Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization Mohamadreza Baghaban Eslaminejad & Samad Nadri Received: 3 April 2008 / Accepted: 12 March 2009 / Published online: 19 May 2009 / Editor: J. Denry Sato # The Society for In Vitro Biology 2009 Abstract Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5×10 6 cells/cm 2 and upon confluency were reseeded as either low density (50 cells/cm 2 ) or high density (8×10 4 cells/cm 2 ) cultures. The cells were expand- ed through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic mor- phology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothe- lial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system. Keywords Murine mesenchymal stem cells . Cell seeding density . Culture mineralization . Surface antigens Introduction MSCs are defined as multipotent cells having two charac- teristics properties: (1) ability to differentiate into mesen- chymal lineages as adipose, cartilage and bone cells and (2) the capacity to undergo extensive replication for a long time while maintaining differentiation potentials (Majumdar et al. 1998; Pittenger et al. 1999). The definitive existence of MSCs was first reported by Friedenstein and co-workers who described that bone marrow contained a population of adherent cells with a capacity to produce small colonies which are similar to small deposits of bone and cartilage (Friedenstein et al. 1974a, b). MSCs have been reported to enjoy more differentiation potential than it was originally thought in giving rise to the cells other than those of mesenchymal origins such as neurons, keratinocytes, lungs, intestines, and renal epithelial cells (Chapel et al. 2003; Sugaya 2003). Such property is referred to as MSCs’ plasticity or transdifferentiation. MSCs are considered to be appropriate cellular materials for reconstruction of a variety of tissue defects (Baksh et al. 2004). Efficacy of the cells has been previously demon- strated in some diseases and tissue damages including osteogenesis imperfecta, bone defects, infarcted heart, and articular cartilage damages (Koe et al. 2000; Petite et al. 2000; Quarto et al. 2001; Horwitz et al. 2002; Barry 2003; In Vitro Cell.Dev.Biol.-Animal (2009) 45:451–459 DOI 10.1007/s11626-009-9198-1 M. B. Eslaminejad (*) Stem Cell Department, Cell Sciences Research Center, Royan Institute, ACECR, P. O. Box 19395-4644, Tehran, Iran e-mail: eslami@royaninstitute.org e-mail: bagesla@yahoo.com S. Nadri Department of Stem Cells and Tissue Engineering, Stem Cell Technology Institute, Tehran, Iran e-mail: eslami@royaninstitute.org M. B. Eslaminejad