Expanding Mouse Ventricular Cardiomyocytes Through GSK‐3 Inhibition

Jan W. Buikema1, Peter‐Paul M. Zwetsloot1, Pieter A. Doevendans1, Joost P.G. Sluijter1, Ibrahim J. Domian2

1 Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands, 2 Harvard Stem Cell Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 23.9
DOI:  10.1002/0471143030.cb2309s61
Online Posting Date:  December, 2013
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Abstract

Controlled proliferation of cardiomyocytes remains a major limitation in cell biology and one of the main underlying hurdles for true modern regenerative medicine. Here, a technique is described for robust expansion of early fetal‐derived mouse ventricular cardiomyocytes on a platform usable for high‐throughput molecular screening, tissue engineering and, potentially, in vivo translational experiments. This method provides a small‐molecule approach to control proliferation or differentiation of early beating cardiomyocytes through modulation of the Wnt/β‐catenin signaling pathway. Moreover, isolation and expansion of fetal cardiomyocytes takes less than 3 weeks, yields a relatively pure (∼70%) functional myogenic population, and is highly reproducible. Curr. Protoc. Cell Biol. 61:23.9.1‐23.9.10. © 2013 by John Wiley & Sons, Inc.

Keywords: cardiomyocyte proliferation; differentiation; isolation; expansion; GSK‐3 inhibitor; Wnt/β‐catenin signaling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Fetal Ventricular Cardiomyocytes
  • Basic Protocol 2: Two‐Dimensional Culture of Fetal Ventricular Cardiomyocytes
  • Alternate Protocol 1: Three‐Dimensional Culture of Fetal Ventricular Cardiomyocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Fetal Ventricular Cardiomyocytes

  Materials
  • Pregnant C57BL/6 or CD1 mouse, E11.5 to E14.5
  • 1× phosphate‐buffered saline (PBS), sterile and ice cold
  • Collagenase digestion solution (see recipe)
  • 0.25% trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • Fetal bovine serum (FBS; Gemini Bioproducts, cat. no. 100‐500)
  • Cardiomyocyte culture medium (see recipe)
  • Scissors
  • Forceps
  • 10‐cm cell culture dish
  • Scalpel
  • 15‐ml centrifuge tubes (BD, cat. no. 352097)
  • 37°C water bath
  • Additional reagents and equipment for counting cells (unit )

Basic Protocol 2: Two‐Dimensional Culture of Fetal Ventricular Cardiomyocytes

  Materials
  • 0.1% (w/v) gelatin (Sigma, cat. no. G1890) in phosphate‐buffered saline (PBS)
  • Cardiomyocyte culture medium (see recipe)
  • 10 mM 6‐bromoindirubin‐3′‐oxime (BIO, a GSK‐3 inhibitor; Sigma, cat. no. B1686) in DMSO (store up to 1 year at −80°C)
  • 10 mM inhibitor of Wnt response‐1 (IWR, an Axin inhibitor; Sigma, cat. no. I0161) in DMSO (store up to 1 year at −80°C)
  • Dimethyl sulfoxide (DMSO)
  • 4% paraformaldehyde (PFA) solution
  • Primary antibodies against:
    • Cardiac troponin T (cTnT, mouse monoclonal, NeoMarkers, Ms‐295, 0.2 mg/ml, 1:250)
    • Ki67 (rabbit monoclonal, Abcam, cat. no. 16667, 1 mg/ml, 1:300)
    • α‐Sarcomeric actinin (α‐SA, mouse monoclonal, Sigma, cat. no. A7811, 1 mg/ml, 1:250)
    • Connexin‐43 (Cx43, rabbit polyclonal, Sigma, cat. no. C6219, 1 mg/ml, 1:150)
  • Secondary antibodies:
    • Donkey anti‐mouse, Alexa 488 (Invitrogen, cat. no. A‐21202, 1:400)
    • Donkey anti‐rabbit, Alexa 594 (Invitrogen, cat. no. A‐21206, 1:400)
  • Saponin (Sigma, cat. no. 47036)
  • 4′,6‐Diamidino‐2‐phenylindole, dihydrochloride (DAPI, Invitrogen, cat. no. D1306, 1:10,000)
  • TRIzol reagent (Invitrogen, cat. no. 15596‐026)
  • Qiagen RNeasy Mini Kit (Qiagen, cat. no. 74104)
  • iScript cDNA Synthesis Kit (Bio‐Rad, cat. no. 170‐8891)
  • 24‐, 96‐, or 384‐well cell culture plate(s)
  • 37°C/5% CO 2 culture incubator
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Figures

Videos

Literature Cited

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Internet Resources
   http://pga.mgh.harvard.edu/primerbank/
  PCR primer sequences are available online at PrimerBank.
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