(a) Brightfield and fluorescence microscopy of a representative beating embryoid body at day 14 of differentiation. (b) Immunostaining of isolated single cells confirms the expression of cardiac specific markers such as troponin-T and connexin-43. Green, GFP; Red, TRITC-antibody stain; Blue, DAPI-stained nuclei. (c) Histology of ES cells injected subcutaneously showing in vivo differentiation into striated cardiomyocytes. (d) RT-PCR comparison of Bi-Tet (−Dox) and Bi-Tet (+Dox) cells at day 0 and day 14 of differentiation. There is no significant difference in expression of ES cell (Oct4), endoderm (AFP), mesoderm (Flk-1), ectoderm (Ncam), and cardiac specific markers (MLC2v, Nkx2.5, β-MHC) between the 2 groups, suggesting that reporter gene and Bi-Tet systems do not affect ES cell pluripotency.