Abstract
BACKGROUND: Mesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca(2+)) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs. METHODS: hUCB-MSCs were maintained under hypoxic conditions (3% O(2)) with 1.8 mM Ca(2+) during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs. RESULTS: The proliferation of Ca(2+)/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca(2+) and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca(2+)/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca(2+)/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs. CONCLUSIONS: These findings suggest that a Ca(2+)/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.