Abstract
Osteoclasts are cells specialized to resorb bone (1,2). They originate from hematopoietic progenitors (3,4), which are widely distributed throughout the body. Osteoclast progenitors differentiate into cells that are positive for calcitonin receptor and tartrate-resistant acid phosphatase (TRAP), which are specific markers for this lineage, and then they fuse with each other and form fully functional multinucleated large cells (1,2) (Fig. 1). TRAP-positive osteoclasts are found tightly attached to bone matrix (1,2). Two cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB (RANK) ligand (RANKL; also known as OPGL, ODF, and TRANCE), are known to be responsible for this differentiation (5–7). In this chapter, we describe the procedure for the induction of differentiation into the osteoclast lineage from murine embryonic stem (ES) cells (8). We utilize co-culture systems with stromal cell lines, that is, bone marrow-derived ST2 cells (9) or newborn calvaria-derived M-CSF-deficient OP9 cells (10). M-CSF is produced constitutively by ST2, and the expression of RANKL is induced on ST2 by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex) (6).
Scheme describing the development of osteoclast lineage. The essential molecules expressed in this lineage are indicated at the stage where maturation is arrested by the loss of function. The cells positive for TRAP are shaded.
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References
Mundy, G. R. and Roodman, G. D. (1987) Osteoclast ontogeny and function. Bone Miner. Res. 5, 209–279.
Suda, T., Udagawa, N., and Takahashi, N. (1996) Cells of bone: osteoclast generation, in Principles of bone biology (Bilezikian, J. P., Raisz, L. G., and Roden, G. A., eds.), Academic Press, New York, NY, pp. 87–102.
Ash, P., Loutit, J. F., and Townsend, K. M. (1980) Osteoclasts derived from haematopoietic stem cells. Nature 283, 669–670.
Hayashi, S.-I., Yamane, T., Miyamoto, A., Hemmi, H., Tagaya, H., Tanio, Y, et al. (1998) Commitment and differentiation of stem cells to the osteoclast lineage. Biochem. Cell Biol. 76, 911–922.
Yoshida, H., Hayashi, S.-I., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., et al. (1990) The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442–444.
Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S.-I., et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95, 3597–3602.
Lacey, D. L., Timms, E., Tan, H.-L., Kelley, M. J., Dunstan, C. R., Burgess, T., et al. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176.
Yamane, T, Kunisada, T., Yamazaki, H., Era, T, Nakano, T, and Hayashi, S. I. (1997) Development of osteoclasts from embryonic stem cells through a pathway that is c-fms but not c-kit dependent. Blood 90, 3516–3523.
Ogawa, M., Nishikawa, S., Ikuta, K., Yamamura, F., Naito, M., Takahashi, K., and Nishikawa, S.-I. (1988) B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac. EMBO J. 7, 1337–1343.
Kodama, H., Nose, M., Niida, S., Nishikawa, S., and Nishikawa, S.-I. (1994) Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells. Exp. Hematol. 22, 979–984.
Nakano, T., Kodama, H., and Honjo, T. (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101.
Yamane, T, Kunisada, T, Yamazaki, H., Nakano, T, Orkin, S. H., and Hayashi, S.-I. (2000) Sequential requirements for SCL/tal-1, GATA-2, M-CSF, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development. Exp. Hematol. 28, 833–840.
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.
Li, E., Bestor, T. H., and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926.
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.
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© 2002 Humana Press Inc., Totowa, NJ
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Yamane, T., Kunisada, T., Hayashi, SI. (2002). Embryonic Stem Cells as a Model for Studying Osteoclast Lineage Development. In: Turksen, K. (eds) Embryonic Stem Cells. Methods in Molecular Biology™, vol 185. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-241-4:97
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DOI: https://doi.org/10.1385/1-59259-241-4:97
Publisher Name: Springer, Totowa, NJ
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