Characterisation and Developmental Potential of Ovine Bone Marrow Derived Mesenchymal Stem Cells ROSA C. MCCARTY, 1,2,3 STAN GRONTHOS, 3,4 ANDREW C. ZANNETTINO, 3,4 BRUCE K. FOSTER, 1,2 AND CORY J. XIAN 1,2,5 * 1 Department of Orthopaedic Surgery, Women’s & Children’s Hospital, Adelaide, Australia 2 Department of Paediatrics, University of Adelaide, Adelaide, Australia 3 Division of Haematology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia 4 Department of Medicine, University of Adelaide, Adelaide, Australia 5 Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia Since discovery, significant interest has been generated in the potential application of mesenchymal stem cells or multipotential stromal cells (MSC) for tissue regeneration and repair, due to their proliferative and multipotential capabilities. Although the sheep is often used as a large animal model for translating potential therapies for musculoskeletal injury and repair, the characteristics of MSC from ovine bone marrow have been inadequately described. Histological and gene expression studies have previously shown that ovine MSC share similar properties with human and rodents MSC, including their capacity for clonogenic growth and multiple stromal lineage differentiation. In the present study, ovine bone marrow derived MSCs positively express cell surface markers associated with MSC such as CD29, CD44 and CD166, and lacked expression of CD14, CD31 and CD45. Under serum-deprived conditions, proliferation of MSC occurred in response to EGF, PDGF, FGF-2, IGF-1 and most significantly TGF-a. While subcutaneous transplantation of ovine MSC in association with a ceramic HA/TCP carrier into immunocomprimised mice resulted in ectopic osteogenesis, adipogenesis and haematopoietic-support activity, transplantation of these cells within a gelatin sponge displayed partial chondrogenesis. The comprehensive characterisation of ovine MSC described herein provides important information for future translational studies involving ovine MSC. J. Cell. Physiol. 219: 324–333, 2009. ß 2008 Wiley-Liss, Inc. Mesenchymal stem cells or multipotential stromal cells (MSC) are self renewing multipotential progenitor cells with the capacity to differentiate toward numerous cell types of, and beyond those of mesodermal origin, including osteoblasts, adipocytes, chondrocytes, myocytes, fibroblasts, marrow stroma and cells of neural phenotype upon exposure to the appropriate stimuli (Bruder et al., 1994, 1998; Prockop, 1997; Johnstone et al., 1998; Yoo et al., 1998; Pittenger et al., 1999; Woodbury et al., 2000; Bianco et al., 2001; Zhao et al., 2002; Gronthos et al., 2003; Xu et al., 2004). The potential of mesenchymal stem cell populations to differentiate into a multitude of tissue types in vitro and in vivo has prompted considerable interest in their application to repair or regenerate musculoskeletal tissues. Characterisation of the immunophenotype of MSC has facilitated more precise and consistent identification and purification of MSC. Cultured human MSC express a repertoire of cell surface antigens including STRO-1, CD105, CD90, CD73, CD29, CD44, CD166, CD146 and conversely lack expression of the haematopoietic marker CD45 and endothelial marker CD31 (Simmons and Torok-Storb, 1991; Gronthos and Simmons, 1995; Gronthos et al., 2003; Shi and Gronthos, 2003). In addition to humans, MSC have been identified in large and small animals including goat (Fischer et al., 2003; Murphy et al., 2003), rat (Javazon et al., 2001; Lennon and Caplan, 2006; Yoshimura et al., 2007), rabbit (Awad et al., 1999; Tsutsumi et al., 2001), mouse (Phinney et al., 1999; Tropel et al., 2004), horse (Fortier et al., 1998), cow (Bosnakovski et al., 2005), dog (Kadiyala et al., 1997), cat (Martin et al., 2002), sheep (Jessop et al., 1994; Rhodes et al., 2004; Zhang et al., 2004), pig (Ringe et al., 2002; Moscoso et al., 2005; Vacanti et al., 2005) and non-human primates (Devine et al., 2003). Interestingly, bone marrow-derived MSC (BM MSC) from virtually all species examined are heterogeneous in size, morphology, proliferation and differentiation potential (Friedenstein et al., 1987; Owen, 1988; Kuznetsov et al., 1997; Gronthos et al., 2003). The use of sheep as a large animal model for orthopaedic research continues to increase in popularity due to similarities with humans in weight, size, joint structure, bone/cartilage regenerative processes, and thus the potential in translational research. Although knowledge regarding human MSC properties and characteristics has been comprehensively investigated, very little characterisation of ovine MSC has been undertaken; this includes the cell surface immunophenotype, the mitogens they respond to under serum deprived conditions during ex vivo expansion, and their multiple differentiation potentials. Thus, any similarities and differences between ovine and human MSC remain unknown, the outcomes of which may Contract grant sponsor: Bone Growth Foundation, Australia. Contract grant sponsor: NHMRC, Australia; Contract grant numbers: 242804, 258700, 453443, 453497. *Correspondence to: Cory J. Xian, Sansom Institute, University of South Australia, City East Campus, GPO Box 2471, Adelaide 5001, Australia. E-mail: cory.xian@unisa.edu.au Received 14 November 2008; Accepted 17 November 2008 Published online in Wiley InterScience (www.interscience.wiley.com.), 29 December 2008. DOI: 10.1002/jcp.21670 ORIGINAL ARTICLE 324 Journal of Journal of Cellular Physiology Cellular Physiology ß 2008 WILEY-LISS, INC.