Abstract
Mesenchymal stem cells (MSCs) are defined as self-renewing and multipotent cells capable of differentiating into multiple cell types, including osteocytes, chondrocytes, adipocytes, hepatocytes, myocytes, neurons, and cardiomyocytes. MSCs were originally isolated from the bone marrow stroma but they have recently been identified also in other tissues, such as fat, epidermis, and cord blood. Several methods have been used for MSC isolation. The most common method is based on the ability of the MSCs to selectively adhere to plastic surfaces. Phenotypic characterization of MSCs is usually carried out using immunocytochemical detection or fluorescence-activated cell sorting (FACS) analysis of cell surface molecule expression. However, the lack of specific markers renders the characterization of MSCs difficult and sometimes ambiguous. MSCs posses remarkable expansion potential in culture and are highly amenable to genetic modification with various viral vectors rendering them optimal vehicles for cell-based gene therapy. Most importantly, MSC plasticity and the possibility to use them as autologous cells render MSCs suitable for cell therapy and tissue engineering. Furthermore, it is known that MSCs produce and secrete a great variety of cytokines and chemokines that play beneficial paracrine actions when MSCs are used for tissue repair. In this chapter, we describe methods for isolation, ex vivo expansion, phenotypic characterization, and viral infection of MSCs from mouse bone marrow. We also describe a method for preparation of conditioned and concentrated conditioned medium from MSCs. The conditioned medium can be easily tested both in vitro and in vivo when a particular paracrine effect (i.e., cytoprotection) is hypothesized to be an important mechanism of action of the MSCs and/or screened to identify a target paracrine/autocrine mediator.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dexter, T.M., Allen, T.D., Lajtha, L.G. (1977) Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 91, 335–344.
Becker, A.J., McCulloch, E.A., Till, J.E. (1963) Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197, 452–454.
Siminovitch, L., McCulloch, E.A., Till, J.E. (1963) The distribution of colony-forming cells among spleen colonies. J Cell Physiol 62, 327–336.
Friedenstein, A.J., Chailakhjan, R.K., Lalykina, K.S. (1970) The development of fibroblast colonies in monolayer cultures of guinea pig bone marrow and spleen cells. Cell Tissue Kinet 3, 393–403.
Friedenstein, A.J., Deriglasova, U.F., Kulagina, N.N., Panasuk, A.F., Rudakowa, S.F., Luria, E.A., Ruadkow, I.A. (1974) Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2, 83–92.
Friedenstein, A.J., Chailakhyan, R.K., Gerasimov, U.V. (1987) Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 20, 263–272.
Aston, B.A., Allen, T.D., Howlett, C.R., Eaglesom, C.C., Hattori, A., Owen, M. (1980) Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop Relat Res 151, 294–307.
Owen, M. (1988) Marrow stromal stem cells. J Cell Sci Suppl 10, 63–76.
Caplan, A.I. (1991) Mesenchymal stem cells. J Orthop Res 9, 641–650.
Minguelll, J.J., Erices, A., Conget, P. (2001) Mesenchymal stem cells. Exp Biol Med 226, 507–520.
He, Q., Wan, C., Li, G. (2007) Concise review: multipotent mesenchymal stromal cells in blood. Stem cells 25, 69–77.
Prockop, D.J. (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74.
Nuttall, M.E., Patton, A.J., Olivera, D.L., Nadeau, D.P., Gowen, M. (1998) Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 13, 371–382.
Wakitani, S., Saito, T., Caplan, A.I. (1995) Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18, 1417–1426.
Kopen, G.C., Prockop, D.J., 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 U S A 96, 10711–10716.
Makino, S., Fukuda, K., Miyoshi, S., Kodama, H., Pan, J., Sano, M., Takahashi, T., Hori, S., Abe, H., Hata, J., Umezawa, A., Ogawa, S. (1999) Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103, 697–705.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Limoneti, D.W., Carig, S., Marshak, D.R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
Bianco, P., Riminucci, M., Kuznetsov, S., and Robey, P.G. (1999) Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Exp 9, 159–173.
Liechty, K.W., MacKenzie, T.C., Shaaban, A.F., Radu, A., Moseley, A. M., Deans, R., Marshak, D. R., and Flake, A. W. (2000) Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 6, 1282–1286.
Pochampally, R.R., Neville, B.T., Schwarz, E.J., Li, M. M., and Prockop, D. J. (2004) Rat adult stem cells (marrow stromal cells) engraft and differentiate in chick embryos without evidence of cell fusion. Proc Natl Acad Sci USA 101, 9282–9285.
Horwitz, E.M., Prockop, D.J., Fitzpatrick, L.A., Koo, W.W., Gordon, P.L., Neel, M., (1999) Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta, Nat Med 5, 262–264.
Caplan A.I., Bruden, S.P. (2001) Mesenchymal stem cells: building blocks for molecular medicine in the 21th century. Trends Mol Med 7, 259–264.
Melo, L.G., Pachori, A.S., Kong, D., Gnecchi, M., Wang, K., Pratt, R. E., and Dzau, V. J. (2004) Molecular and cell-based therapies for protection, rescue, and repair of ischemic myocardium: reasons for cautious optimism. Circulation 109, 2386–2393.
Ryan, J.M., Barry, F.P., Murphy, J.M., Mahorn, B.P. (2005) Mesenchymal stem cells avoid allogeneic rejection. J Inflamm 26, 2–8.
Dzau, V.J., Gnecchi, M., Pachori, A.S. (2005) Enhancing stem cell therapy through genetic modification. J Am Coll Cardiol 46, 1351–1353.
Caplan, A.I., Dennis, J.E. (2006) Mesenchymal Stem Cells as Trophic Mediators. J Cell Biochem 98, 1076–1084.
Gnecchi, M., He, H., Liang, O.D., Melo, L. G., Morello, F., Mu, H., Noiseux, N., Zhang, L., Pratt, R. E., Ingwall, J. S., et al. (2005) Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med 11, 367–368.
Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L., Morello, F., Mu, H., Melo, L. G., Pratt, R. E., Ingwall, J. S., Dzau, V. J. (2006) Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 20, 661–669.
Noiseux, N., Gnecchi, M., Lopez-Ilasca, M., Zhang, L., Solomon, S.D., Deb, A., Dzau, V.J., Pratt, R.E. (2006) Mesenchymal stem cells overexpressing Akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther 14, 840–850.
Iso, Y., Spees, J.L., Serrano, C., Bakondi, B., Pochampally, R., Song, Y.H., Sobel, B.E., Delafontaine, P., Prockop, D.J. (2007) Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment. Biochem Biophys Res Commun 354, 700–706.
Honma, T., Honmou, O., Iihoshi, S., Harada, K., Houkin, K., Hamada, H., Kocsis, J.D. (2005) Intravenous infusion of immortalized human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Exp Neurol 199, 56–66.
Togel, F., Weiss, K., Yang, Y., Hu, Z., Zhang, P., Westenfelder, C. (2007) Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury. Am J Physiol Renal Physiol 292, F1626–F1635.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gnecchi, M., Melo, L.G. (2009). Bone Marrow-Derived Mesenchymal Stem Cells: Isolation, Expansion, Characterization, Viral Transduction, and Production of Conditioned Medium. In: Audet, J., Stanford, W.L. (eds) Stem Cells in Regenerative Medicine. Methods in Molecular Biology, vol 482. Humana Press. https://doi.org/10.1007/978-1-59745-060-7_18
Download citation
DOI: https://doi.org/10.1007/978-1-59745-060-7_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-797-6
Online ISBN: 978-1-59745-060-7
eBook Packages: Springer Protocols