Differentiation of Embryonic Stem Cells

Ahmed Mansouri1, Hidefumi Fukumitsu2, Jan Schindehuette3, Kerstin Krieglstein1

1 DFG Center for Molecular Physiology of the Brain, Göttingen, Germany, 2 Gifu Pharmaceutical University, Gifu, Japan, 3 University of Göttingen, Department of Clinical Neurophysiology, Göttingen, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.6
DOI:  10.1002/0471142301.ns0306s47
Online Posting Date:  April, 2009
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Abstract

Mouse embryonic stem (ES) cells are derived from mouse blastocyst and are able to generate all embryonic tissues in vitro. This propensity of ES cells has acquired considerable attention in recent years due to the promising potential for future cell replacement–based therapies. Therefore, it is of fundamental interest to establish protocols that allow the differentiation of ES cells into specific cell types. In recent years, several such differentiation procedures have been described for mouse and human embryonic stem cells. This unit describes a simple procedure that promotes the neuronal differentiation of mouse embryonic stem cells and yields a high proportion of midbrain dopaminergic neurons. Furthermore, this procedure permits the isolation of neural stem cell lines from mouse ES cells. Curr. Protoc. Neurosci. 47:3.6.1‐3.6.21. © 2009 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; dopaminergic neurons; SDIA; neural stem cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Mouse Embryonic Stem Cells on Embryonic Fibroblasts
  • Basic Protocol 2: Differentiation of Mouse ES Cells into Neurons with Dopaminergic Characteristics Using PA6 Stromal Cells (Stromal‐Cell Derived Inducing Activity; SDIA)
  • Alternate Protocol 1: Generation of Neural Stem Cells from Mouse Embryonic Stem Cells Using SDIA
  • Support Protocol 1: Preparation of Embryonic Fibroblasts (EMFIS)
  • Support Protocol 2: Inactivation of Embryonic Fibroblasts Using Mitomycin C
  • Support Protocol 3: Maintenance of Stromal Cells PA6
  • Support Protocol 4: Testing Serum Batches for ES Cell Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of Mouse Embryonic Stem Cells on Embryonic Fibroblasts

  Materials
  • Gelatin solution (see recipe for gelatinized plates)
  • Plated feeder layer of mitomycin C–inactivated mouse embryonic fibroblasts (emfis; see protocol 5)
  • Embryonic fibroblast medium (emfis medium; see recipe)
  • ES cells (the authors use own cells MPI II; can be purchased from ATCC), frozen
  • Embryonic stem cell culture medium (ES medium; see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • ES trypsin solution (see recipe)
  • G‐MEM‐based ES medium (see recipe)
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • 3.5‐, 8.5‐, and 14.5‐cm petri dishes (Becton Dickinson)
  • 37°C water bath
  • 2‐ml pipet
  • Pasteur pipet
  • 1‐ml cryotubes (Nunc, cat. no. 368632)
  • Styrofoam box
  • −70°C freezer
  • Additional reagents and equipment for performing a cell count ( appendix 3B)

Basic Protocol 2: Differentiation of Mouse ES Cells into Neurons with Dopaminergic Characteristics Using PA6 Stromal Cells (Stromal‐Cell Derived Inducing Activity; SDIA)

  Materials
  • Frozen ES cells cultured on gelatinized plates in G‐MEM‐based medium ( protocol 1)
  • G‐MEM‐based ES medium (see recipe)
  • Frozen mitomycin C–inactivated PA6 cells ( protocol 6)
  • PA6 medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • ES trypsin solution (see recipe)
  • Differentiation medium (see recipe)
  • Induction medium (see recipe)
  • Glial Cell line‐derived neurotrophic factor GDNF (see recipe)
  • Brain‐derived neurotrophic factor (BDNF; see recipe)
  • FGF8b (see recipe), optional
  • Sonic hedgehog (SHH; see recipe), optional
  • 37°C water bath
  • 14‐ml and 50‐ml conical tubes (e.g., Falcon)
  • 8.5‐cm gelatinized plates (see recipe)
  • 2‐ml pipet
  • Slide flasks (e.g., Nunc, cat. no. 170920)
  • Pasteur pipet
  • Additional reagents and equipment for performing a cell count ( appendix 3B)

Alternate Protocol 1: Generation of Neural Stem Cells from Mouse Embryonic Stem Cells Using SDIA

