Low oxygen tension modulates the osteogenic differentiation of mouse embryonic stem cells

This study examined the effects of low oxygen tension on the osteogenic differentiation of embryonic stem cells (ESCs) in a three-dimensional culture system. The high expression levels of hypoxia-related proteins hypoxia-inducible factor-1α and vascular endothelial growth factor were first validated in ESCs subjected to hypoxic conditions compared with normoxic controls. The osteogenic differentiation of hypoxic ESCs with either osteogenic or osteogenic factor-free media was subsequently evaluated by measuring alkaline phosphatase activity, intracellular calcium levels, matrix mineralization, and the protein levels of osteogenic markers Runt-related transcription factor 2 and osterix. We confirmed that hypoxia significantly stimulated ESC osteogenic activity; the strongest stimulation of ESC osteogenesis was exerted when cells were grown in osteogenic media. To identify differentially expressed genes associated with hypoxia-induced ESC differentiation, we performed microarray analysis of ESCs cultured in osteogenic media under normoxic and hypoxic conditions. This study demonstrated that differences in oxygen tension induced the differential expression of genes known to play roles in such processes as skeletal system development and signaling pathways for bone morphogenetic protein, Wnt, Notch, mitogen-activated protein kinase, and integrin. These findings reveal the effects of low oxygen tension on osteogenic progression in ESCs and provide insight into the molecular pathways that regulate ESC differentiation following exposure to hypoxia.