Abstract
Differentiation procedures leading to osteogenic and chondrogenic differentiation of embryonic stem cells (ESCs) have been established and well upgraded over the past decade. Novel cell-culture conditions, signaling inducers, and chemical modifications of cellular environment have been found and optimized for use as steering or supporting modules in ESC differentiation.
While most of the novel studies of osteoblasts or chondrocytes differentiated from ESCs deal with their regenerative potential, the “childhood diseases” of basic differentiation have not yet been quite solved. Purification procedures are still facing a lack of exclusive markers for osteogenic progenitors and a collateral development of other cell types at the end points of differentiation that possibly lead to teratomas.
This chapter discusses the role of novel markers and inducers in osteogenic and chondrogenic differentiation, their effect on signaling pathways, particularly on that of Wnt/beta-catenin, and the time-specific manner of their action. We present an improved osteogenic differentiation protocol based on the hanging drop method and a time-optimized use of 1α,25-(OH)2 vitamin D3, all-trans retinoic acid, and bone morphogenetic protein 2 (BMP-2) with an end point efficiency increased up to 90% and a protocol for chondrogenic differentiation, which employs BMP-2 and transforming growth factor β1 as chondrogenic inducers, with 60% chondrogenic end point efficiency.
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Duplomb, L., Dagouassat, M., Jourdon, P., and Heymann, D. (2007) Concise review: embryonic stem cells: a new tool to study osteoblast and osteoclast differentiation. Stem Cells. 25(3), 544–552.
Buttery, L.D.K., Bourne, S., Xynos, J.D., Wood, H., Hughes, F.J., Hughes, S.P.F., et al. (2001) Differentiation of osteoblasts and in vitro bone formation from murine embryonic stem cells. Tissue Eng. 7, 89–99.
zur Nieden, N. I., Kempka, G., and Ahr, H. J. (2003) In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation. 71, 18–27.
Phillips, B.W., Belmonte, N., Vernochet, C., Ailhaud, G., and Dani, C. (2001) Compactin enhances osteogenesis in murine embryonic stem cells. Biochem. Biophys. Res. Commun. 284(2), 478–484.
Sottile, V., Thomson, A., and McWhir, J. (2003) In vitro osteogenic differentiation of human ES cells. Cloning Stem Cells. 5, 149–155.
Tremoleda, J.L., Forsyth, N.R., Khan, N.S., Wojtacha, D., Christodoulou, I., Tye, B.J., et al. (2008) Bone tissue formation from human embryonic stem cells in vivo. Cloning Stem Cells. 10(1), 119–132.
Arpornmaeklong, P., Brown, S.E., Wang, Z., and Krebsbach, P.H. (2009) Phenotypic characterization, osteoblastic differentiation, and bone regeneration capacity of human embryonic stem cell-derived mesenchymal stem cells. Stem Cells Dev. 18(7), 955–968.
Duester, G. (2008) Retinoic acid synthesis and signaling during early organogenesis. Cell. 134(6), 921–931.
zur Nieden, N.I., Price, F.D., Davis, L.A., Everitt, R.E., and Rancourt, D.E. (2007) Gene profiling on mixed embryonic stem cell populations reveals a biphasic role for beta-catenin in osteogenic differentiation. Mol. Endocrinol. 21(3), 674–685.
Doss, M.X., Chen, S., Winkler, J., Hippler-Altenburg, R., Odenthal, M., Wickenhauser, C., et al. (2007) Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2+ lineage cells: an insight into mesodermal patterning. Genome Biol. 8(9), R184.
Davis, L.A. and zur Nieden, N.I. (2008) Mesodermal fate decisions of a stem cell: the Wnt switch. Cell. Mol. Life Sci. 65(17), 2658–2674.
Bourne, S., Polak, J.M., Hughes, S.P., and Buttery, L.D. (2004) Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting. Tissue Eng. 10(5–6), 796–806.
Okazaki, M., Takeshita, S., Kawai, S., Kikuno, R., Tsujimura, A., Kudo, A., et al. (1994) Molecular cloning and characterization of OB-cadherin, a new member of cadherin family expressed in osteoblasts. J. Biol. Chem. 269(16), 12092–12098.
