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Embryonic Stem Cell Protocols pp 171–190Cite as

Derivation and Characterization of Chondrocytes From Embryonic Stem Cells In Vitro

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Part of the Methods in Molecular Biology™ book series (MIMB,volume 330)

Abstract

The model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates (embryoid bodies, EBs) can be used to analyze cell differentiation from a pluripotent stem cell via progenitor cells up to terminally differentiated cell types. ES cells are known to be pluripotent; they have the capacity to differentiate into any cell lineage of the three germ layers. Using various ES cell lines, we characterized chondrogenic and osteogenic differentiation in EBs by histochemical staining, immunostaining, mRNA-in situ hybridization, and reverse transcriptase polymerase chain reaction analysis. Here, we describe in detail our established protocols to analyze chondrogenic differentiation of ES cells. We summarize different ways to modulate ES cell-derived chondrogenic differentiation.

Key Words

  • Chondrogenic differentiation
  • embryonic stem cells
  • osteogenic differentiation

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References

  1. Evans M. J. and Kaufman M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.

    CAS  CrossRef  PubMed  Google Scholar 

  2. 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. USA 78, 7634–7638.

    CAS  CrossRef  PubMed  Google Scholar 

  3. Rathjen P. D., Lake J., Whyatt L. M., Bettess M. D., and Rathjen J. (1998) Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. Reprod. Fertil. Dev. 10, 31–47.

    CAS  CrossRef  PubMed  Google Scholar 

  4. Kramer J., Hegert C., Guan K., Wobus A. M., Müller 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.

    CAS  CrossRef  PubMed  Google Scholar 

  5. Hegert C., Kramer J., Hargus G., et al. (2002) Differentiation plasticity of chondrocytes derived from mouse embryonic stem cells. J. Cell Sci. 115, 4617–4628.

    CAS  CrossRef  PubMed  Google Scholar 

  6. Guan K., Rohwedel J., and Wobus A. M. (1999) Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro. Cytotechnology 30, 211–226.

    CAS  CrossRef  PubMed  Google Scholar 

  7. Rohwedel J., Guan K., Hegert C., and Wobus A. M. (2001) Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol. In Vitro 15, 741–753.

    CAS  CrossRef  PubMed  Google Scholar 

  8. Kramer, J. Unpublished results.

    Google Scholar 

  9. Kramer J., Hegert C., Hargus G., and Rohwedel J. (2007) Mouse ES cell lines show a variable degree of chondrogenic differentiation in vitro. Cell Biol. Int. 29, 139–146.

    CrossRef  Google Scholar 

  10. Rohwedel J., Maltsev V., Bober E., Arnold H. H., Hescheler J., and Wobus A. M. (1994) Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164, 87–101.

    CAS  CrossRef  PubMed  Google Scholar 

  11. Rohwedel J., Kleppisch T., Pich U., et al. (1998) Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro. Exp. Cell Res. 239, 214–225.

    CAS  CrossRef  PubMed  Google Scholar 

  12. Wobus A. M., Rohwedel J., Maltsev V., and Hescheler J. (1994) In vitro differentiation of embryonic stem cells into cardiomyocytes or skeletal muscle cells is specifically modulated by retinoic acid. Roux’s Arch. Dev. Biol. 204, 36–45.

    CAS  CrossRef  Google Scholar 

  13. Dani C., Smith A. G., Dessolin S., et al. (1997) Differentiation of embryonic stem cells into adipocytes in vitro. J. Cell Sci. 110, 1279–1285.

    CAS  PubMed  Google Scholar 

  14. Rohwedel J., Guan K., and Wobus A. M. (1999) Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs 165, 190–202.

    CAS  CrossRef  PubMed  Google Scholar 

  15. Yamada G., Kioussi C., Schubert F. R., et al. (1994) Regulated expression of Brachyury(T), Nkx1.1 and Pax genes in embryoid bodies. Biochem. Biophys. Res. Commun. 199, 552–563.

    CAS  CrossRef  PubMed  Google Scholar 

  16. Rohwedel J., Guan K., Zuschratter W., et al. (1998) Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro. Dev. Biol. 201, 167–184.

