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Human mesenchymal stem cells (hMSCs) derived from adult bone marrow are multipotent cells shown to have increased differentiation potential when cultured in reduced-oxygen (2% O2) environments. Hepatocyte growth factor (HGF) is a mesodermal cytokine critical for organ formation during embryogenesis, including cardiogenesis. In this work, we tested the hypothesis that culturing hMSCs in reduced O2 conditions with media supplemented with HGF increases the expression of cardiac myocyte markers. RT-PCR demonstrated expression of myocyte specific alpha-Actinin (ACTN2) and myocyte enhancement factor 2C (MEF2C) in reduced-O2/HGF+ treated cells. Real time PCR showed an 11-fold increase in ACTN2 and 2-fold increase in MEF2C in reduced-O2/HGF+ treated cells compared to hMSCs cultured in atmospheric-O2/HGF-conditions. These results suggest that the combination of low oxygen and HGF may increase the cardiac differentiation capabilities of hMSCs.