P19 embryonal carcinoma stem cells have been used as a model system in studies on the molecular mechanisms underlying cardiomyogenic differentiation. Recent studies have shown that the immunosuppressive drug, Cyclosporin A(CsA) would affect cell fate, proliferation and survival. However, little is known about the effects of CsA on the fate of stem cells. Here, we investigated the effects of CsA on the cardiac differentiation of P19 embryonal carcinoma stem cells. P19 cells were treated with different concentrations of CsA in the absence or presence of 1% dimethyl sulfoxide(DMSO) during embryoid body formation in bacterial dishes. Alternatively, different concentrations of CsA were also added to the preformed embryoid bodies which were treated without or with 1% DMSO during embryoid body formation. Quantitative real-time PCR analysis showed that exposure of embryoid bodies to concentrations in the range from 0.15 to 0.3 µM CsA caused about a 1.5- and 3.2-fold increase of GATA4 and α-myosin heavy chain(α-MHC) mRNA expression in the absence of DMSO during embryoid body formation, respectively. Combined treatment of both CsA and DMSO to P19 cells during embryoid body formation caused a 1.4- and 2.4-fold increase of GATA4 and α-MHC mRNA expression compared with only DMSO-treated group during embryoid body formation, showing the synergistic effect on the combined use of DMSO and CsA. The expressions of the bone morphogenetic protein(BMP) and Wnt signaling molecules were modulated in CsA-treated P19 cells. The increased expression of cardiac markers in CsA-treated P19 cells during embryoid body formation was also confirmed by immunostaining. Furthermore, beating cells were observed in CsA-treated cells during embryoid body formation at day 10 of differentiation, but not in the control group. However, enhancement of cardiac differentiation was not observed when CsA was added to the preformed embryoid bodies without or with 1% DMSO. This study demonstrates that CsA induces cardiac differentiation of P19 cells during embryoid body formation, depending in part on alteration of the BMP and Wnt signaling molecules.
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