Abstract
The STRO-1 antibody can be used as a single reagent to isolate human bone marrow stromal stem cells (BMSSC), owing to its restricted specificity to a cell surface molecule expressed by clonogenic BMSSC, with little or no reactivity to hematopoietic stem/progenitor populations or mature stromal elements. The present protocol uses a combination of two different immunoselection methodologies in an attempt to generate highly purified preparations of BMSSC. This process involves the initial isolation of a minor subpopulation of bone marrow mononuclear cells (approx 10%) expressing the STRO-1 antigen, by means of magnetic activated cell sorting. Dual-color fluorescence activated cell sorting is then used as a secondary step to further purify the rare STRO-1bright expressing fraction that contains all of the colony-forming BMSSC, based on their co-expression of a secondary cell surface marker, CD106 (VCAM-1).
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References
1. Castro-Malaspina, H., Gay, R. E., Resnick, G., Kapoor, N., Meyers, P., Chiarieri, D., McKenzie, S., Broxmeyer, H. E., and Moore, M. A. (1980) Characterization of human bone marrow fibroblast colony-forming cells (CFU-F) and their progeny. Blood 56(2):289–301.
2. Friedenstein, A. J., Chailakhjan, R. K., and Lalykina, K. S. (1970) The development of fibroblast colonies in monolayer cultures of guinea- pig bone marrow and spleen cells. Cell Tissue Kinet 3(4):393–403.
3. Owen, M., and Friedenstein, A. J. (1988) Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 136(29):42–60.
4. Gronthos, S., Zannettino, A. C., Hay, S. J., Shi, S., Graves, S. E., Kortesidis, A., and Simmons, P. J. (2003) Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 116(Pt 9):1827–1835.
5. Simmons, P. J., and Torok-Storb, B. (1991) Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 78(1):55–62.
6. Waller, E. K., Olweus, J., Lund-Johansen, F., Huang, S., Nguyen, M., Guo, G. R., and Terstappen, L. (1995) The “common stem cell” hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors. Blood 85(9):2422–2435.
7. Castro-Malaspina, H., Rabellino, E. M., Yen, A., Nachman, R. L., and Moore, M. A. (1981) Human megakaryocyte stimulation of proliferation of bone marrow fibroblasts. Blood 57(4):781–787.
8. Boiret, N., Rapatel, C., Veyrat-Masson, R., Guillouard, L., Guerin, J. J., Pigeon, P., Descamps, S., Boisgard, S., and Berger, M. G. (2005) Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow. Exp Hematol 33(2):219–225.
9. Deschaseaux, F., Gindraux, F., Saadi, R., Obert, L., Chalmers, D., and Herve, P. (2003) Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype. Br J Haematol 122(3):506–517.
10. Filshie, R. J., Zannettino, A. C., Makrynikola, V., Gronthos, S., Henniker, A. J., Bendall, L. J., Gottlieb, D. J., Simmons, P. J., and Bradstock, K. F. (1998) MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies. Leukemia 12(3):414–421.
11. Gronthos, S., Graves, S. E., Ohta, S., and Simmons, P. J. (1994) The STRO−1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 84(12):4164–4173.
12. Gronthos, S., and Simmons, P. J. (1995) The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood 85(4):929–940.
13. Joyner, C. J., Bennett, A., and Triffitt, J. T. (1997) Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies. Bone 21(1):1–6.
14. Miura, M., Chen, X. D., Allen, M. R., Bi, Y., Gronthos, S., Seo, B. M., Lakhani, S., Flavell, R. A., Feng, X. H., Robey, P. G., Young, M., and Shi, S. (2004) A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells. J Clin Invest 114(12):1704–1713.
15. Quirici, N., Soligo, D., Bossolasco, P., Servida, F., Lumini, C., and Deliliers, G. L. (2002) Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol, 30(7):783–791.
16. Shi, S., and Gronthos, S. (2003) Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 18(4):696–704.
17. Simmons, P. J., Gronthos, S., Zannettino, A., Ohta, S., and Graves, S. (1994) Isolation, characterization and functional activity of human marrow stromal progenitors in hemopoiesis. Prog Clin Biol Res 389:271–280.
18. Gronthos, S., Graves, S. E., and Simmons, P. J. (1998) Isolation, purification and in vitro manipulation of human bone marrow stromal precursor cells. In: Beresford JNaO, M.E. (ed.) Marrow stromal cell culture. Cambridge University Press, Cambridge, pp. 26–42.
19. Lansdorp, P. M., and Dragowska, W. (1992) Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. J Exp Med 175(6):1501–1509.
20. Migliaccio, G., Migliaccio, A. R., and Adamson, J. W. (1988) In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum-deprived cultures. Blood 72(1):248–256.
21. Reyes, M., Dudek, A., Jahagirdar, B., Koodie, L., Marker, P. H., and Verfaillie, C. M. (2002) Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109(3):337–346.
22. Reyes, M., and Verfaillie, C. M. (2001) Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells. Ann N Y Acad Sci 938:231–233; discussion 233–235.
Acknowledgments
The authors wish to acknowledge the technical assistance of Ms. Fiona Kohr and Ms. Sharon Paton. This work was supported by National Health and Medical Council of Australia grants (A Zannettino, S Gronthos).
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Gronthos, S., Zannettino, A.C.W. (2008). A Method to Isolate and Purify Human Bone Marrow Stromal Stem Cells. In: Prockop, D.J., Bunnell, B.A., Phinney, D.G. (eds) Mesenchymal Stem Cells. Methods in Molecular Biology™, vol 449. Humana Press. https://doi.org/10.1007/978-1-60327-169-1_3
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DOI: https://doi.org/10.1007/978-1-60327-169-1_3
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