Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells.

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony-forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia-dependent migration of bone marrow-derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boyden's chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia-dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c-Jun N-terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor-1 (HIF-1)-dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia-dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF-1-dependent expression and release of VEGF.