Abstract
Knowledge of the molecular and cellular events characterizing osteoblast development is growing as new markers, including important classes of regulatory molecules such as transcription factors (e.g., Cbfa-1 [1]), are elucidated. Nevertheless, a paucity of definitive and specific markers, especially for the more primitive progenitors and the hemopoietic lineages (2). One useful model, however, has been culture of mixed populations of freshly isolated cells derived from a variety of bones (e.g., 21-d fetal rat calvaria [RC]) or bone marrow stroma under conditions that favor osteoblast development (2). For example, when such heterogeneous primary cultures are grown long-term (approx 3 wk) in medium supplemented with ascorbic acid and α-glycerophosphate, a low frequency (about 0.00001–1% of unfractionated freshly isolated populations) of osteoprogenitor cells present divide and differentiate to form 3-dimensional mineralized bone nodules (3,4). These infrequent cells comprise the colony forming units or colony forming cells-osteoprogenitor (CFU-Os or CFC-Os, respectively) in populations from the whole tissue and appear analogous to the nonstem cell CFU/CFCs in lineages such as the hemopoietic. Notably, the frequency of such cells can be determined by limiting dilution, and they appear to have limited capacity for self-renewal (3,4). On the other hand, morphological, immunohistochemical, and molecular analyses have confirmed that differentiation of CFU-Os and formation of bone nodules reproducibly recapitulates a proliferation-differentiation sequence from an early precursor cell to a mature osteoblast (2).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ducy, P. (2000) CBFA1: a molecular switch in osteoblast biology. Dev. Dyn. 219, 461–471.
Aubin, J. E. (1998) Bone stem cells. 25th Anniversary Issue: new directions and dimensions in cellular biochemistry. Invited chapter. J. Cell Biochem. Suppl. 30/31, 73–82.
Bellows, C. G. and Aubin, J. E. (1989) Determination of numbers of osteoprogenitors present in isolated fetal rat calvaria cells in vitro. Dev. Biol. 133, 8–13.
Aubin, J. E. (1999) Osteoprogenitor cell frequency in rat bone marrow stromal cell populations: role for heterotypic cell-cell interactions in osteoblast differentiation. J. Cell Biochem. 72, 396–410.
Brady, G., Barbara, M., and Iscove, N. N. (1990) Representative in vitro cDNA amplification from individual hemopoietic cells and colonies. Methods Mol. Cell Biol. 2, 17–25.
Brady, G. and Iscove, N. N. (1993) Construction of cDNA libraries from single cells. Methods Enzymol. 225, 611–623.
Liu, F., Malaval, L., Gupta, A., and Aubin, J. E. (1994) Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level. Dev. Biol. 166, 220–234.
Liu, F., Malaval, L., and Aubin, J. E. (1997) The mature osteoblast phenotype is characterized by extensive palsticity. Exp. Cell Res. 232, 97–105.
Brady, G., Billia, F., Knox, J., Hoang, T., Kirsch, I. R., Voura, E., et al. (1995) Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr. Biol. 5, 909–922.
Cheng, T., Shen, H., Giokas, D., Gere, J., Tenen, D. G., and Scadden, D. T. (1996) Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells. Proc. Nat. Acad. Sci. USA 93, 13,158–13,163.
Cumano, A., Paige, C. J., Iscove, N. N., and Brady, G. (1992) Bipotential precursors of B cells and macrophages in murine fetal liver. Nature 356, 612–615.
Billia, F., Barbara, M., McEwen, J., Trevisan, M., and Iscove, N. N. (2001) Resolution of pluripotential intermediates in murine hematopoietic differentiation by global complementary DNA amplification from single cells: Confirmation of assignments by expression profiling of cytokine receptor transcripts. Blood 97, 2257–2268.
Trumper, L. H., Brady, G., Bagg, A., Gray, D., Loke, S. L., Griesser, H., et al. (1993) Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations. Blood 81, 3097–3115.
Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S., Kabrun, N., and Keller, G. (1997) A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature 386, 488–493.
Robertson, S. M., Kennedy, M., Shannon, J. M., and Keller, G. (2000) A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1. Development 127, 2447–2459
Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R., et al. (1996) Laser capture microdissection. Science 274, 998–1001.
Raetz, C. R., Wermuth, M. M., McIntyre, T. M., Esko, J. D., and Wing, D. C. (1982) Somatic cell cloning in polyester stacks. Proc. Natl. Acad. Sci. USA 79, 3223–3227.
Esko, J. D. (1989) Replica plating of animal cells. Methods Cell Biol. 32, 387–422.
Jensen, R. A., Page, D. L., and Holt, J. T. (1997) RAP-PCR using RNA from tissue microdissection. Methods Mol. Biol. 85, 277–283.
Candeliere, G. A., Rao, Y., Floh, A., Sandler, S. D., and Aubin, J. E. (1999) cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes. Nucleic Acids Res. 27, 1079–1083.
McClelland, M., Arensdorf, H., Cheng, R. and Welsh, J. (1994) Arbitrarily primed PCR fingerprints resolved on SSCP gels. Nucleic Acids Res. 22, 1770–1771.
Liang, P. and Pardee, A. B. (1998) Differential display. A general protocol. Mol. Biotechnol. 10, 261–267.
Brail, L. H., Jang, A., Billia, F., Iscove, N. N., Klamut, H. J., and Hill, R. P. (1999) Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR). Mutat. Res. 406, 45–54.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Aubin, J.E., Liu, F., Candeliere, G.A. (2002). Single-Cell PCR Methods for Studying Stem Cells and Progenitors. In: Turksen, K. (eds) Embryonic Stem Cells. Methods in Molecular Biology™, vol 185. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-241-4:403
Download citation
DOI: https://doi.org/10.1385/1-59259-241-4:403
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-881-3
Online ISBN: 978-1-59259-241-8
eBook Packages: Springer Protocols