Murine Embryonic Stem Cell Derivation, In Vitro Pluripotency Characterization, and In Vivo Teratoma Formation

Yu‐Fen Chou1, Akiko Yabuuchi2

1 Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York, 2 Advanced Medical Research Institute of Fertility, Kato Ladies Clinic, Tokyo, Japan
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.22
DOI:  10.1002/0471140856.tx0222s50
Online Posting Date:  November, 2011
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Abstract

The derivation of embryonic stem (ES) cells represents one of the most important breakthroughs in mammalian developmental biology. In addition to their utility in a wide array of in vitro studies, ES cells are also one of the most useful starting materials for the generation of mutants by homologous recombination in mice (Thomson and Solter, 1988). When ES cells are injected into host blastocysts and transferred to the uterus of a pseudo‐pregnant mouse, they can contribute to different types of tissues in chimeric mice, including the germ line (Bradley et al., 1984). Hundreds of genes have been studied through genetic manipulation of ES cells to model human genetic diseases. In this unit, the ES cell lines are derived from the 129SvEv mice strain, which has a high probability of promoting germ line transmission. Procedures for validating and characterizing ES cell pluripotency are also described in detail. Curr. Protoc. Toxicol. 50:2.22.1‐2.22.13. © 2011 by John Wiley & Sons, Inc.

Keywords: embryonic stem (ES) cell; blastocyst; derivation; pluripotency; teratoma

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Murine Embryonic Stem (ES) Cell Derivation
  • Basic Protocol 2: In Vitro Pluripotency Characterization
  • Basic Protocol 3: In Vivo Teratoma Formation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Murine Embryonic Stem (ES) Cell Derivation

  Materials
  • 129SvEv female and male mice (3‐ to 4‐week–old females and 8‐week‐old males; Taconic)
  • Pregnant mare serum gonadotropin (PMSG; see recipe)
  • Human chorionic gonadotropin (hCG; see recipe)
  • Mouse embryonic stem (mES) cell culture medium (see recipe)
  • Mouse embryonic fibroblast (MEF) culture medium (see recipe)
  • 0.2% (w/v) gelatin solution in Milli‐Q water (see recipe)
  • Mouse embryonic fibroblasts (MEFs) treated with mytomycin C from CF1 strain (Chemicon/Millipore)
  • 1× EmbryoMax M2 medium with phenol red (Millipore)
  • Iodine or combination of 70% ethanol/surfactant
  • Acid tyrode solution (Millipore)
  • 1× FHM HEPES–buffered medium without phenol red (Millipore)
  • 0.25% (1×) trypsin/EDTA (Invitrogen)
  • Phosphate buffered saline without Ca2+ and Mg2+ (PBS; Invitrogen)
  • C57BL/6‐TgN(ACTbEGFP) male mice (Jackson Laboratories); only for generation of GFP‐positive ES cell lines (ES cell lines are F1 cross between C57BL/6 and 129SvEv; B6/129 F1 hybrid; also called V6.5), optional
  • 1‐ml syringes (BD Biosciences)
  • 27‐G needles (BD Biosciences)
  • Fine forceps and fine scissors
  • IVF 4‐well plates (Nunc/Fisher)
  • 37°C, 5% CO 2 incubator with water tray
  • 37°C water bath
  • 35‐ and 60‐mm tissue culture dishes (Corning/Fisher)
  • Inverted light microscope
  • 0.22‐µm bottle top filter (Corning/Fisher)

Basic Protocol 2: In Vitro Pluripotency Characterization

  Materials
  • ES cells
  • Phosphate buffered saline (PBS; Invitrogen)
  • 4% paraformaldehyde (PFA; Sigma)
  • 0.05% (w/w) Tween‐20 (Sigma or Bio‐Rad) in PBS
  • Alkaline phosphatase (AP) staining kit (Chemicon)
  • 3% BSA (Sigma) and 0.1% Triton X‐100 (Sigma) in PBS
  • Primary antibodies:
    • Oct4 antibody (Santa Cruz)
    • Sox2 antibody (Chemicon)
    • Nanog antibody (Abcam)
  • Secondary antibodies conjugated with desired fluorochromes (Molecular Probe)
  • Vectashield mounting medium with DAPI (Vector Laboratories)
  • RPMI medium (Invitrogen) containing 0.5% FBS, ice cold
  • Anti‐SSEA1 antibody (Hybridoma bank at the University of Iowa)
  • PE‐conjugated rat anti‐mouse IgM (BD Biosciences)
  • 35‐mm tissue culture dishes or 6‐well plates (Corning/Fisher)
  • Fluorescent microscope with digital camera
  • Coverslips
  • FACSCalibur analyzer or FACSAria cell sorter (BD Biosciences)

Basic Protocol 3: In Vivo Teratoma Formation

  Materials
  • ES cells
  • Bovine collagen (StemCell Technologies)
  • Matrigel (Sigma)
  • MEF medium (see recipe)
  • NOD.CB17‐Prkdcscid/J male mice (3 to 5 weeks old; Jackson Laboratories)
  • Isoflurane (Fisher)
  • 4% paraformaldehyde (PFA; Sigma)
  • 70% ethanol
  • 100‐mm tissue culture dish (Corning/Fisher)
  • Hemacytometer
  • 50‐ml tubes (Falcon)
  • 1‐ml syringe (BD Biosciences)
  • 23‐G needle (BD Biosciences)
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Figures

Videos

Literature Cited

Literature Cited
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