Using ES Cells Labeled with GFP for Analyzing Cell Behavior During Differentiation

Zhonghua Liu1, Queenie P. Vong2, Yixian Zheng1

1 Carnegie Institution for Science, Department of Embryology, Baltimore, Maryland, 2 St. Jude Children's Research Hospital, Memphis, Tennessee
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1D.8
DOI:  10.1002/9780470151808.sc01d08s22
Online Posting Date:  August, 2012
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Abstract

Mouse embryonic stem (ES) cell is a unique yet highly successful system to study stem cell maintenance and differentiation. In this protocol, we describe the generation of engineered mouse ES cells that stably express GFP alone or GFP fused with histone H2B, which allow the cells to be traced live or in fixed samples. This system can be used to study cell behavior changes as ES cells differentiate into different lineages. Curr. Protoc. Stem Cell Biol. 22:1D.8.1‐1D.8.9. © 2012 by John Wiley & Sons, Inc.

Keywords: mouse embryonic stem cell; live imaging; time‐lapse movie; differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishment of ES Cells Stably Expressing Histone H2B‐GFP
  • Basic Protocol 2: Monitoring ES Cell Migration by Time‐Lapse Movie During Trophectoderm Differentiation
  • Support Protocol 1: Measurement of Cell Motility by Nuclear Displacement
  • Basic Protocol 3: Analyzing Lineage Segregation by Cell Sorting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Establishment of ES Cells Stably Expressing Histone H2B‐GFP

  Materials
  • Plate of low‐passage E14 mouse embryonic stem (ES) cells (BayGenomics) without feeder
  • 0.1% gelatin (Millipore)
  • GMEM basal medium (Invitrogen)
  • Lipofectamine 2000 (Invitrogen)
  • hEF‐1α promoter driven H2B‐GFP expression construct with neomycin resistance, linearized (available upon request)
  • Phosphate‐buffered saline (PBS), calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen)
  • 0.05% Trypsin‐EDTA (Invitrogen)
  • Complete ES cell medium (see recipe)
  • G418 (Invitrogen)
  • Gelatinized 96‐well plate
  • 1.5‐ml microcentrifuge tubes, sterile
  • Inverted fluorescent microscope
  • 1000‐µl pipets
  • Hemacytometer
  • 37°C CO 2 incubator

Basic Protocol 2: Monitoring ES Cell Migration by Time‐Lapse Movie During Trophectoderm Differentiation

  Materials
  • E14 ES cells stably expressing histone H2B‐GFP ( protocol 1) and parental E14 ES cells (BayGenomics)
  • GMEM basal medium (Invitrogen)
  • Lipofectamine 2000 (Invitrogen)
  • Stealth Oct3/4, Nanog, Sox2 siRNA oligos (Invitrogen)
  • Stealth RNAi negative control medium GC (Invitrogen)
  • Complete ES cell medium (see recipe)
  • Gelatinized 12‐well plates
  • 37°C CO 2 incubator
  • Inverted fluorescent microscope (Nikon TE2000) equipped with CoolSNAP HQ CCD camera (Photometrics), 3‐axis stage controller (Prior Scientific), and LiveCell chamber (Pathology Devices)
  • Computer with imaging software (MetaMorph)

Support Protocol 1: Measurement of Cell Motility by Nuclear Displacement

  Materials
  • ZHBTc4 mouse ES cell (generously provided by Dr. Hitoshi Niwa) that harbor the tetracycline‐regulated Oct3/4 transgene (Niwa et al., )
  • E14 mouse embryonic stem cell (Baygenomics)
  • ES cell medium (see recipe)
  • Phosphate‐buffered saline, calcium‐ and magnesium‐free (CMF‐PBS; Invitrogen, cat. no. 20012‐050)
  • Gelatinized 35‐mm culture dishes
  • Tetracycline (10 mg/ml in H 2O; filter sterilized and store up to 1 year at −20°C)
  • 4% Paraformaldehyde in PBS (diluted from 16% stock; Electron Microscopy Sciences, cat. no. 15710)
  • 0.5% Triton‐X in PBS (Sigma‐Aldrich, cat. no. T9284)
  • 4% BSA in PBS (Sigma‐Aldrich, cat. no. A4503)
  • Mouse anti‐Oct3/4 antibody (BD Biosciences)
  • Goat anti‐GFP antibody (Abcam)
  • PBS with 0.05% Tween 20
  • Alexa‐594 donkey anti‐mouse IgG (Molecular Probes)
  • Alexa‐488 donkey anti‐goat IgG (Molecular Probes)
  • Hemacytometer
  • Multichannel pipets
  • 100‐mm petri dishes
  • 37°C incubator
  • Coverslips
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Figures

Videos

Literature Cited

   Conner, D.A. 2001. Mouse embryonic stem (ES) cell culture. Curr. Protoc. Mol. Biol. 51:23.3.1‐23.3.6.
   Murry, C.E. and Keller, G. 2008. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661‐680.
   Nagy, A., Gertsenstein, M., and Vintersten, K. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Niwa, H., Miyazaki, J., and Smith, A.G. 2000. Quantitative expression of Oct‐3/4 defines differentiation, dedifferentiation or self‐renewal of ES cells. Nat. Genet. 24:372‐376.
   Rossant, J. 2008. Stem cells and early lineage development. Cell 132:527‐531.
   Schmidt, J.V. 2001. Embryonic stem (ES) cell culture basics. Curr. Protoc. Toxicol. 9:15.1.1‐15.1.15.
   Vong, Q.P., Liu, Z., Yoo, J.G., Chen, R., Xie, W., Sharov, A.A., Fan, C.M., Liu, C., Ko, M.S., and Zheng, Y. 2010. A role for borg5 during trophectoderm differentiation. Stem Cells 28:1030‐1038.
Internet Resources
   http://baygenomics.ucsf.edu/protocols/index.html
  BayGenomics
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