  • Serum‐containing medium (e.g., ES, fibroblast, or PA6 medium)
  • NS medium (see recipe)
  • Accutase (PAA, cat. no. L11‐007; store up to 1 year at −20°C)
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • Neurobasal differentiation medium (see recipe)
  • Tetrahydrobiopterin (see recipe)
  • Ascorbate (see recipe)
  • Brain‐derived neurotrophic factor (BDNF; see recipe)
  • Epidermal growth factor (EGF; see recipe)
  • Basic Fibroblast Growth Factor 8b (FGF8b; see recipe)
  • Sonic hedgehog (SHH; see recipe)
  • Glial Cell line‐derived neurotrophic factor GDNF (R&D; see recipe)
  • 3.5‐cm plate
  • 1‐ml micropipettor
  • Gelatinized plates (see recipe)
  • Cryotubes
  • Polyornithine/laminin‐coated slide flasks (see recipe)
  • Additional reagents and equipment for differentiation of mouse ES cells ( protocol 2)

Support Protocol 1: Preparation of Embryonic Fibroblasts (EMFIS)

  Materials
  • Pregnant mice (E13‐E15)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% Trypsin/EDTA (Invitrogen, cat. no. 25300‐054; store 10‐ml aliquots up to 2 years at −20°C)
  • Emfis medium (see recipe)
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • Dissection equipment
  • Tweezers and scissors
  • 6‐cm culture dish (Greiner)
  • 500‐ml Erlenmeyer flask, containing 50 ml of glass beads (3‐mm diameter) and a magnetic stirring bar
  • Magnetic stirring device
  • 10‐ and 25‐ml pipets
  • V‐bottomed 14‐ml and 50‐ml Falcon tubes
  • Gelatinized 14.5‐cm and 8.5‐cm dishes (Becton Dickinson; see recipe)
  • Incubator 37°C, 5% CO 2
  • Cryotubes
  • Additional reagents and equipment for removing embryos from pregnant mice (unit 3.2)
NOTE: All solutions, equipment, and instruments that are directly used to isolate the embryonic fibroblasts should be sterilized by filtration or autoclaving.NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 2: Inactivation of Embryonic Fibroblasts Using Mitomycin C

  Materials
  • Frozen fibroblasts ( protocol 4)
  • Embryonic fibroblast (emfis) medium
  • 0.25% Trypsin/EDTA (Invitrogen, cat. no. 25300‐054; store 10‐ml aliquots up to 2 years at −20°C)
  • Mitomycin C solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • ES medium (see recipe)
  • Freezing medium (see recipe)
  • Liquid nitrogen
  • 14‐ml Falcon tubes
  • 8.5‐cm and 14.5‐cm gelatinized culture plates (see recipe)
  • 37°C, 5% CO 2 incubator
  • Gelatinized plates
  • 10‐ml pipets
  • 50‐ml tubes

Support Protocol 3: Maintenance of Stromal Cells PA6

  Materials
  • Stromal cells PA6 (RIKEN Bio Resource Center Cell Bank, Material Transfer agreement required)
  • PA6 medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.25% Trypsin/EDTA (Invitrogen, cat. no. 25300‐054; store 10‐ml aliquots up to 2 years at −20°C )
  • Mitomycin C (see recipe)
  • V‐bottomed 14‐ml and 50‐ml Falcon tubes (Becton Dickinson)
  • 14.5‐cm and 8.5‐cm dishes (Becton Dickinson)
  • Gelatinized plates (see recipe)
  • Additional reagents and equipment for performing a cell count ( appendix 3B)

Support Protocol 4: Testing Serum Batches for ES Cell Culture

  Materials
  • ES cells (the authors use own cells MPI II; can be purchased from ATCC), frozen
  • ES medium (see recipe)
  • Fetal bovine serum (FBS; Invitrogen)
  • Methylene blue (Sigma Aldrich, cat. no. M9140)
  • Basic fuchsin (Sigma Aldrich, cat. no. 47860)
  • Methanol
  • Phosphate‐buffered saline (PBS; see recipe)
  • 3.5‐cm plates
  • Gelatinized 6‐well plates (see recipe)
  • Additional reagents and equipment for culturing ES cells ( protocol 1) and counting cells ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
   Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli, M., Biella, G., Sun, Y., Sanzone, S., Ying, Q.L., Cattaneo, E., and Smith, A. 2005. Niche‐independent symmetrical self‐renewal of a mammalian tissue stem cell. PLoS Biol. 3:e283.
   Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W., and Kemler, R. 1985. The in vitro development of blastocyst‐derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27‐45.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S. I., and Sasai, Y. 2000. Induction of midbrain dopaminergic neurons from ES cells by stromal cell‐derived inducing activity. Neuron 1:31‐40.
   Nagy, A., Gerstsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo, A Laboratory Manual (Third Edition). Cold Spring Harbor Laboratory Press, New York.
   Thomson, J.A., Itskovitz‐Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145‐1157.
   Wichterle, H., Lieberam, I., Porter, J.A., and Jessell, T.M. 2002. Directed differentiation of embryonic stem cells into motor neurons. Cell 110:385‐397.
   Ying, Q.L. and Smith, A.G. 2003. Defined conditions for neural commitment and differentiation. Methods Enzymol. 365:327‐341.
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