Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells

Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.

Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.

After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation. Conclusions: Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.