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In Vitro Differentiation of Embryonic Stem Cells as a Model of Early Hematopoietic Development

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Leukemia

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 538))

Summary

Embryonic Stem (ES) are pluripotent cells derived from the inner cell mass of blastocysts. ES cells differentiate in vitro into all kind of cells and the development of endothelial and hematopoietic cells from mouse ES cells has been especially established. As such, the in vitro differentiation of ES cells provides a powerful experimental model to study and determine the role of specific genes in the development of the hematopoietic system. Using this approach we have demonstrated the critical function of the transcription factor AML1/Runx1 at the onset of hematopoietic development (Blood 100:458–466, 2002; Blood 103:886–889, 2004). In this chapter, we will describe our protocols and methods for the culture of healthy ES cells, their effective differentiation toward hematopoiesis, and the quantitative analysis of their hematopoietic potential by replating or gene expression analyses.

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References

  1. Doetschman T.C., Eistetter H., Katz M., Schmidt W., 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.

    PubMed  CAS  Google Scholar 

  2. Wiles M., Keller G. (1991). Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111, 259–267.

    PubMed  CAS  Google Scholar 

  3. Schmitt R., Bruyns E., Snodgrass H. (1991). Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev 5, 728–740.

    Article  PubMed  CAS  Google Scholar 

  4. Burkert U., von Ruden T., Wagner E.F. (1991). Early fetal haematopoietic development from in vitro differentiated embryonic stem cells. New Biol 3, 698–708.

    PubMed  CAS  Google Scholar 

  5. Keller G., Kennedy M., Papayannopoulou T., Wiles M. (1993). Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol Cell Biol 13, 473–486.

    PubMed  CAS  Google Scholar 

  6. Nakano T., Kodama H., Honjo T. (1994). Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101.

    Article  PubMed  CAS  Google Scholar 

  7. Keller G. (1995). In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 7, 862–869.

    Article  PubMed  CAS  Google Scholar 

  8. Kennedy M., Firpo M., Choi K., Wall C., Robertson S., Kabrun N., Keller G. (1997). A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386, 488–493.

    Article  PubMed  CAS  Google Scholar 

  9. Kabrun N., Buhring H.J., Choi K., Ullrich A., Risau W., Keller G. (1997). Flk-1 expression defines a population of early embryonic hematopoietic precursors. Development 124, 2039–2048.

    PubMed  CAS  Google Scholar 

  10. Choi K., Kennedy M., Kazarov A., Papadimitriou J.C., Keller G. (1998). A common precursor for hematopoietic and endothelial cells. Development 125, 725–732.

    PubMed  CAS  Google Scholar 

  11. Nishikawa S.I., Nishikawa S., Hirashima M., Matsuyoshi N., Kodama H. (1998). Progressive lineage analysis by cell sorting and culture identifies FLK1+ VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125, 1747–1757.

    PubMed  CAS  Google Scholar 

  12. Fehling H.J., Lacaud G., Kubo A., Kennedy M., Robertson S., Keller G., Kouskoff V. (2003). Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227.

    Article  PubMed  CAS  Google Scholar 

  13. Lacaud G., Keller G., Kouskoff V. (2004). Tracking mesoderm formation and specification to the hemangioblast in vitro. Trends Cardiovasc Med 14, 314–317.

    Article  PubMed  Google Scholar 

  14. Russel E. (1979). Hereditary anemias of the mouse: a review for geneticists. Adv Genet 20, 357–459.

    Article  Google Scholar 

  15. Moore M., Metcalf D. (1970). Ontogeny of the hematopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Hematol 18, 279–296.

    Article  CAS  Google Scholar 

  16. Haar J.L., Ackerman G.A. (1971). Ultrastructural changes in mouse yolk sac associated with the initiation of vitelline circulation. Anat Rec 170, 437–456.

    Article  PubMed  CAS  Google Scholar 

  17. Sabin F.R. (1920). Studies on the origin of blood vessels and of red corpuscles as seen in the living blastoderm of the chick during the second day of incubation. Contrib Embryol 9, 213–262.

    Google Scholar 

  18. Murray P.D.F. (1932). The development in vitro of the blood of the early chick embryo. Proc R Soc London 11, 497–521.

    Article  Google Scholar 

  19. Lacaud G., Gore L., Kennedy M., Kouskoff V., Kingsley P., Hogan C., Carlsson L., Speck N., Palis J., Keller G. (2002). Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 100, 458–466.

    Article  PubMed  CAS  Google Scholar 

  20. Lacaud G., Kouskoff V., Trumble A., Schwantz S., Keller G. (2004). Haploinsufficiency of Runx1 results in the acceleration of mesodermal development and hemangioblast specification upon in vitro differentiation of ES cells. Blood 103, 886–889.

    Article  PubMed  CAS  Google Scholar 

  21. Lacaud G., Robertson S., Palis J., Kennedy M., Keller G. (2001). Regulation of hemangioblast development. Ann N Y Acad Sci 938, 96–107.

    Article  PubMed  CAS  Google Scholar 

  22. Pearson S., Sroczynska P., Lacaud G., Kouskoff V. (2008). The step-wise specification of embryonic stem cells to hematopoietic fate is driven by sequential exposure to BMP4, Activin A, FGF and VEGF. Development 135, 1525–1535.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by Cancer Research UK.

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Correspondence to Georges Lacaud .

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

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Sroczynska, P., Lancrin, C., Pearson, S., Kouskoff, V., Lacaud, G. (2009). In Vitro Differentiation of Embryonic Stem Cells as a Model of Early Hematopoietic Development. In: Eric So, C.W. (eds) Leukemia. Methods in Molecular Biology™, vol 538. Humana Press. https://doi.org/10.1007/978-1-59745-418-6_16

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  • DOI: https://doi.org/10.1007/978-1-59745-418-6_16

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-989-5

  • Online ISBN: 978-1-59745-418-6

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