Differentiation and Contractile Analysis of GFP‐Sarcomere Reporter hiPSC‐Cardiomyocytes

Arun Sharma1, Christopher N. Toepfer2, Manuel Schmid3, Amanda C. Garfinkel1, Christine E. Seidman4

1 Department of Genetics, Harvard Medical School, Boston, Massachusetts, 2 Radcliffe Department of Medicine, University of Oxford, Oxford, 3 Deutsches Herzzentrum München, Technische Universität München, Munich, 4 Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.12
DOI:  10.1002/cphg.53
Online Posting Date:  January, 2018
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Human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful cellular platform for illuminating mechanisms of human cardiovascular disease and for pharmacological screening. Recent advances in CRISPR/Cas9‐mediated genome editing technology underlie this profound utility. We have generated hiPSC‐CMs harboring fluorescently‐tagged sarcomeric proteins, which provide a tool to non‐invasively study human sarcomere function and dysfunction. In this unit, we illustrate methods for conducting high‐efficiency, small molecule‐mediated differentiation of hiPSCs into cardiomyocytes, and for performing non‐invasive contractile analysis through direct sarcomere tracking of GFP‐sarcomere reporter hiPSC‐CMs. We believe that this type of analysis can overcome sensitivity problems found in other forms of contractile assays involving hiPSC‐CMs by directly measuring contractility at the fundamental contractile unit of the hiPSC‐CM, the sarcomere. © 2018 by John Wiley & Sons, Inc.

Keywords: cardiomyocytes; contractility; CRISPR; drug screening; fluorescent reporter; iPSCs; pluripotent stem cells; sarcomere

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Differentiation of Genome‐Edited, Fluorescent Sarcomere Reporter hiPSCs Into Cardiomyocytes
  • Basic Protocol 2: Non‐Invasive, Functional Assessement of Sarcomere Dynamics in Genome‐Edited, GFP‐Sarcomere Reporter hiPSC‐CMs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Differentiation of Genome‐Edited, Fluorescent Sarcomere Reporter hiPSCs Into Cardiomyocytes

  • GFP‐sarcomere reporter hiPSCs (unit 21.11; Sharma et al., )
  • mTeSR1 feeder‐free pluripotent stem cell maintenance medium (StemCell Technologies, cat. no. 05850)
  • Matrigel (hESC‐qualified; BD Biosciences, cat. no. 354277)
  • DMEM/F12 medium (Thermo Fisher Scientific, cat. no. 11320033)
  • hiPSC dissociation buffer (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 20012‐050)
  • hiPSC passaging medium (see recipe)
  • Cardiomyocyte differentiation, maintenance, and purification media (see recipes):
    • CHIR99021 (Tocris, cat. no. 4423)
    • Wnt‐C59 (Biorbyt, cat. no. orb181132)
    • DMSO (Sigma‐Aldrich, cat. no. D‐2650)
    • 6‐well tissue culture‐treated plates

Basic Protocol 2: Non‐Invasive, Functional Assessement of Sarcomere Dynamics in Genome‐Edited, GFP‐Sarcomere Reporter hiPSC‐CMs

  Materials (also see protocol 1)
  • Genome‐edited, GFP‐sarcomere reporter hiPSC‐CMs ( protocol 1), no older than day 20
  • Cardiomyocyte passaging medium (see recipe)
  • TrypLE express enzyme (Thermo Fisher Scientific, cat. no. 12605010)
  • Steriflip‐GP, 0.22‐µm (Millipore, cat. no. SCGP00525)
  • 12‐well glass bottom imaging plate (MatTek, cat. no. P12G‐1.0‐10‐F)
  • 15‐ and 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Fluorescence plate microscope with 40×, 60×, or 100× objectives
  • SarcOptiM ImageJ analysis software (https://pccv.univ‐tours.fr/ImageJ/SarcOptiM/)
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Literature Cited

Literature Cited
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