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Internal Standards in Differentiating Embryonic Stem Cells In Vitro

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Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 329))

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Abstract

Embryonic stem (ES) cell lines are important for use in developmental biology studies, and because these cells are totipotent, they may provide a much-needed source of differentiated cells for certain therapeutic applications. The phenotype of the ES cell in culture is often assessed by (semi)quantitative RNA analyses. In such cases, it is critical to use appropriate internal standards to correct for experimentally induced sources of error. This is particularly true for ES cell differentiation because it is heterogeneous in nature. We describe protocols for determining the suitability of housekeeping genes to act as internal controls in differentiating ES cell cultures. Such assessment is needed for every experimental condition under investigation. The protocol focuses on polymerase chain reaction; however, the principle and experimental design are applicable to any (semi)quantitative RNA assay.

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References

  1. Smith A. G. (2001) Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462.

    Article  CAS  PubMed  Google Scholar 

  2. Bradley A., Evans M., Kaufman M. H., and Robertson E. (1984) Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256.

    Article  CAS  PubMed  Google Scholar 

  3. Boheler K. R. and Fiszman M. Y. (1999) Can exogenous stem cells be used in transplantation? Cells Tissues Organs 165, 237–245.

    Article  CAS  PubMed  Google Scholar 

  4. D'Amour K. and Gage F. H. (2001) New tools for human developmental biology. Nat. Biotechnol. 18, 381–382.

    Article  Google Scholar 

  5. Odorico J. S., Kaufman D. S., and Thomson J. A. (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19, 193–204.

    Article  CAS  PubMed  Google Scholar 

  6. Abe K., Niwa H., Iwase K., et al. (1996) Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp. Cell Res. 229, 27–34.

    Article  CAS  PubMed  Google Scholar 

  7. Leahy A., Xiong J. W., Kuhnert F., and Stuhlmann H. (1999) Use of developmental marker genes to define temporal and spatial patterns of differentiation during mbryoid body formation. J. Exp. Zool. 284, 67–81.

    Article  CAS  PubMed  Google Scholar 

  8. Itskovitz-Eldor J., Schuldiner M., Karsenti D., et al. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6 88–95.

    CAS  PubMed  Google Scholar 

  9. Buttery L. D., Bourne S., Xynos J. D., et al. (2001) M. Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 7, 89–99.

    Article  CAS  PubMed  Google Scholar 

  10. Gualandris A., Annes J. P., Arese M., Noguera I., Jurukovski V., and Rifkin D. B. (2000) The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium. Mol. Biol. Cell. 11, 4295–4308.

    CAS  PubMed  Google Scholar 

  11. Kramer J., Hegert C., Guan K., Wobus A. M., Muller P. K., and Rohwedel J. (2000) Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mech. Dev. 92, 193–205.

    Article  CAS  PubMed  Google Scholar 

  12. Schuldiner M., Yanuka O., Itskovitz-Eldor J., Melton D. A., and Benvenisty N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA. 97, 11,307–11,312.

    Article  CAS  PubMed  Google Scholar 

  13. Wobus A. M., Kaomei G., Shan J., et al. (1997) Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J. Mol. Cell Cardiol. 29, 1525–1539.

    Article  CAS  PubMed  Google Scholar 

  14. Thellin O., Zorzi W., Lakaye B., et al. (1999) Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75, 291–295.

    Article  CAS  PubMed  Google Scholar 

  15. Spanakis E. (1993) Problems related to the interpretation of autoradiographic data on gene expression using common constitutive transcripts as controls. Nucleic Acids Res. 21, 3809–3819.

    Article  CAS  PubMed  Google Scholar 

  16. Vassilieva S., Guan K., Pich U., and Wobus A. M. (2000) Establishment of SSEA-1-and Oct-4-expressing rat embryonic stem-like cell lines and effects of cytokines of the IL-6 family on clonal growth. Exp. Cell Res. 258, 361–373.

    Article  CAS  PubMed  Google Scholar 

  17. Phillips B. W., Belmonte N., Vernochet C., Ailhaud G., and Dani C. (2001) Compactinenhances osteogenesis in murine embryonic stem cells. Biochem. Biophys. Res. Commun. 284, 478–484.

    Article  CAS  PubMed  Google Scholar 

  18. Weinhold B., Schratt G., Arsenian S., et al. (2000) Srf(-/-)ES cells display non-cellautonomous impairment in mesodermal differentiation. EMBO J. 19, 5835–5844.

    Article  CAS  PubMed  Google Scholar 

  19. Wiles M. V. and Johansson B. M. (1999) Embryonic stem cell development in a chemically defined medium. Exp. Cell Res. 247, 241–248.

    Article  CAS  PubMed  Google Scholar 

  20. Murphy C. L. and Polak J. M. (2002) Differentiating embryonic stem cells: GAPDH, but neither HPRT nor β-tubulin is suitable as an internal standard for measuring RNA levels. Tissue Eng. 8, 551–559.

    Article  CAS  PubMed  Google Scholar 

  21. Robertson E., Bradley A., Kuehn M., and Evans M. (1986) Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445–448.

    Article  CAS  PubMed  Google Scholar 

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© 2006 Humana Press Inc.

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Murphy, C.L. (2006). Internal Standards in Differentiating Embryonic Stem Cells In Vitro. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 329. Humana Press. https://doi.org/10.1385/1-59745-037-5:101

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  • DOI: https://doi.org/10.1385/1-59745-037-5:101

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-498-2

  • Online ISBN: 978-1-59745-037-9

  • eBook Packages: Springer Protocols

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