • Dimethylsulfoxide (DMSO, Sigma, cat. no. 2650)
  • MSD 96‐Well MULTI‐ARRAY Human Cardiac Troponin T Assay Kit (Meso Scale Discovery, cat. no. K151EFC)
  • Tween 20
  • Dulbecco's phosphate‐buffered saline without Ca2+/Mg2+ (CMF‐DPBS; Lonza, cat. no. 17‐512)
  • 325/455 nm (excitation/emission) filter set
  • IN Cell Analyzer 2000 (GE Healthcare)
  • TECAN Infinite M1000 Plate Reader (TECAN)
  • 96‐well E‐plate Cardio 96 (ACEA Biosciences, cat. no. 64170350001) or uClear optical bottom plates; Greiner Bio‐One, cat. no. 655090)
  • 96‐well white opaque‐walled, flat‐bottom plate (Greiner Bio‐One, cat. no. 655904)
  • 96‐well polypropylene V bottom plate (Greiner Bio‐One, cat. no. 651261)
  • MSD SECTOR Imager 6000 (Meso Scale Discovery)
  • Refrigerated centrifuge with microtiter plate carrier
  • Adhesive plate seals (VWR, cat. no. 60941‐126)
  • Plate shaker
  • Additional reagents and equipment for staining nuclear DNA in cells fixed with paraformaldehyde ( protocol 1, steps 13 to 21)

Basic Protocol 3: Real‐Time Monitoring of Cardiomyocyte Function by Impedance‐Based Contractility and Electrical Activity by Field Potential Measurement

  Materials
  • Human induced pluripotent stem cell–derived cardiomyocytes (iCell Cardiomyocytes, Cellular Dynamics International) and plating/thawing and maintenance media (Cellular Dynamics International)
  • Doxorubicin‐HCl (Enzo Life Sciences, cat. no. BML‐GR319)
  • Neuregulin‐1β (NRG) [Recombinant human neuregulin‐1β (NRG1‐β1/HRG1‐β1 EGF domain), R&D Systems, cat. no. 396‐HB‐050]
  • Dimethylsulfoxide (DMSO, Sigma, cat. no. 2650−5 × 5 ml)
  • Dulbecco's phosphate‐buffered saline with Ca2+/Mg2+ (DPBS; Lonza, cat. no. 17‐513)
  • Bovine serum albumin (BSA, Sigma, cat. no. A9418)
  • Trastuzumab (Genentech, Inc.)
  • E‐4031 dihydrochloride (Tocris, cat. no. 1808)
  • Blebbistatin (Sigma, cat. no. B0560)
  • xCELLigence RTCA Cardio instrument (ACEA Biosciences, cat. no. 380601060)
  • E‐Plate cardio 96 (ACEA Biosciences, cat. no. 6417035001), freshly coated with gelatin as in protocol 1
  • Spreadsheet software, e.g., MS Excel
  • E‐Plate CardioECR 48 (ACEA Biosciences, cat. no. 300600950), freshly coated with fibronectin as in protocol 1
  • xCELLigence RTCA CardioECR system (ACEA Biosciences, cat. no. 380601210)
  • Additional reagents and equipment for plating cells on fibronectin‐coated plates ( protocol 1) and quantification of cellular ATP content, LDH release, caspase‐3/7 activity, and cardiac troponin release ( protocol 2)

Basic Protocol 4: Analysis of ErbB, AKT, AND Erk1/2 Protein Expression and Pathway Modulation in Human Induced Pluripotent Stem Cell–Derived Cardiomyoctyes

