The L-type Ca2+ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells
(A) RT-PCR for L-type Ca2+ channel α-subunit isoforms in undifferentiated ES cells and CMs at day 12 of differentiation, demonstrating (i) presence of L-type Ca2+ channel mRNA even in undifferentiated ES cells and (ii) an increase in mRNA encoding the CaV1.2 subunit of the channel. (B) RT-PCR analyses of cardiac-specific and transcription factors α-MHC/Myh6, ANF/Nppa, MLC2v/Myl2, NKx2.5 and GATA4 in beating cluster at day 12 of both ES and iPS differentiation. (C) RT-PCR analyses of representative markers, PECAM-1 (mesoderm, endothelial cell), AFP (endoderm, early hepatocyte progenitor), Nestin and NF-H (ectoderm, neural progenitor) and α-SMA (mesoderm) in ES and iPS beating cluster at day 12 of differentiation. (D) Quantitative real-time PCR analyses of mesoderm and cardiac markers and specific transcript (Brachyury T, Mesp1, NKx2.5, Tbx5, Mest, Myocd, Myh6/α-MHC, Myl2/MLC2v, and Nppa/ANF), as well as cardiac ionic channels (CACNA1c, KCNH2 and SCN5A) expression at different stage of ES cell differentiation. GAPDH was used as housekeeping gene and served to normalize the result. Results are reported as the means±SEM (n = 3). *P<0.05 vs. control CMs.