Download PDF
Thromb Haemost 2005; 94(04): 719-727
DOI: 10.1160/TH05-05-0338
Theme Issue Article
Schattauer GmbH

An optimized embryonic stem cell model for consistent gene expression and developmental studies

A fundamental study
Cornelia Gissel   *
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
,
Chris Voolstra   *
2   Institute for Genetics, University of Cologne, Cologne, Germany
,
Michael Xavier Doss
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
,
Christoph I. Koehler
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
,
Johannes Winkler
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
,
Jürgen Hescheler
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
,
Agapios Sachinidis
1   Center of Physiology and Pathophysiology, Institute of Neurophysiology and Centre of Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
› Author AffiliationsFinancial support: This work was supported by a grant from the European Community (6th Framework Programme, Thematic Priority: Life sciences, genomics and biotechnology for health, Contract No: FunGenES LSHG-CT-2003-503494)
Further Information

Publication History

Received17 May 2005

Accepted after resubmission31 August 2005

Publication Date:
07 December 2017 (online)

    Permissions and Reprints

Summary

In vitro differentiation of embryonic stem (ES) cells results in generation of tissue-specific somatic cells and may represent a powerful tool for general understanding of cellular differentiation and developmentin vivo. Culturing of most ES cell lines requires murine embryonic fibroblasts (MEF), which may influence adventitiously the genetic differentiation program of ES cells. We compared the expression profile of key developmental genes in the MEF-independent CGR8 ES cell line and in the MEFdependent D3 ES cell line. Using neomycin-resistant MEFs we demonstrated that MEFs are able to contaminate the D3 ES cells even after removing the MEFs. Subsequently, optimal differentiation conditions were established for the differentiation of CGR8 ES cells into various germ layer cells. Detailed gene expression studies in differentiating CGR8 cells were done by RTPCR analysis and by microarray analysis demonstrating a general trend of the assessed genes to be expressed either in 3 days- or 10-days old embryoid bodies (EBs) when compared to undifferentiated ES cells. Subsets within the various functional gene classes were defined that are specifically up- or down-regulated in concert. Interestingly, the present results demonstrate that developmental processes toward germ layer formation are irreversible and mostly independent of the culture conditions. Notably, apoptotic and mitochondrial ribosomal genes were downand up-regulated in 10-days old EBs, respectively, whereas compared to the 3-days old EBs whereas the activity of the extracellular signal-regulated kinase (ERK)1/2 decreased with progressive development. This article defines a platform for ES cell differentiation and gene expression studies.

Supplementary information available online atwww.uni-koeln.de/med-fak/physiologie/np/sachinidis.htm

Keywords

CGR8 embryonic stem cells - gene expression - microarrays - development

* Contributed equally


 

