Abstract
Gene expression profiling using microarrays is a powerful method for studying the biology of hematopoietic stem cells (HSCs). Here, we present methods for activating HSCs with the chemotherapeutic drug 5-Fluorouracil, isolating HSCs from whole bone marrow, and performing microarray analysis. We also discuss quality control criteria for identifying good arrays and bioinformatics strategies for analyzing them. Using these methods, we have characterized the gene expression signatures of HSC quiescence and proliferation and have constructed a molecular model of HSC activation and self-renewal.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Goodell, M. A., Brose, K., Paradis G., Conner, A. S., and Mulligan, R. C. (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797–1806.
Bradford, G. B., Williams, B., Rossi, R., and Bertoncello, I. (1997) Quiescence, cycling, and turnover in the primitive hematopoietic stem cell compartment. Exp. Hematol. 25, 445–453.
Morrison, S. J. and Weissman, I. L. (1994) The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1, 661–673.
Cheshier, S. H., Morrison, S. J., Liao, X., and Weissman, I. L. (1999) In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 96, 3120–3125.
Arai, F., Hirao, A., Ohmura, M., et al. (2004) Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118, 149–161.
Van Zant, G. (1984) Studies of hematopoietic stem cells spared by 5-fluorouracil. J. Exp. Med. 159, 679–690.
Harrison, D. E. and Lerner, C. P. (1991) Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 78, 1237–1240.
Randall, T. D. and Weissman, I. L. (1997) Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 89, 3596–3606.
Hart, C., Schochetman, G., Spira, T., et al. (1988) Direct detection of HIV RNA expression in seropositive subjects. Lancet 2, 596–599.
Eikhom, T. S., Abraham, K. A., and Dowben, R. M. (1975) Ribosomal RNA metabolism in synchronized plasmacytoma cells. Exp. Cell Res. 91, 301–309.
Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.
Lisitsyn, N. and Wigler, M. (1993) Cloning the differences between two complex genomes. Science 259, 946–951.
Diatchenko, L., Lau, Y. F., Campbell, A. P., et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025–6030.
Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995) Serial analysis of gene expression. Science 270, 484–487.
Datson, N. A., van der Perk-de Jong, J., van den Berg, M. P., de Kloet, E. R., and Vreugdenhil, E. (1999) MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue. Nucleic Acids Res. 27, 1300–1307.
Adams, M. D., Soares, M. B., Kerlavage, A. R., Fields, C., and Venter, J. C. (1993) Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat. Genet. 4, 373–380.
Lockhart, D. J., Dong, H., Byrne, M. C., et al. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat. Biotechnol. 14, 1675–1680.
Pietu, G., Alibert, O., Guichard, V., et al. (1996) Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array. Genome Res. 6, 492–503.
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Venezia, T. A., Merchant, A. A., Ramos, C. A., et al. (2004) Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol. 2, e301.
Lambert, J. F., Liu, M., Colvin, G. A., et al. (2003) Marrow stem cells shift gene expression and engraftment phenotype with cell cycle transit. J. Exp. Med. 197, 1563–1572.
Goodell, M. A., McKinney-Freeman, S., and Camargo, F. D. (2005) Isolation and characterization of side population cells. Methods Mol. Biol. 290, 343–352.
Young, A., Whitehouse, N., Cho, J., and Shaw, C. (2005) Ontology Traverser: an R package for GO analysis. Bioinformatics 21, 275–276.
Affymetrix. (2004) GeneChip Expression Analysis: Data Analysis Fundamentals.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Bowman, T.V., Merchant, A.A., Goodell, M.A. (2007). Molecular Profiling of Hematopoietic Stem Cells. In: Beksac, M. (eds) Bone Marrow and Stem Cell Transplantation. Methods in Molecular Medicine, vol 134. Humana Press. https://doi.org/10.1007/978-1-59745-223-6_1
Download citation
DOI: https://doi.org/10.1007/978-1-59745-223-6_1
Publisher Name: Humana Press
Print ISBN: 978-1-58829-595-8
Online ISBN: 978-1-59745-223-6
eBook Packages: Springer Protocols