    CAS  CrossRef  PubMed  Google Scholar 

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

    CAS  CrossRef  PubMed  Google Scholar 

  18. Amit M., Shariki C., Margulets V., and Itskovitz-Eldor J. (2004) Feeder layer-and serumfree culture of human embryonic stem cells. Biol. Reprod. 70, 837–845.

    CAS  CrossRef  PubMed  Google Scholar 

  19. Wakitani S., Takaoka K., Hattori T., et al. (2003) Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology (Oxford) 42, 162–165.

    CAS  CrossRef  Google Scholar 

  20. Thomas K. R. and Capecchi M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.

    CAS  CrossRef  PubMed  Google Scholar 

  21. Bi W., Huang W., Whitworth D. J., et al. (2001) Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc. Natl. Acad. Sci. USA 98, 6698–6703.

    CAS  CrossRef  PubMed  Google Scholar 

  22. Bi W., Deng J. M., Zhang Z., Behringer R. R., and de Crombrugghe B. (1999) Sox9 is required for cartilage formation. Nat. Genet. 22, 85–89.

    CAS  CrossRef  PubMed  Google Scholar 

  23. Akiyama H., Chaboissier M. C., Martin J. F., Schedl A., and de Crombrugghe B. (2002) The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 16, 2813–2828.

    CAS  CrossRef  PubMed  Google Scholar 

  24. Wobus A. M., Grosse R., and Schöneich J. (1988) Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro. Biomed. Biochim. Acta 47, 965–973.

    CAS  PubMed  Google Scholar 

  25. 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.

    CAS  PubMed  Google Scholar 

  26. Hooper M., Hardy K., Handyside A., Hunter S., and Monk M. (1987) HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature 326, 292–295.

    CAS  CrossRef  PubMed  Google Scholar 

  27. Nagy A., Rossant J., Nagy R., Abramow-Newerly W., and Roder J. C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428.

    CAS  CrossRef  PubMed  Google Scholar 

  28. Linsenmayer T. F. and Hendrix M. J. (1980) Monoclonal antibodies to connective tissue macromolecules: type II collagen. Biochem. Biophys. Res. Commun. 92, 440–446.

    CAS  CrossRef  PubMed  Google Scholar 

  29. Dorheim M. A., Sullivan M., Dandapani V., et al. (1993) Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells. J. Cell Physiol. 154, 317–328.

    CAS  CrossRef  PubMed  Google Scholar 

  30. Schmid T. M. and Linsenmayer T. F. (1985) Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J. Cell Biol. 100, 598–605.

    CAS  CrossRef  PubMed  Google Scholar 

  31. Hedbom E., Antonsson P., Hjerpe A., et al. (1992) Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage. J. Biol. Chem. 267, 6132–6136.

    CAS  PubMed  Google Scholar 

  32. Wobus A. M., Holzhausen H., Jäkel P., and Schöneich J. (1984) Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res. 152, 212–219.

    CAS  CrossRef  PubMed  Google Scholar 

  33. Wong H., Anderson W. D., Cheng T., and Riabowol K. T. (1994) Monitoring mRNA expression by polymerase chain reaction: the &quote;primer-dropping&quote; method. Anal. Biochem. 223, 251–258.

    CAS  CrossRef  PubMed  Google Scholar 

  34. 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.

    CAS  CrossRef  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Medical Molecular Biology, University of Lübeck, Lübeck, Germany

    Jan Kramer, Gunnar Hargus & Jürgen Rohwedel

Authors
  1. Jan Kramer
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  2. Gunnar Hargus
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  3. Jürgen Rohwedel
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Editor information

Editors and Affiliations

  1. Ottawa Health Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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© 2006 Humana Press Inc.

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Kramer, J., Hargus, G., Rohwedel, J. (2006). Derivation and Characterization of Chondrocytes From Embryonic Stem Cells In Vitro. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology™, vol 330. Humana Press. https://doi.org/10.1385/1-59745-036-7:171

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  • DOI: https://doi.org/10.1385/1-59745-036-7:171

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-784-6

  • Online ISBN: 978-1-59745-036-2

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