Chapter 18 Bone Marrow-Derived Mesenchymal Stem Cells: Isolation, Expansion, Characterization, Viral Transduction, and Production of Conditioned Medium Massimiliano Gnecchi and Luis G. Melo y 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. How- ever, 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. Introduction The bone marrow stroma was originally thought to function mainly as a structural support for the hematopoietic stem and y Luis G. Melo passed away suddenly on September 26, 2007. This work is dedicated to his courage. Julie Audet, William L. Stanford (eds.), Stem Cells in Regenerative Medicine: Methods and Protocols, vol. 482 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media DOI 10.1007/978-1-59745-060-7_18 Springerprotocols.com 281