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Hypoxic culture has been shown to delay premature senescence occurring during in vitro culture. Human mesenchymal stem cells (hMSCs) cultured under hypoxia have been reported to maintain their stemness properties and delay senescence compared to the cells cultured under normoxia. However, the molecular mechanism by which hypoxia regulates premature senescence has not been fully revealed. In this study, hMSCs were cultured under the conditions of 21% (normoxia) and 1% O2 (hypoxia) tension and analyzed for cell growth, expression of MSC surface markers, multilineage differentiation, and cellular senescence. Our results showed that more cells retained MSC surface markers in hypoxic culture than those in normoxic culture, and hypoxia was able to enhance multilineage differentiation of hMSCs. The hypoxic condition also delayed cellular senescence of hMSCs, increased activation of AKT signaling, and upregulated both intra- and extracellular levels of macrophage migration inhibitory factor (MIF) compared to the normoxic condition. Inhibition of AKT activity in hypoxic culture increased the number of cells with positive staining for senescence-associated β-galactosidase activity, upregulated expression levels of senescence-associated markers p16 and p21 mRNA transcripts, and decreased expression levels of potency-associated markers, including NANOG, OCT3/4, and SOX2. On the other hand, upregulated intra- and extracellular levels of MIF by stable MIF overexpression in normoxic culture increased the activation of AKT while decreasing mRNA expression of senescence-associated markers and increasing expression of potency-associated markers. Taken together, our findings suggest that hMSCs in hypoxic culture produce endogenous MIF to activate AKT signaling to delay the progression of cellular senescence.

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