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Stem cells transplanted to the ischemic myocardium usually encounter massive cell death within a few days of therapy. Hypoxic preconditioning (HPC) is currently employed as a strategy to prepare stem cells for increased survival and engraftment in the heart. However, HPC of stem cells has provided varying results, supposedly due to the differences in the oxygen concentration, duration of exposure, and passage conditions. In the present study, we determined the effect of HPC on rat mesenchymal stem cells (MSCs) exposed to 0.5% oxygen concentration for 24, 48, or 72 h. We evaluated the expression of prosurvival, proangiogenic, and functional markers such as hypoxia-inducible factor-1α, VEGF, phosphorylated Akt, survivin, p21, cytochrome c, caspase-3, caspase-7, CXCR4, and c-Met. MSCs exposed to 24-h hypoxia showed reduced apoptosis on being subjected to severe hypoxic conditions. They also had significantly higher levels of prosurvival, proangiogenic, and prodifferentiation proteins when compared with longer exposure (72 h). Cells taken directly from the cryopreserved state did not respond effectively to the 24-h HPC as those that were cultured under normoxia before HPC. Cells cultured under normoxia before HPC showed decreased apoptosis, enhanced expression of connexin-43, cardiac myosin heavy chain, and CD31. The preconditioned cells were able to differentiate into the cardiovascular lineage. The results suggest that MSCs cultured under normoxia before 24-h HPC are in a state of optimal expression of prosurvival, proangiogenic, and functional proteins that may increase the survival and engraftment in the infarct heart. These results could provide further insights into optimal preparation of MSCs which would greatly influence the effectiveness of cell therapy in vivo.

Hypoxic preconditioning induces the expression of prosurvival and proangiogenic markers in mesenchymal stem cells