  • References

  • 1 Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-6.
    CrossrefPubMedGoogle Scholar
  • 2 Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 7634-8.
    CrossrefPubMedGoogle Scholar
  • 3 Boeuf H, Hauss C, Graeve F D. et al. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol 1997; 138: 1207-17.
    CrossrefPubMedGoogle Scholar
  • 4 Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24: 372-6.
    CrossrefPubMedGoogle Scholar
  • 5 Chambers I, Colby D, Robertson M. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003; 113: 643-55.
    CrossrefPubMedGoogle Scholar
  • 6 Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 1995; 7: 862-9.
    CrossrefPubMedGoogle Scholar
  • 7 Rodaway A, Patient R. Mesendoderm. an ancient germ layer? Cell 2001; 105: 169-72.
    PubMedGoogle Scholar
  • 8 Sachinidis A, Fleischmann BK, Kolossov E. et al. Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res 2003; 58: 278-91.
    CrossrefPubMedGoogle Scholar
  • 9 Trounson A. Human embryonic stem cells: mother of all cell and tissue types. Reprod Biomed Online. 2002; 4 (Suppl. 01) 58-63.
    PubMedGoogle Scholar
  • 10 Desbaillets I, Ziegler U, Groscurth P. et al. Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol 2000; 85: 645-51.
    CrossrefPubMedGoogle Scholar
  • 11 Oettgen P. Transcriptional regulation of vascular development. Circ Res 2001; 89: 380-8.
    CrossrefPubMedGoogle Scholar
  • 12 Doss MX, Koehler CI, Gissel C. et al. Embryonic stem cells: a promising tool for cell replacement therapy. J Cell Mol Med 2004; 8: 465-73.
    CrossrefPubMedGoogle Scholar
  • 13 Bain G, Ray WJ, Yao M. et al. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem Biophys Res Commun 1996; 223: 691-4.
    CrossrefPubMedGoogle Scholar
  • 14 Karbanova J, Mokry J. Histological and histochemical analysis of embryoid bodies. Acta Histochem 2002; 104: 361-5.
    CrossrefPubMedGoogle Scholar
  • 15 Kawase E, Suemori H, Takahashi N. et al. Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int J Dev Biol 1994; 38: 385-90.
    PubMedGoogle Scholar
  • 16 Baharvand H, Matthaei KI. Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains. In Vitro Cell Dev Biol Anim 2004; 40: 76-81.
    CrossrefPubMedGoogle Scholar
  • 17 Schoonjans L, Kreemers V, Danloy S. et al. Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains. Stem Cells 2003; 21: 90-7.
    CrossrefPubMedGoogle Scholar
  • 18 Nichols J, Evans EP, Smith AG. Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 1990; 110: 1341-8.
    CrossrefPubMedGoogle Scholar
  • 19 Mehlen P, Mehlen A, Godet J. et al. hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells. J Biol Chem 1997; 272: 31657-65.
    CrossrefPubMedGoogle Scholar
  • 20 Wakayama T, Rodriguez I, Perry A C. et al. Mice cloned from embryonic stem cells. Proc Natl Acad Sci USA 1999; 96: 14984-9.
    CrossrefPubMedGoogle Scholar
  • 21 Toumadje A, Kusumoto K, Parton A. et al. Pluripotent differentiation in vitro of murine ES-D3 embryonic stem cells. In Vitro Cell Dev Biol Anim 2003; 39: 449-53.
    CrossrefPubMedGoogle Scholar
  • 22 Prelle K, Wobus AM, Krebs O. et al. Overexpression of insulin-like growth factor-II in mouse embryonic stem cells promotes myogenic differentiation. Biochem Biophys Res Commun 2000; 277: 631-8.
    CrossrefPubMedGoogle Scholar
  • 23 Wobus AM, Guan K, Yang H T. et al. Embryonic stem cells as a model to study cardiac, skeletal muscle, and vascular smooth muscle cell differentiation. Methods Mol Biol 2002; 185: 127-56.
    PubMedGoogle Scholar
  • 24 Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001; 19: 271-8.
    CrossrefPubMedGoogle Scholar
  • 25 Grabel L, Becker S, Lock L. et al. Using EC and ES cell culture to study early development: recent observations on Indian hedgehog and Bmps. Int J Dev Biol 1998; 42: 917-25.
    PubMedGoogle Scholar
  • 26 Izumi M, Fujio Y, Kunisada K. et al. Bone morphogenetic protein-2 inhibits serum deprivation-induced apoptosis of neonatal cardiac myocytes through activation of the Smad1 pathway. J Biol Chem 2001; 276: 31133-41.
    CrossrefPubMedGoogle Scholar
  • 27 Conlon FL, Smith JC. Interference with brachyury function inhibits convergent extension, causes apoptosis, and reveals separate requirements in the FGF and activin signalling pathways. Dev Biol 1999; 213: 85-100.
    CrossrefPubMedGoogle Scholar
  • 28 Showell C, Binder O, Conlon FL. T-box genes in early embryogenesis. Dev Dyn 2004; 229: 201-18.
    CrossrefPubMedGoogle Scholar
  • 29 Itskovitz-Eldor J, Schuldiner M, Karsenti D. et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000; 6: 88-95.
    CrossrefPubMedGoogle Scholar
  • 30 Becker S, Casanova J, Grabel L. Localization of endoderm-specific mRNAs in differentiating F9 embryoid bodies. Mech Dev 1992; 37: 3-12.
    CrossrefPubMedGoogle Scholar
  • 31 Hipfner DR, Cohen SM. Connecting proliferation and apoptosis in development and disease. Nat Rev Mol Cell Biol 2004; 5: 805-15.
    CrossrefPubMedGoogle Scholar
  • 32 Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995; 80: 179-85.
    CrossrefPubMedGoogle Scholar
  • 33 Gissel C, Nierhoff D, Fleischmann B K. et al. Culture of embryoid bodies. Practical Methods in Cardiovascular Research. Springer Verlag; 2005: 577-91.
    Google Scholar
  • 34 Sachinidis A, Gissel C, Nierhoff D. et al. Identification of plateled-derived growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell Physiol Biochem 2003; 13: 423-9.
    CrossrefPubMedGoogle Scholar
  • 35 Sachinidis A, Gouni-Berthold I, Seul C. et al. Early intracellular signalling pathway of ethanol in vascular smooth muscle cells. Br J Pharmacol 1999; 128: 1761-71.
    CrossrefPubMedGoogle Scholar
  • 36 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.
    CrossrefPubMedGoogle Scholar
  • 37 Sachinidis A, Skach RA, Seul C. et al. Inhibition of the PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins. FASEB J 2002; 16: 893-5.
    CrossrefPubMedGoogle Scholar
  • 38 Saeed AI, Sharov V, White J. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 2003; 34: 374-8.
    CrossrefPubMedGoogle Scholar
  • 39 Sturn A, Quackenbush J, Trajanoski Z. Genesis: cluster analysis of microarray data. Bioinformatics 2002; 18: 207-8.
    CrossrefPubMedGoogle Scholar
  • 40 Eisen MB, Spellman PT, Brown P O. et al. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 1998; 95: 14863-8.
    CrossrefPubMedGoogle Scholar
  • 41 Zeeberg BR, Feng W, Wang G. et al. GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 2003; 4: R28.
    CrossrefPubMedGoogle Scholar
  • 42 Breitling R, Amtmann A, Herzyk P. Graph-based iterative Group Analysis enhances microarray interpretation. BMC Bioinformatics 2004; 5: 100.
    CrossrefPubMedGoogle Scholar
  • 43 Diehn M, Sherlock G, Binkley G. et al. SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data. Nucleic Acids Res 2003; 31: 219-23.
    CrossrefPubMedGoogle Scholar
  • 44 el Meziane A, Callen JC, Mounolou JC. Mitochondrial gene expression during Xenopus laevis development: a molecular study. EMBO J 1989; 8: 1649-55.
    CrossrefPubMedGoogle Scholar