Background. The interests in mesenchymal stem cells (MSCs) and their application in cell therapy have resulted in a better understanding of the basic biology of these cells. Recently hypoxia has been indicated as crucial for complete chondrogenesis. We aimed at analyzing bone marrow MSCs (BM-MSCs) differentiation capacity under normoxic and severe hypoxic culture conditions. Methods. MSCs were characterized by flow cytometry and differentiated towards adipocytes, osteoblasts, and chondrocytes under normoxic or severe hypoxic conditions. The differentiations were confirmed comparing each treated point with a control point made of cells grown in DMEM and fetal bovine serum (FBS). Results. BM-MSCs from the donors displayed only few phenotypical differences in surface antigens expressions. Analyzing marker genes expression levels of the treated cells compared to their control point for each lineage showed a good differentiation in normoxic conditions and the absence of this differentiation capacity in severe hypoxic cultures. Conclusions. In our experimental conditions, severe hypoxia affects the in vitro differentiation potential of BM-MSCs. Adipogenic, osteogenic, and chondrogenic differentiations are absent in severe hypoxic conditions. Our work underlines that severe hypoxia slows cell differentiation by means of molecular mechanisms since a decrease in the expression of adipocyte-, osteoblast-, and chondrocyte-specific genes was observed. 1. Introduction Mesenchymal stem cells (MSCs) are multipotent cells that can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into multiple lineages [1–5]. These cells are located in bone marrow (BM), around blood vessels, in fat, skin, muscle, and other tissues, and their presence contributes to the reparative capacity of these tissues. MSCs from different tissue sources can have biologic distinctions. In this way, MSCs derived from bone marrow show a higher potential for osteogenic differentiation , while MSCs of synovial origin show a greater tendency toward chondrogenic differentiation . Moreover, under identical culture conditions of differentiation, MSCs isolated from the synovial membrane show more chondrogenic potential than those derived from bone marrow, periosteum, skeletal muscle, or adipose tissue . The recent use of autologous or allogenic stem cells has been suggested as an alternative therapeutic approach for treatment of cartilage defects , with these cells representing a promising resource for different tissue engineering and cell-based therapies
S. Hombach-Klonisch, S. Panigrahi, I. Rashedi et al., “Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications,” Journal of Molecular Medicine, vol. 86, no. 12, pp. 1301–1314, 2008.
G. Pasquinelli, P. Tazzari, F. Ricci et al., “Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta,” Ultrastructural Pathology, vol. 31, no. 1, pp. 23–31, 2007.
M. Rydén, A. Dicker, C. G？therstr？m, et al., “Functional characterization of human mesenchymal stem cell-derived adipocytes,” Biochemical and Biophysical Research Communications, vol. 311, no. 2, pp. 391–397, 2003.
A. Muraglia, R. Cancedda, and R. Quarto, “Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model,” Journal of Cell Science, vol. 113, no. 7, pp. 1161–1166, 2000.
F. Djouad, C. Bony, T. H？upl et al., “Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells,” Arthritis Research & Therapy, vol. 7, no. 6, pp. R1304–R1315, 2005.
Y. Sakaguchi, I. Sekiya, K. Yagishita, and T. Muneta, “Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source,” Arthritis and Rheumatism, vol. 52, no. 8, pp. 2521–2529, 2005.
M. Sato, K. Uchida, H. Nakajima et al., “Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis,” Arthritis Research and Therapy, vol. 14, article R31, 2012.
G. P. Lasala, J. A. Silva, B. A. Kusnick, and J. J. Minguell, “Combination stem cell therapy for the treatment of medically refractory coronary ischemia: A Phase I study,” Cardiovascular Revascularization Medicine, vol. 12, no. 1, pp. 29–34, 2011.
J. S. Lee, J. M. Hong, G. J. Moon et al., “A long-term follow-up study of intravenous autologous mesenchymal stem cell transplantation in patients with ischemic stroke,” Stem Cells, vol. 28, no. 6, pp. 1099–1106, 2010.
N. K. Satija, V. K. Singh, Y. K. Verma et al., “Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine,” Journal of Cellular and Molecular Medicine, vol. 13, no. 11-12, pp. 4385–4402, 2009.
K. L. Talks, H. Turley, K. C. Gatter et al., “The expression and distribution of the hypoxia-inducible factors HIF-1α and HIF-2α in normal human tissues, cancers, and tumor-associated macrophages,” American Journal of Pathology, vol. 157, no. 2, pp. 411–421, 2000.
A. J. Giaccia, M. C. Simon, and R. Johnson, “The biology of hypoxia: the role of oxygen sensing in development, normal function, and disease,” Genes and Development, vol. 18, no. 18, pp. 2183–2194, 2004.
S. Carrancio, N. López-Holgado, F. M. Sánchez-Guijo et al., “Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification,” Experimental Hematology, vol. 36, no. 8, pp. 1014–1021, 2008.
M. Kanichai, D. Ferguson, P. J. Prendergast, and V. A. Campbell, “Hypoxia promotes chondrogenesis in rat mesenchymal stem cells: a role for AKT and hypoxia-inducible factor (HIF)-1α,” Journal of Cellular Physiology, vol. 216, no. 3, pp. 708–715, 2008.