Sui, Y., Clarke, T., and Khillan, J.S. (2003) Limb bud progenitor cells induce differentiation of pluripotent embryonic stem cells into chondrogenic lineage. Differentiation. 71, 578–585.
Vats, A., Bielby, R.C., Tolley, N., Dickinson, S.C., Boccaccini, A.R., Hollander, A.P., et al. (2006) Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng. 12(6), 1687–1697.
Worster, A.A., Brower-Toland, B.D., Fortier, L.A., Bent, S.J., Williams, J., and Nixon, A.J. (2001) Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a threedimensional matrix. J. Orthop. Res. 19, 738–749.
Estes, B.T., Wu, A.W., and Guilak, F. (2006) Potent induction of chondrocytic differentiation of human adipose-derived adult stem cells by bone morphogenetic protein 6. Arthritis Rheum. 54, 1222–1232.
Wang, W.-G., Loua, S.-Q., Ju, X.-D., Xia, K., and Xia, J.-H. (2003) In vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells in monolayer culture: activation by transfection with TGF-β2. Tissue Cell. 35, 69–77.
Schonherr, E. and Hausser, H.J. (2000) Extracellular matrix and cytokines: a functional unit. Dev. Immunol. 7, 89–101.
Majumdar, M.K., Wang, E., and Morris, E.A. (2001) BMP-2 and BMP-9 promotes chondrogenic differentiation of human multipotential mesenchymal cells and overcomes the inhibitory effect of IL-1. J. Cell. Physiol. 189, 275–284.
Hatakeyama, Y., Tuan, R.S., and Shum, L. (2004) Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis. J. Cell. Biochem. 91, 1204–1217.
zur Nieden, N.I., Kempka, G., Rancourt, D.E., and Ahr, H.J. (2005) Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: effect of cofactors on differentiating lineages. BMC Dev. Biol. 5, 1.
Ballock, R.T. and Reddi, A.H. (1994) Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J. Cell. Biol. 126(5), 1311–1318.
Johnstone, B., Hering, T.M., Caplan, A.I., Goldberg, V.M., and Yoo, J.U. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell. Res. 238, 265–272.
Mackay, A.M., Beck, S.C., Murphy, J.M., Barry, F.P., Chichester, C.O., and Pittenger, M.F. (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4(4), 415–428.
Denker, A.E., Nicoll, S.B., and Tuan, R.S. (1995) Formation of cartilage-like spheroids by micromass cultures of murine C3H10T1/2 cells upon treatment with transforming growth factor-beta 1. Differentiation. 59, 25–34.
Tanaka, H., Murphy, C.L., Murphy, C., Kimura, M., Kawai, S., and Polak, J.M. (2004) Chondrogenic differentiation of murine embryonic stem cells: effects of culture conditions and dexamethasone. J. Cell. Biochem. 93(3), 454–462.
Toh, W.S., Yang, Z., Liu, H., Heng, B.C., Lee, E.H., and Cao, T. (2007) Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells. 25(4), 950–960.
Hwang, N.S., Kim, M.S., Sampattavanich, S., Baek, J.H., Zhang, Z., and Elisseeff, J. (2006) Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells. Stem Cells. 24(2), 284–291.
Hwang, N.S., Varghese, S., Zhang, Z., and Elisseeff, J. (2006) Chondrogenic differentiation of human embryonic stem cell-derived cells in arginine-glycine-aspartate-modified hydrogels. Tissue Eng. 12(9), 2695–2706.
Acknowledgment
NzN acknowledges the support of the German Ministry for Science, Education and Research (grant 0312314). NzN was funded through a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research while developing the improved osteogenic protocol.
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Kuske, B., Savkovic, V., zur Nieden, N.I. (2011). Improved Media Compositions for the Differentiation of Embryonic Stem Cells into Osteoblasts and Chondrocytes. In: Nieden, N. (eds) Embryonic Stem Cell Therapy for Osteo-Degenerative Diseases. Methods in Molecular Biology, vol 690. Humana Press. https://doi.org/10.1007/978-1-60761-962-8_14
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DOI: https://doi.org/10.1007/978-1-60761-962-8_14
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