Bone Marrow-Derived Mesenchymal Stem Cells: Isolation, Expansion, Characterization, Viral Transduction, and Production of Conditioned Medium

  • Massimiliano Gnecchi
  • Luis G. Melo
Part of the Methods in Molecular Biology book series (MIMB, volume 482)


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.

Key words

Mesenchymal stem cells cell surface markers flow cytometry gene transfer conditioned medium soluble factors paracrine effect drug discovery 


  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    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.CrossRefPubMedGoogle Scholar
  3. 3.
    Siminovitch, L., McCulloch, E.A., Till, J.E. (1963) The distribution of colony-forming cells among spleen colonies. J Cell Physiol 62, 327–336.CrossRefPubMedGoogle Scholar
  4. 4.
    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.PubMedGoogle Scholar
  5. 5.
    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.PubMedGoogle Scholar
  6. 6.
    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.PubMedGoogle Scholar
  7. 7.
    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.Google Scholar
  8. 8.
    Owen, M. (1988) Marrow stromal stem cells. J Cell Sci Suppl 10, 63–76.PubMedGoogle Scholar
  9. 9.
    Caplan, A.I. (1991) Mesenchymal stem cells. J Orthop Res 9, 641–650.CrossRefPubMedGoogle Scholar
  10. 10.
    Minguelll, J.J., Erices, A., Conget, P. (2001) Mesenchymal stem cells. Exp Biol Med 226, 507–520.Google Scholar
  11. 11.
    He, Q., Wan, C., Li, G. (2007) Concise review: multipotent mesenchymal stromal cells in blood. Stem cells 25, 69–77.CrossRefPubMedGoogle Scholar
  12. 12.
    Prockop, D.J. (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74.CrossRefPubMedGoogle Scholar
  13. 13.
    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.CrossRefPubMedGoogle Scholar
  14. 14.
    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.CrossRefPubMedGoogle Scholar
  15. 15.
    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.CrossRefPubMedGoogle Scholar
  16. 16.
    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.CrossRefPubMedGoogle Scholar
  17. 17.
    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.CrossRefPubMedGoogle Scholar
  18. 18.
    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.Google Scholar
  19. 19.
    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.CrossRefPubMedGoogle Scholar
  20. 20.
    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.CrossRefPubMedGoogle Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    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.CrossRefPubMedGoogle Scholar
  23. 23.
    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.CrossRefPubMedGoogle Scholar
  24. 24.
    Ryan, J.M., Barry, F.P., Murphy, J.M., Mahorn, B.P. (2005) Mesenchymal stem cells avoid allogeneic rejection. J Inflamm 26, 2–8.Google Scholar
  25. 25.
    Dzau, V.J., Gnecchi, M., Pachori, A.S. (2005) Enhancing stem cell therapy through genetic modification. J Am Coll Cardiol 46, 1351–1353.CrossRefPubMedGoogle Scholar
  26. 26.
    Caplan, A.I., Dennis, J.E. (2006) Mesenchymal Stem Cells as Trophic Mediators. J Cell Biochem 98, 1076–1084.CrossRefPubMedGoogle Scholar
  27. 27.
    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.CrossRefPubMedGoogle Scholar
  28. 28.
    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.CrossRefPubMedGoogle Scholar
  29. 29.
    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.CrossRefPubMedGoogle Scholar
  30. 30.
    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.CrossRefPubMedGoogle Scholar
  31. 31.
    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.CrossRefPubMedGoogle Scholar
  32. 32.
    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.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Massimiliano Gnecchi
    • 1
  • Luis G. Melo
    • 2
  1. 1.Department of CardiologyFondazione IRCCS Policlinico San Matteo and University of PaviaItaly
  2. 2.Department of PhysiologyQueen’s UniversityKingstonCanada

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