Abstract
Mesenchymal stem cells are typically enriched from bone marrow via their preferential attachment to tissue culture plastic. However, this isolation method has proven ineffective for murine MSCs because various hematopoietic cell lineages survive and/or proliferate on stromal layers in the absence of exogenous cytokines and therefore constitute a large percentage of the plastic adherent population. Although various methods have been described to remove contaminating hematopoietic populations from these cultures none have gained widespread acceptance. Consequently, we developed a method based on immunodepletion to fractionate hematopoietic and endothelial lineages from plastic adherent fibroblastoid (stromal) cells elaborated from murine bone marrow. Colony-forming assays, immunostaining and flow cytometry has been used to validate the effectiveness of this method. Moreover, immunodepleted populations have been shown to exhibit the capacity for multilineage differentiation in vitro and in vivo and therefore retain the characteristics of MSCs. Most recently, we also catalogued the transcriptome of immunodepleted populations via serial analysis of gene expression. Therefore, our immunodepletion scheme provides a means to enrich from murine bone marrow MSCs that's molecular and biological characteristics are well described. Importantly, this immunodepletion method does not employ long-term expansion of plastic adherent cells ex vivo, thereby avoiding the generation of immortalized cell lines.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
1. Phinney, D. G. (2002) Building a consensus regarding the nature and origin of mesenchymal stem cells. J. Cell. Biochem. Suppl 38, 7–12.
2. Dennis, J. E., Merriam, A., Awadallah, A., Yoo, J. U., Johnstone, B., and Caplan, A. I. (1999) A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J. Bone Min. Res. 14, 700–709.
3. Pittenger, M. F., MacKay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
4. Mauraglia, A., Cancedda, R., and Quarto, R. (2000) Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J. Cell Sci. 113, 1161–1166.
5. Kuznetsov, S. A., Krebsbach, P. H., Satomura, K., Kerr, J., Riminucci, M., Benayahu, D., et al. (1997) Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo. J. Bone Min. Res. 12, 1335–1347.
6. Caplan, A. I. (1994) The mesengenic process. Clin. Plastic Surg. 21, 429–435.
7. Bearpark, A. D. and Gordon, M. Y. (1989) Adhesive properties distinguish sub-populations of hematopoietic stem cells with different spleen colony-forming and marrow repopulating capacities. Bone Marrow Transpl. 4, 625–628.
8. Kerk, D. K., Henry, E. A., Eaves, A. C., and Eaves, C. J. (1985) Two classes of primitive pluripotent hematopoietic progenitor cells: Separation by adherence. J. Cell Physiol. 125, 127–134.
9. Deryugina, E. I. and Muller-Sieburg, C. E. (1993) Stromal cells in long-term cultures: Keys to the elucidation of hematopoietic development. Crit. Rev. Immun. 13, 115–150.
10. Simmons, P. J., Masinovsky, B., Longenecker, B. M., Berenson, R., Torok-Storb, B., and Gallatin, W. M. (1992). Vascular cell adhesion molecule-1 expressed by bone marrow stromal cells mediates the binding of hematopoietic progenitor cells. Blood 80, 388–395.
11. Witte, P. L., Robinson, M., Henley, A., Low, M. G., Stiers, D. L., Perkins, S., et al. (1987) Relationship between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures. Eur. J. Immunol. 17, 1473–1484.
12. Phinney, D. G., Kopen, G., Isaacson, R. L., and Prockop, D. J. (1999) Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: Variations in yield, growth, and differentiation. J. Cell. Biochem. 72, 570–585.
13. Gordon, M. Y., Bearpark, A. D., Clarke, D., and Dowding, C. R. (1990) Haemopoietic stem cell subpopulations in mouse and man: Discrimination by differential adherence and marrow repopulating ability. Bone Marrow Transpl. 5(Suppl), 6–8.
Kiefer, F., Wagner, E. F., and Keller, G. Fractionation of mouse bone marrow by adherence separates primitive hematopoietic stem cells from in vitro colony-forming cells and spleen colony-forming cells. Blood 78, 2577–2582.
15. Winemann, J. P., Nishikawa, S-I., and Muller-Sieburg, C. E. (1993) Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit. Blood 81, 365–372.
16. Wang, Q-R. and Wolf, N. S. (1990) Dissecting the hematopoietic microenvironment. VIII. Clonal isolation and identification of cell types in murine CFU-F colonies by limiting dilution. Exp. Hematol. 18, 355–359.
17. Modderman, W. E., Vrijheid-Lammers, T., Lowik, C. W., and Nijweide, P. J. (1994) Removal of hematopoietic cells and macrophages from mouse bone marrow cultures: isolation of fibroblast-like stromal cells. Expt. Hematol. 22, 194–201.
18. Van Vlasselaer, P., Falla, N., Snoeck, H., and Mathieu, E. (1994) Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin. Blood 84, 753–763.
19. Kopen, G., Prockop, D. J., and Phinney, D. G. (1999) Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. Acad. Sci. USA. 96, 10,711–10,716.
20. Baddoo, M., Hill, K., Wilkinson, R., Gaupp, D., Hughes, C., Kopen G. C., et al. (2003) Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J. Cell. Biochem. 89, 1235–1249.
21. Phinney, D. G., Hill, K., Michelson, C., Dutreil, M., Hughes, C., Humphries, S., et al. (2006) Biological activities encoded by the murine mesenchymal stem cell transcriptome provide a basis for their developmental plasticity and broad clinical efficacy. Stem Cells 24, 186–198.
22. Meirelles, L. S. and Nardi, N. B. (2003) Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion and characterization. Brit. J. Hematol. 123, 702–712.
23. Sun, S., Guo, Z., Xiao, X., Liu, B., Liu, X., Tang, P-H., et al. (2003) Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 21, 527–535.
24. Peister, A., Mellad, J. A., Larson, B. L., Hall, B. M., Gibson, L. F., and Prockop, D. J. (2004) Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–1668.
25. Prowse, K. R. and Greider, C. W. (1995) Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc. Natl. Acad. Sci. USA 92, 4818–4822.
26. Dobson, K. R., Reading, L., Haberey, M., Marine, X., and Scutt, A. (1999) Centrifugal isolation of bone marrow from bone: an improved method for the recovery and quantification of bone marrow osteoprogenitor cells from rat tibiae and femurae. Calc. Tissue Internat. 65, 411–413.
27. Johnstone, B, Hering, T. M., Caplan, A. I., Goldberg, V. M., and Yoo, J. U. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272.
28. MacKay, A. M., Beck, S. C., Murphy, J. M., Barry, F. P., Chichester, C. O., and Pittenger, M. F. (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4, 415–428.
29. Gregory, C. A., Gunn, W. G., Peister, A., and Prockop, D. J. (2004) An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal. Biochem. 329, 77–84.
Acknowledgments
The author would like to thank Maria DuTreil and Dina Gaupp for assistance in preparing this chapter.
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
Phinney, D.G. (2008). Isolation of Mesenchymal Stem Cells from Murine Bone Marrow by Immunodepletion. 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-60327-169-1_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-771-6
Online ISBN: 978-1-60327-169-1
eBook Packages: Springer Protocols