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.
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References
Evans M. J. and Kaufman M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156.
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.
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.
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.
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.
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.
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.
Kramer, J. Unpublished results.
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.
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.
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.
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.
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.
Rohwedel J., Guan K., and Wobus A. M. (1999) Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs 165, 190–202.
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.
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.
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.
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.
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.
Thomas K. R. and Capecchi M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.
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.
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.
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.
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.
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.
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.
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.
Linsenmayer T. F. and Hendrix M. J. (1980) Monoclonal antibodies to connective tissue macromolecules: type II collagen. Biochem. Biophys. Res. Commun. 92, 440–446.
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.
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.
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.
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.
Wong H., Anderson W. D., Cheng T., and Riabowol K. T. (1994) Monitoring mRNA expression by polymerase chain reaction: the "e;primer-dropping"e; method. Anal. Biochem. 223, 251–258.
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.
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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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