Differentiation of Human Embryonic Stem Cells to Cardiomyocytes by Coculture with Endoderm in Serum‐Free Medium

Christine L. Mummery1, Dorien Ward1, Robert Passier1

1 University of Utrecht Medical Centre, Utrecht, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.2
DOI:  10.1002/9780470151808.sc01f02s2
Online Posting Date:  July, 2007
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Many of the applications envisaged for human embryonic stem cells (hESC) undergoing cardiomyogenesis require that the differentiation procedure is robust and high yield. For many hESC lines currently available this is a challenge; beating areas are often obtained but subsequent analysis shows only few (<1%) cardiomyocytes actually present. Here the authors provide a protocol based on serum‐free coculture with a mouse endoderm‐like cell line (END2), which yields cultures containing on average 25% cardiomyocytes for two widely available hESC lines, hES2 and hES3. The authors also provide a variant on the protocol based on growth of hESC aggregates/embryoid bodies in END2‐conditioned medium and a method for dissociating beating aggregates without compromising cardiomyocyte viability so that they can be used for transplantation into animals or further (electrophysiological) analysis. Curr. Protoc. Stem Cell Biol. 2:1F.2.1‐1F.2.14. © 2007 by John Wiley & Sons, Inc.

Keywords: cardiomyocytes; human embryonic stem cells; coculture; hES2; hES3; visceral endoderm

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Table of Contents

  • Introduction
  • Basic Protocol 1: Setting Up hESC and END2 Cocultures and Maintenance of Culture During Differentiation
  • Support Protocol 1: Culture of END2 Cells and Preparation as Feeder Cells
  • Basic Protocol 2: Selection and Dissociation of Beating Areas
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Setting Up hESC and END2 Cocultures and Maintenance of Culture During Differentiation

  • Culture plates (12‐well) with mitomycin C–treated END2 cells ( protocol 2)
  • hESC medium (for hES2 and hES3 cells; see recipe) containing FBS for undifferentiated cell growth
  • hESC medium without FBS, for differentiation
  • Colonies of hESC (unit 1.1) in organ culture dishes Phosphate‐buffered saline calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen), sterile, 37°C
  • Dispase solution, 10 mg/ml in standard hESC medium, freshly prepared and filter sterilized (using a 0.22‐µm filter)
  • Antibody against α‐actinin (mouse monoclonal; Sigma), optional
  • Antibody against tropomyosin (mouse monoclonal; Sigma), optional
  • 37°C, 5% CO 2 incubator
  • 35‐mm tissue culture dishes
  • Organ culture dishes
  • 1000‐µl pipet
  • 200‐ to 1000‐µl pipet tips
  • Stereo dissecting microscope at 4× magnification (with heated stage if possible)
  • Additional reagents and equipment for obtaining culture plates with mitomycin C–treated END2 cells ( protocol 2) and obtaining organ dishes containing colonies of hESC (unit 1.1)

Support Protocol 1: Culture of END2 Cells and Preparation as Feeder Cells

  • 0.1% (w/v) gelatin (see recipe)
  • END2 culture medium (see recipe)
  • END2 cells ( )
  • Phosphate‐buffered saline, calcium and magnesium free (CMF‐PBS; Invitrogen), sterile
  • Mitomycin C stock (see recipe)
  • Trypsin/EDTA (Invitrogen, no. 25300‐054)
  • 25‐, 75‐, and 175‐cm2 tissue culture flasks coated with 0.1% gelatin
  • 37°C incubator
  • 12‐well plates coated with 0.1% gelatin
  • Cover slips treated with 0.1% gelatin, in a 12‐well plate
  • Additional reagents and equipment for counting cells (Phelan, )

Basic Protocol 2: Selection and Dissociation of Beating Areas

  • 12‐day cocultures of hES2 or hES3 cells displaying beating areas ( protocol 1)
  • hESC medium with FBS (see recipe)
  • Low‐calcium buffer (buffer 1; see recipe)
  • Enzyme buffer (buffer 2; see recipe)
  • KB buffer (buffer 3; see recipe)
  • Scissors
  • 12‐well plate
  • 1000‐µl pipet
  • 200‐ to 1000‐µl pipet tips
  • Nonpivoting shaker1.5‐ml microcentrifuge tube, optional
  • Gelatin‐coated (0.1%) coverslips
  • Additional reagents and equipment for obtaining 12‐day cocultures of hES2 or hES3 cells displaying beating areas ( protocol 1)
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Literature Cited

Literature Cited
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