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Differentiation and Dynamic Analysis of Primitive Vessels from Embryonic Stem Cells

  • Gefei Zeng
  • Victoria L. Bautch
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 482)

Abstract

Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the ability to differentiate into various cell types in vitro. When placed in basal medium with added serum, mouse ES cells undergo a programed differentiation favoring formation of cell types that are found in the embryonic yolk sac, including vascular endothelial cells. These in vitro differentiated endothelial cells form primitive blood vessels, analogous to the first vessels that form in the embryo and the yolk sac. This differentiation model is ideal for both genetic and pharmacological manipulation of early vascular development. We have made mouse ES cell lines that express endothelial-specific GFP or H2B-GFP and used these lines to study the processes of mammalian vessel development by real-time imaging. Here we describe protocols for making transgenic ES cells and imaging the processes of blood vessel development. We also provide methods for ES cell maintenance and differentiation, and methods for analysis of vascular marker expression.

Key words

Angiogenesis in vitro differentiation murine embryonic stem cell Histone-2B time-lapse imaging cell division orientation immunofluorescence GFP 

References

  1. 1.
    Poole, T. J., and Coffin, J. D. (1989) Vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern. J Exp Zool 251, 224–231.CrossRefPubMedGoogle Scholar
  2. 2.
    Risau, W. (1997) Mechanism of angiogenesis. Nature 386, 671–674.CrossRefPubMedGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    Risau, W., Sariola, H., Zerwes, H.G., Sasse, J., Ekblom, P., Kemler, R., and Doetschman, T. (1988) Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development 102, 471–478.PubMedGoogle Scholar
  5. 5.
    Wang, R., Clark, R., Bautch, V.L. (1992) Embryonic stem cell-derived cystic embryoid bodies from vascular channels: an in vitro model of blood vessel development. Development 114, 303–316.PubMedGoogle Scholar
  6. 6.
    Keller, G.M. (1995) In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 7, 862–869.CrossRefPubMedGoogle Scholar
  7. 7.
    Vittet, D., Prandini, M.H., Berthier, R., Schweitzer, A., Martin-Sisteron, H., Uzan, G., and Dejana, E. (1996) Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood 88, 3424–3431.PubMedGoogle Scholar
  8. 8.
    Bautch, V.L. (2002) Embryonic stem cell differentiation and the vascular lineage. Methods Mol Biol 185, 117–125.PubMedGoogle Scholar
  9. 9.
    Bautch, V.L., Stanford, W.L., Rapoport, R., Russell, S., Byrum, R. S., and Futch, T. A. (1996) Blood island formation in attached cultures of murine embryonic stem cells. Dev Dyn 205, 1–12.CrossRefPubMedGoogle Scholar
  10. 10.
    Kearney, J.B., Bautch, V.L. (2003) In vitro differentiation of mouse ES cells: hematopoietic and vascular development. Meth Enzymol 365, 83–98.CrossRefPubMedGoogle Scholar
  11. 11.
    Jones, E.A., Baron, M.H., Fraser, S.E., and Dickinson, M. E. (2005) Dynamic in vivo imaging of mammalian hematovascular development using whole embryo culture. Meth Mol Med 105, 381–394.Google Scholar
  12. 12.
    Kearney, J.B., Kappas, N.C., Ellerstrom, C., DiPaola, F. W., and Bautch, V. L. (2004) The VEGF receptor flt-1 (VEGFR-1) is a positive modulator of vasular sprout formation and branching morphogenesis. Blood 103, 4527–4535.CrossRefPubMedGoogle Scholar
  13. 13.
    Zeng G., Taylor S.M., McColm J.R., Kappas N.C., Kearney J. B., Williams L. H., Hartnett M. E., Bautch V. L. (2007) Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation. Blood 109, 1345–1352.Google Scholar
  14. 14.
    Zwaginga, J.J., Doevendans, P. (2003) Stem cell-derived angiogenic/vasculogenic cells: possible therapies for tissue repair and tissue engineering. Clin Exp Pharmacol Physiol 30, 900–908.CrossRefPubMedGoogle Scholar
  15. 15.
    Alessandri, G., Emanueli, C., Madeddu, P. (2004) Genetically engineered stem cell therapy for tissue regeneration. Ann N Y Acad Sci 1015, 271–284.CrossRefPubMedGoogle Scholar
  16. 16.
    Madeddu, P. (2005) Therapeutic angiogenesis and vasculogenesis for tissue regeneration. Exp Physiol 90, 315–326.CrossRefPubMedGoogle Scholar
  17. 17.
    Feraud, O., Prandini, M.H., Vittet, D. (2003) Vasculogenesis and angiogenesis from in vitro differentiation of mouse embryonic stem cells. Meth Enzymol 365, 214–228.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Gefei Zeng
    • 1
  • Victoria L. Bautch
    • 1
  1. 1.Department of Biology, Carolina Cardiovascular Biology CenterUniversity of North Carolina at Chapel HillChapel HillUSA

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