Introduction: The capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has\r\ndrawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow\r\nstromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on\r\nhow BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic\r\ndevelopment pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.\r\nMethods: Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under\r\neither hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay\r\nwas used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were\r\ncharacterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells.\r\nBMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free\r\nchondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was\r\ncharacterized by biochemical and histological methods and by quantitative gene-expression analysis.\r\nResults: After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8%\r\nto 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29,\r\nCD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet\r\nculture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC\r\npellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining,\r\nalong with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA\r\nexpression of hypoxia inducible factor-2 alpha (HIF-2a) but suppressed the mRNA expression of collagen X in\r\nBMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.\r\nConclusions: Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic\r\nconditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and\r\nexpansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.
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