  Materials
  • Human induced pluripotent stem cell–derived cardiomyocytes (iCell Cardiomyocytes, Cellular Dynamics International) and plating/thawing and maintenance media (Cellular Dynamics International); growing in culture in 6‐well plates (see protocol 1)
  • Dulbecco's phosphate‐buffered saline with Ca2+/Mg2+ (DPBS; Lonza, cat. no. 17‐513) and without Ca2+/Mg2+ (CMF‐DPBS; Lonza, cat. no. 17‐512)
  • Complete RIPA lysis buffer (see recipe)
  • Complete M‐PER lysis buffer (see recipe)
  • NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, cat. no. 28833)
  • Phosphatase Inhibitor Cocktail (Sigma, cat. no. P0044)
  • Halt Protease Inhibitor Cocktail, EDTA‐free (Thermo Scientific, cat. no. 87785)
  • Protease Inhibitor Cocktail (Sigma, cat. no. P8340)
  • Pierce BCA Protein Assay Kit (Thermo Scientific, cat. no. 23225)
  • pI Standard Ladder 3 (ProteinSimple, cat. no. 040‐646)
  • XDR Charge Separation Master Kit for Peggy Sue, Peggy, or NanoPro 1000 (ProteinSimple, cat. no. CBS 2001)
  • Wes 12‐230 kDa Master Kit (ProteinSimple, cat. no. PS‐MK01 or PS‐MK02)
  • Antibodies:
    • EGF/ErbB1 Receptor (D38B1) XP rabbit monoclonal antibody (Cell Signaling, cat. no. 4267 S)
    • ErbB2 Receptor (M45) rabbit polyclonal antibody (Cell Signaling, cat. no. 3250 S)
    • ErbB4 Receptor (111B2) rabbit monoclonal antibody (Cell Signaling, cat. no. 4795 S)
    • AKT (pan; C67E7) rabbit monoclonal antibody (Cell Signaling, cat. no. 4691 S)
    • Phospho‐AKT (Ser473) rabbit polyclonal antibody (Cell Signaling, cat. no. 9271)
    • p44/42 MAPK (Erk1/2) rabbit polyclonal antibody (Cell Signaling cat. no. 9102)
    • Phospho‐ p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal (Cell Signaling, cat. no. 9101)
    • Alpha‐tubulin (11H10) rabbit monoclonal antibody (Cell Signaling, cat. no. 2125)
    • Lamin B1(D4Q4Z) rabbit monoclonal antibody (Cell Signaling, cat. no. 12586)
    • FOXO3a (D19A7) rabbit monoclonal antibody (Cell Signaling, cat. no. 12829)
    • Phospho‐FOXO3A (S253) rabbit polyclonal antibody (Abcam, cat. no. ab47285)
    • CREB (48H2) rabbit monoclonal antibody (Cell Signaling, cat. no. 9197)
    • Phospho‐CREB (Ser133) (87G3) rabbit monoclonal (Cell Signaling, cat. no. 9198)
  • Amplified Rabbit or Mouse Secondary Antibody Detection Kit (ProteinSimple, cat. no. 041‐126 or 041‐127, respectively)
  • Goat anti‐rabbit antibody conjugated with Alexa Fluor 488 (Life Technologies, cat. no. A11008)
  • Lipofectamine RNAiMAX transfection reagent (Life Technologies, cat. no. 13778)
  • Opti‐MEM I Reduced Serum Medium (Life Technologies, cat. no. 31985‐062)
  • ON‐TARGETplus SMARTpool Human ErbB2 siRNAs (GE Dharmacon, cat. no. L‐003126‐00‐0005)
  • Non‐targeting pool control siRNAs (GE Dharmacon, cat. no. D‐00180‐10‐05)
  • Lapatinib (National Cancer Institute compound repository, cat. no. NSC 727989)
  • Dimethylsulfoxide (DMSO, Sigma, cat. no. 2650−5 × 5 ml)
  • Neuregulin‐1β (NRG) [Recombinant human neuregulin‐1β (NRG1‐β1/HRG1‐β1 EGF domain), R&D Systems, cat. no. 396‐HB‐050]
  • Phosphate‐buffered saline (PBS; Lonza, cat. no. 17‐517Q)
  • Bovine serum albumin (BSA, Sigma, cat. no. A9418)
  • Bicine/CHAPS Lysis Buffer and Sample Diluent (ProteinSimple, cat. no. 040‐764)
  • DMSO Inhibitor Mix (ProteinSimple, cat. no. 040‐510)
  • Trastuzumab (Genentech, Inc.)
  • Serum‐free DMEM: DMEM (no glucose; Life Technologies, cat. no. 11966‐025) supplemented with 1 mM sodium pyruvate (Life Technologies, cat. no. 11360‐070) and 10 mM D‐(+)‐galactose (Sigma‐Aldrich, cat. no. G5388‐100 G)
  • Premix G2, Pharmalyte pH 5–8 Separation Gradient (ProteinSimple, cat. no. 040‐973)
  • Paraformaldehyde (Electron Microscopy Science, cat. no. 15714)
  • Odyssey Blocking Buffer (LI‐COR, cat. no. 927‐40003)
  • Goat serum (Rockland, cat. no. D204‐00‐0050)
  • Sytox red (Life Technologies, cat. no. 34859)
  • Cell scrapers, sterile
  • Tilting plate holder (Thomas Scientific, cat. no. 1226C51)
  • 1.5‐ml microcentrifuge tubes, ice‐cold
  • Sonic Dismembrator (Fisher Scientific, model 100)
  • Infinite M1000 plate reader (TECAN)
  • MS Excel (optional)
  • Wes size‐based electrophoresis immunoassay Simple Western system (ProteinSimple) and Wes Compass software (provided with the instrument or offered as free download from ProteinSimple Web site)
  • Centrifuge with microtiter plate carrier
  • 6‐ or 12‐well culture plates
  • NanoPro 1000 charge‐based nanofluidic isoelectric focusing proteomic immunoassay (NIA) Simple Western system (ProteinSimple)
  • 96‐well Acoustic uClear Flat‐Bottom Black‐Wall Plates (Greiner, cat. no. 655090)
  • IN Cell Analyzer 2000 (GE Healthcare)
  • Additional reagents and equipment for culturing cardiomyocytes ( protocol 1)
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Figures

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Literature Cited

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