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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


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

  • 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

  • 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

  • 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)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Bradley, A., Evans, M., Kaufman, M.H., and Robertson, E. 1984. Formation of germ‐line chimaeras from embryo‐derived teratocarcinoma cell lines. Nature 309:255‐256.
   Brüstle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiestler, O.D., Duncan, I.D., and McKay, R.D. 1999. Embryonic stem cell‐derived glial precursors: A source of myelinating transplants. Science 285:754‐756.
   Cerdan, C., Hong, S.H., and Bhatia, M. 2007. Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures. Curr. Protoc. Stem Cell Biol. 3:1D.2.1‐1D.2.16.
   Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J.C., and Keller, G. 1998. A common precursor for hematopoietic and endothelial cells. Development 125:725‐732.
   Chou, Y.F., Chen, H.H., Eijpe, M., Yabuuchi, A., Chenoweth, J.G., Tesar, P., Lu, J., McKay, R.D., and Geijsen, N. 2008. The growth factor environment defines distinct pluripotent ground states in novel blastocyst‐derived stem cells. Cell 135:449‐461.
   Evans, M.J. and Kaufman, M.H. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154‐156.
   Fuegemann, C.J., Samraj, A.K., Walsh, S., Fleischmann, B.K., Jovinge, S., and Breitbach, M. 2010. Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging‐drop and mass culture methods. Curr. Protoc. Stem Cell Biol. 15:1F.11.1‐1F.11.13.
   Gertow, K., Przyborski, S., Loring, J.F., Auerbach, J.M., Epifano, O., Otonkoski, T., Damjanov, I., and Ährlund‐Richter, L. 2007. Isolation of human embryonic stem cell‐derived teratomas for the assessment of pluripotency. Curr. Protoc. Stem Cell Biol. 3:1B.4.1‐1B.4.29.
   Gorba, T. and Allsopp, T.E. 2003. Pharmacological potential of embryonic stem cells. Pharmacol. Res. 47:269‐278.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Klug, M.G., Soonpaa, M.H., Koh, G.Y., and Field, L.J. 1996. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J. Clin. Invest. 98:216‐224.
   Martin, G.R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U.S.A. 78:7634‐7638.
   Martin, G.R. and Evans, M.J. 1975. Differentiation of clonal lines of teratocarcinoma cells: Formation of embryoid bodies in vitro. Proc. Natl. Acad. Sci. U.S.A. 72:1441‐1445.
   Pease, S., Braghetta, P., Gearing, D., Grail, D., and Williams, R.L. 1990. Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF). Dev. Biol. 141:344‐352.
   Scholz, G., Genschow, E., Pohl, I., Bremer, S., Paparella, M., Raabe, H., Southee, J., and Spielmann, H. 1999. Prevalidation of the embryonic stem cell test (EST)—A new in vitro embryotoxicity test. Toxicol. In Vitro 13:675‐681.
   Schmidt, J.V. 2001. Embryonic stem (ES) cell culture basics. Curr. Protoc. Toxicol. 9:15.1.1‐15.1.15.
   Thomson, J.A. and Solter, D. 1988. The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos. Genes Dev. 2:1344‐1351.
   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 292:1145‐1147.
   Turksen, K. 2006a. Embryonic Stem Cell Protocols. Volume 1: Isolation and Characterization. Second Edition. Humana Press, Totowa, New Jersey.
   Turksen, K. 2006b. Embryonic Stem Cell Protocols. Volume 2: Differentiation Models. Second Edition. Humana Press, Totowa, New Jersey.
   Yabuuchi, A., Lerou, P.H., and Daley, G.Q. 2008. Derivation of Mouse Parthenogenetic Embryonic Stem Cells. Methods in Bioengineering. pp. 23‐27. (M.L. Yarmush and R.S. Langer, eds.). Artech House, Boston.
PDF or HTML at Wiley Online Library