Differentiation of Human Embryonic Stem Cells in Adherent and in Chemically Defined Culture Conditions

Ludovic Vallier1, Roger Pedersen1

1 Department of Surgery and Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom.
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.4
DOI:  10.1002/9780470151808.sc01d04s4
Online Posting Date:  March, 2008
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Generating fully functional differentiated cells from human embryonic stem cells and achieving this goal using clinically compatible conditions remain major challenges for the stem cell field. The presence of undefined components in standard culture media and protocols (including animal‐derived serum, feeder cells, and extracellular matrices) has significantly impeded the achievement of these objectives. Here, we describe culture conditions to differentiate pluripotent cells in adherent conditions and in the absence of stroma cells, feeder cells, conditioned medium, serum, or complex matrices. Importantly, these defined culture conditions are devoid of animal products, thereby eliminating factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Curr. Protoc. Stem Cell Biol. 4:1D.4.1‐1D.4.7. © 2008 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; differentiation; pluripotency; chemically defined

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1:

  • Cultures of hESC cells (grown on feeder or in feeder‐free conditions, confluent)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (CMF‐PBS; Invitrogen, cat. no. 14190‐094)
  • 10 mg/ml human fibronectin (Chemicon, cat. no. FC010)
  • Chemically defined medium (CDM; see recipe)
  • Activin (R&D Systems)
  • FGF2 (R&D Systems)
  • Collagenase solution (see recipe)
  • Growth factors (see recipe)
  • 6‐ or 12‐well plastic plate, tissue culture treated (Corning)
  • 5‐ml pipet
  • 15‐ml conical centrifuge tube
  • Additional reagents and equipment for immunofluorescence, FACS, and PCR analyses (unit 1.3)
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Literature Cited

Literature Cited
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