Abstract
Hematopoietic stem cell (HSC) subsets are defined by the capacity to which their offspring can contribute to the various mature blood-cell lineages. However, the proliferative potential of stem cells is highly dependent on the environment in which they reside, and it is only in retrospect that the characteristics of a stem cell can be identified. Partly because of these elusive properties of stem cells, various functional assays to measure their frequency have been developed, both in vivo and in vitro. Various chapters in this volume each describe one of these assays. All these assays have the ability to quantify stem cells with certain properties, but not others. Therefore, the determination of which stem-cell assay will be most appropriate to use is highly dependent on a particular experimental setting. This chapter describes the cobblestone-areaforming cell (CAFC) assay (1).
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References
Ploemacher, R. E., van der Sluijs, J. P., van Beurden, C. A., Baert, M. R., and Chan, P. L. (1991) Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 78, 2527–2533.
Deryugina, E. I. and Muller-Sieburg, C. E. (1993) Stromal cells in long-term cultures: keys to the elucidation of hematopoietic development? Crit. Rev. Immunol. 13, 115–150.
Muller-Sieburg, C. E. and Deryugina, E. (1995) The stromal cells’ guide to the stem cell universe. Stem Cells (Dayton) 13, 477–186.
Collins, L. S. and Dorshkind, K. (1987) A stromal cell line from myeloid longterm bone marrow cultures can support myelopoiesis and B lymphopoiesis. J. Immunol. 138, 1082–1087.
Moore, K. A., Ema, H., and Lemischka, I. R. (1997) In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood 89, 4337–1347.
Wineman, J., Moore, K., Lemischka, I., and Muller-Sieburg, C. (1996) Functional heterogeneity of the hematopoietic microenvironment: rare stromal elements maintain long-term repopulating stem cells. Blood 87, 4082–4090.
Neben, S., Anklesaria, P., Greenberger, J., and Mauch, P. (1993) Quantitation of murine hematopoietic stem cells in vitro by limiting dilution analysis of cobble stone area formation on a clonal stromal cell line. Exp. Hematol. 21, 438–143.
Down, J. D. and Ploemacher, R. E. (1993) Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: differential effects of host conditioning with gamma radiation andcytotoxic drugs. Exp. Hematol. 21, 913–921.
Down, J. D., Boudewijn, A., Vanos, R., Thames, H. D., and Ploemacher, R. E. (1995) Variations in radiation sensitivity and repair among different hematopoietic stem cell subsets following fractionated irradiation. Blood 86, 122–127.
Ploemacher, R. E., van der Loo, J. C., van Beurden, C. A., and Baert, M. R. (1993) Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo. Leukemia 7, 120–130.
Down, J. D., de Haan, G., Dillingh, J. H., Dontje, B., and Nijhof, W. (1997) Stem cell factor has contrasting effects in combination with 5-fluorouracil or total-body irradiation on frequencies of different hemopoietic cell subsets and engraftment of transplanted bone marrow. Radiat. Res. 147, 680–685.
Breems, D. A., Blokland, E. A., Neben, S., and Ploemacher, R. E. (1994) Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukemia 8, 1095–1104.
van Hennik, P. B., Verstegen, M. M., Bierhuizen, M. F., Limon, A., Wognum, A. W., Cancelas, J. A., et al. (1998) Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice. Blood 92, 4013–022.
Verstegen, M. M., van Hennik, P. B., Terpstra, W., van den Bos, C., Wielenga, J. J., van Rooijen, N., et al. (1998) Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38-cells. Blood 91, 1966–1976.
Breems, D. A., van Hennik, P. B., Kusadasi, N., Boudewijn, A., Cornelissen, J. J., Sonneveld, P., et al. (1996) Individual stem cell quality in leukapheresis products is related to the number of mobilized stem cells. Blood 87, 5370–5378.
de Haan, G., Nijhof, W., and Van Zant, G. (1997) Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: correla-tion between lifespan and cycling activity. Blood 89, 1543–1550.
de Haan, G. and Van Zant, G. (1999) Dynamic changes in mouse hematopoietic stem cell numbers during aging. Blood 93, 3294–3301.
de Haan, G. and Van Zant, G. (1997) Intrinsic and extrinsic control of hemopoietic stem cell numbers: mapping of a stem cell gene. J. Exp. Med. 186, 529–536.
de Haan, G. and Van Zant, G. (1999) Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span. FASEB J. 13, 707–713.
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© 2002 Humana Press Inc., Totowa, NJ
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de Haan, G., Ploemacher, R. (2002). The Cobblestone-Area-Forming Cell Assay. In: Klug, C.A., Jordan, C.T. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Medicine, vol 63. Humana Press. https://doi.org/10.1385/1-59259-140-X:143
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DOI: https://doi.org/10.1385/1-59259-140-X:143
Publisher Name: Humana Press
Print ISBN: 978-0-89603-812-7
Online ISBN: 978-1-59259-140-4
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