Ex Vivo Expansion of Human Mesenchymal Stem Cells: A More Effective Cell Proliferation Kinetics and Metabolism Under Hypoxia FRANCISCO DOS SANTOS, 1 PEDRO Z. ANDRADE, 1 JOANA S. BOURA, 1 MANUEL M. ABECASIS, 2 CLA ´ UDIA LOBATO DA SILVA, 1 AND JOAQUIM M.S. CABRAL 1 * 1 IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Te´cnico, Lisboa, Portugal 2 Instituto Portugueˆs de Oncologia de Lisboa, Francisco Gentil (IPOFG), E.P.E., Lisboa, Portugal The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O 2 (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O 2 , resulting in a higher fold increase after 12 days under hypoxia (40  10 vs. 30  6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O 2 and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O 2 presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O 2 favored CFU-F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O 2 , compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010. ß 2009 Wiley-Liss, Inc. In recent years, human mesenchymal stem cells (MSC) have become one of the most promising candidates for tissue engineering and regenerative medicine applications (Caplan, 2007), mostly due to the differentiative potential (Tae et al., 2006) and immunologic properties (Le Blanc and Ringden, 2007; Jones and McTaggart, 2008) of these multipotent cells. Particularly, in the settings of cellular therapy, MSC have been used to prevent or to treat graft-versus-host disease after hematopoietic stem cell transplantation (Ringden et al., 2006; Kebriaei et al., 2009). The frequency of MSC is considered to be as low as 0.01% of BM MNC in a newborn, declining with age to 0.001–0.0005% (Caplan, 2007). Though minimal and maximal doses for therapeutic application have not yet been determined, several million BM MSC per kg of patient body weight have been infused (Subbanna, 2007). Therefore, an efficient and Good Manufacturing Practices (GMP) — compliant ex vivo expansion process is required to achieve MSC clinical relevant numbers. In their BM niche, self-renewal and/or differentiation of MSC are governed by a complex microenvironment signaling that involves cell-to-cell interactions (Ball et al., 2004), soluble factors (proteins or growth factors; Gregory et al., 2003; Choi et al., 2008) and even mechanical forces (Park et al., 2004). The oxygen tension is another important regulator of MSC functions. In fact, Grayson and colleagues showed that, under a 2% O 2 hypoxia, not only the expression of stem cell genes Oct-4 and Rex-1 was up-regulated but also the synthesis of extracellular matrix (ECM) proteins, such as fibronectin, was increased (Grayson et al., 2006, 2007). Moreover, upon induction, levels of differentiation into mesenchymal tissue were increased at lower oxygen tensions (Wang et al., 2005; Grayson et al., 2006, 2007). Apart from the BM, local hypoxia can also occur in certain pathological conditions, for instance ischemia, infection or tissue injury, where MSC are believed to home and have a regenerative effect through a trophic activity (Caplan, 2007). Concomitantly with local nutrient depletion, the low oxygen level in an injured tissue creates a hostile microenvironment that may jeopardize the efficacy of MSC regenerative role. In fact, some authors demonstrated that the combination of hypoxia and serum deprivation induced MSC apoptosis, although the predominant effect was attributed to the absence of growth factors caused by serum deprivation (Zhu et al., 2006; Potier et al., 2007). Nevertheless, Rosova ´ and co-workers showed that hypoxic preconditioning improved the therapeutic potential of human MSC (Rosova et al., 2008). Thus, in order to develop more efficient protocols of cellular therapy using human MSC, it is crucial to perform more detailed studies of MSC performance on hypoxic microenvironments. *Correspondence to: Joaquim M.S. Cabral, IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Te ´cnico, Avenida Rovisco Pais 1049-001 Lisboa, Portugal. E-mail: joaquim.cabral@ist.utl.pt Received 3 August 2009; Accepted 16 October 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 17 December 2009. DOI: 10.1002/jcp.21987 ORIGINAL ARTICLE 27 Journal of Journal of Cellular Physiology Cellular Physiology ß 2009 WILEY-LISS, INC.