Summary
Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone area-forming cell assay; CAFC). These assays measure the potential of a test cell population retrospectively, i.e., at the time its activity is evident when the stem cell itself is often not detectable anymore. Although the in vitro LTC-IC and CAFC assays have been shown to correlate with in vivo activity, in vivo transplantation assays, where it can be shown that cells possess the ability to indefinitely repopulate all blood lineages, are the ultimate proof for HSC activity. Nevertheless, these in vitro assays provide an excellent method to screen for stem cell activity of a putative stem cell population or for screening the effect of a certain treatment on HSCs.
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
Reference
Dexter, T. M., Allen, T. D., and Lajtha, L. G. (1977) Conditionscontrolling the proliferation of haemopoietic stem cells in vitro.J Cell Physiol 91, 335–344.
Ploemacher, R. E., van der Sluijs, J. P., Voerman, J. S. A., and Brons, N. H. C. (1989) An in vitro limiting-dilution assay oflong-term repopulating hematopoietic stem cells in the mouse. Blood 74, 2755–2763.
Ploemacher, R. E., van der Sluijs, J. P., Van Beurden, C. A. J.,Baert, M. R. M., and Chan, P. L. (1991) Use of limiting-dilutiontype long-term marrow cultures in frequency analysis ofmarrow-repopulating and spleen colony-forming hematopoietic stemcells in the mouse. Blood 10, 2527–2533.
Ploemacher, R. E., van der Loo, J. C. M., Van Beurden, C. A. J., and Baert, M. R. M. (1993) Wheat germ agglutinin affinity of murinehemopoietic stem cell subpopulations is an inverse function of theirlong-term repopulating ability in vitro and in vivo. Leukemia 7, 120–130.
Sutherland, H. J., Eaves, C. J., Eaves, A. C., Dragowska, W., andLansdorp, P. M. (1989) Characterization and partial purification ofhuman marrow cells capable of initiating long-term hematopoiesis invitro. Blood 74, 1563–1570.
Lemieux, M. E., Rebel, V. I., Lansdorp, P. M., and Eaves, C. J.(1995) Characterization and purification of a primitivehematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow “switch” cultures. Blood 86, 1339–1347.
Deryugina, E. I., Muller-Sieburg, C. E. (1993) Stromal cells inlong-term cultures: keys to the elucidation of hematopoieticdevelopment? Crit Rev. Immunol. 13, 115–150.
Muller-Sieburg, C. E., Deryugina, E. (1995) The stromal cells’ guideto the stem cell universe. Stem Cells 13, 477–486.
Collins, L. S., Dorshkind, K. (1987) A stromal cell line frommyeloid long-term bone marrow cultures can support myelopoiesis andB lymphopoiesis. J. Immunol. 138, 1082–1087.
Moore, K. A., Ema, H., and Lemischka, I. R. (1997) In vitromaintenance of highly purified, transplantable hematopoietic stemcells. Blood 89, 4337–4347.
Wineman, J., Moore, K., Lemischka, I., and Muller-Sieburg, C. (1996)Functional heterogeneity of the hematopoietic microenvironment: rarestromal 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 limitingdilution analysis of cobblestone area formation on a cloned stromalcell line. Exp Hematol 21, 438–443.
Oostendorp, R. A., Harvey, K. N., Kusadasi, N., de Bruijn, M. F., Saris, C., Ploemacher, R. E., Medvinsky, A. L., and Dzierzak, E. A.(2002) Stromal cell lines from mouse aorta-gonads-mesonephrossubregions are potent supporters of hematopoietic stem cellactivity. Blood 99, 1183–1189.
Down, J. D., Boudewijn, A., van Os, R., Thames, H. D., andPloemacher, R. E. (1995) Variations in radiationsensitivity andrepair among different hematopoietic stem cell subsets followingfractionated irradiation. Blood 86, 122–127.
Wierenga, P. K., Setroikromo, R., Kamps, G., Kampinga, H. H., and Vellenga, E. (2002) Peripheral blood stem cells differ from bonemarrow stem cells in cell cycle status, repopulating potential, andsensitivity toward hyperthermic purging in mice mobilized withcyclophosphamide and granulocyte colony-stimulating factor. J.Hematother. Stem Cell Res. 11, 523–532.
Kamminga, L. M., van Os, R., Ausema, A., Noach, E. J., Dontje, B.,Vellenga, E., and de Haan, G. (2005) Impaired hematopoietic stemcell unctioning after serialtransplantation and during normalaging. Stem Cells 23, 82–92.
van Os, R., Kamminga, L. M., and de Haan, G. (2004) Stem cellassays: something old, something new, something borrowed. StemCells 22, 1181–1190.
Breems, D. A., Blokland, E. A., Neben, S., and Ploemacher, R. E.(1994) Frequency analysis of human primitive haematopoietic stemcell subsets using a cobblestone area-forming cell assay. Leukemia 8, 1095–1104.
Fazekas de St.Groth, S. (1982) The evaluation of limiting dilutionassays. Jmmunol Meth 49, R11–R23.
Acknowledgments
The authors thank Dr. Rob Ploemacher for his support and for introducing the CAFC assay in hematopoietic stem cell research. We also thank Dr. Simon Robinson, Department of Blood and Marrow Transplantation, University of Texas, MD, Anderson Cancer Center, Houston, USA, for critically reading the manuscript.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Os, R.P.v., Dethmers-Ausema, B., Haan, G.d. (2008). In Vitro Assays for Cobblestone Area-Forming Cells, LTC-IC, and CFU-C. In: Bunting, K.D. (eds) Hematopoietic Stem Cell Protocols. Methods in Molecular Biology™, vol 430. Humana Press. https://doi.org/10.1007/978-1-59745-182-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-182-6_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-868-3
Online ISBN: 978-1-59745-182-6
eBook Packages: Springer Protocols