Abstract 15945: An Immunosuppressive Drug, Cyclosporin A Induces Cardiac Differentiation of P19 Embryonic Stem Cells Through Activation of BMP Pathway and Inhibition of TGF-{beta}-Activin Pathway

Background: An Immunosuppressive Drug, Cyclosporin A (CsA) is known to have pleiotropic effects to the proliferation, differentiation and survival of somatic cells as well as stem cells. However, little is known about the mechanisms underlying CsA-induced cardiac differentiation of stem cells. Therefore, the effects of CsA on cardiac differentiation of P19 embryonic stem cells were investigated. Methods: P19 cells were treated with different concentrations of CsA during embryoid body formation in bacterial dishes for 4 days and further cultured on culture dishes for 6 days to induce cardiac differentiation. Results: CsA concentrations ranging from 0.078 to 1.25 μM, with peak value at 0.3125 μM induced specifically cardiac differentiation whereas differentiation into smooth muscle, endothelial, osteogenic, adipogenic and ectodermal cell lineages was not observed in those concentrations of CsA. Furthermore, beating cells were observed in the CsA-treated cells at day 10 of differentiation but not the control group. In real-time PCR and Western blotting experiment, expression of Wnt5a and Wnt8a was significantly upregulated from 48 h to 96 h in the CsA-treated P19 cells but no changes of Wnt2a, Wnt3a, Wnt7a, Wnt7b, Wnt9a and Wnt11. However, WNT/s-catenin signaling inhibitors niclosamide, SB216763 and BIO did not affect the CsA-induced cardiac differentiation of P19 cells. BMP pathway molecules such as Bmp2, Bmp4, Smad1, Smad5, p-Smad1/5 and Smad8 were upregulated in the CsA-treated P19 cells, and a BMP pathway inhibitor dorsomorphin blocked the CsA-induced cardiac differentiation of P19 cells. Interestingly, significant reduction of TGF-β-activin pathway Smads (Smad2 and Smad3) and inhibitory Smads (Smad6 and Smad7) was found in the CsA-treated P19 cells. ERK1/2, PI3K-Akt, JAK-STAT3 pathway molecules were not affected in the CsA-treated P19 cells. Conclusions: Taken together, this result demonstrates that CsA induces the cardiac differentiation of P19 cells via activation of BMP pathway and inhibition of TGF-β-activin pathway.