E. J. Koay and K. A. Athanasiou, “Hypoxic chondrogenic differentiation of human embryonic stem cells enhances cartilage protein synthesis and biomechanical functionality,” Osteoarthritis and Cartilage, vol. 16, no. 12, pp. 1450–1456, 2008.
W. L. Grayson, F. Zhao, R. Izadpanah, B. Bunnell, and T. Ma, “Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs,” Journal of Cellular Physiology, vol. 207, no. 2, pp. 331–339, 2006.
M. Dominici, K. Le Blanc, I. Mueller et al., “Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement,” Cytotherapy, vol. 8, no. 4, pp. 315–317, 2006.
B. Johnstone, T. M. Hering, A. I. Caplan, V. M. Goldberg, and J. U. Yoo, “In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells,” Experimental Cell Research, vol. 238, no. 1, pp. 265–272, 1998.
S.-P. Hung, J. H. Ho, Y.-R. V. Shih, T. Lo, and O. K. Lee, “Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells,” Journal of Orthopaedic Research, vol. 30, no. 2, pp. 260–266, 2012.
J. C. Robins, N. Akeno, A. Mukherjee et al., “Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9,” Bone, vol. 37, no. 3, pp. 313–322, 2005.
C. Holzwarth, M. Vaegler, F. Gieseke et al., “Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells,” BMC Cell Biology, vol. 11, article 11, 2010.
D. C. Chow, L. A. Wenning, W. M. Miller, and E. T. Papoutsakis, “Modeling pO2 distributions in the bone marrow hematopoietic compartment. I. Krogh's model,” Biophysical Journal, vol. 81, pp. 675–684, 2001.
D. C. Chow, L. A. Wenning, W. M. Miller, and E. T. Papoutsakis, “Modeling pO2 distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models,” Biophysical Journal, vol. 81, no. 2, pp. 685–696, 2001.
A. Lavrentieva, I. Majore, C. Kasper, and R. Hass, “Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells,” Cell Communication and Signaling, vol. 8, article 18, 2010.
M. G. Valorani, A. Germani, W. R. Otto et al., “Hypoxia increases Sca-1/CD44 co-expression in murine mesenchymal stem cells and enhances their adipogenic differentiation potential,” Cell and Tissue Research, vol. 341, no. 1, pp. 111–120, 2010.
A. A. M. van Oorschot, A. M. Smits, E. Pardali, P. A. Doevendans, and M.-J. Goumans, “Low oxygen tension positively influences cardiomyocyte progenitor cell function,” Journal of Cellular and Molecular Medicine, vol. 15, no. 12, pp. 2723–2734, 2011.
S. Wang, Y. Zhou, C. N. Seavey et al., “Rapid and dynamic alterations of gene expression profiles of adult porcine bone marrow-derived stem cell in response to hypoxia,” Stem Cell Research, vol. 4, no. 2, pp. 117–128, 2010.
D. P. Lennon, J. M. Edmison, and A. I. Caplan, “Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis,” Journal of Cellular Physiology, vol. 187, no. 3, pp. 345–355, 2001.
S. Parrinello, E. Samper, A. Krtolica, J. Goldstein, S. Melov, and J. Campisi, “Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts,” Nature Cell Biology, vol. 5, no. 8, pp. 741–747, 2003.
G. D'Ippolito, S. Diabira, G. A. Howard, B. A. Roos, and P. C. Schiller, “Low oxygen tension inhibits osteogenic differentiation and enhances stemness of human MIAMI cells,” Bone, vol. 39, no. 3, pp. 513–522, 2006.
P. Malladi, Y. Xu, M. Chiou, A. J. Giaccia, and M. T. Longaker, “Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells,” American Journal of Physiology, vol. 290, no. 4, pp. C1139–C1146, 2006.
T. Ezashi, P. Das, and R. M. Roberts, “Low O2 tensions and the prevention of differentiation of hES cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 13, pp. 4783–4788, 2005.
C. Fehrer, R. Brunauer, G. Laschober et al., “Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan,” Aging Cell, vol. 6, no. 6, pp. 745–757, 2007.
H. Ren, Y. Cao, Q. Zhao, et al., “Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions,” Biochemical and Biophysical Research Communications, vol. 347, no. 1, pp. 12–21, 2006.
D. W. Wang, B. Fermor, J. M. Gimble, H. A. Awad, and F. Guilak, “Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells,” Journal of Cellular Physiology, vol. 204, no. 1, pp. 184–191, 2005.
I. Papandreou, R. A. Cairns, L. Fontana, A. L. Lim, and N. C. Denko, “HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption,” Cell Metabolism, vol. 3, no. 3, pp. 187–197, 2006.
F. dos Santos, P. Z. Andrade, J. S. Boura, M. M. Abecasis, C. L. da Silva, and J. M. S. Cabral, “Ex vivo expansion of human mesenchymal stem cells: a more effective cell proliferation kinetics and metabolism under hypoxia,” Journal of Cellular Physiology, vol. 223, no. 1, pp. 27–35, 2010.
S. Ohnishi, T. Yasuda, S. Kitamura, and N. Nagaya, “Effect of hypoxia on gene expression of bone marrow-derived mesenchymal stem cells and mononuclear cells,” Stem Cells, vol. 25, no. 5, pp. 1166–1177, 2007.