Fig. 2.

Characterization of undifferentiated mESC line E14TG2A, embryoid body formation and cardiac differentiation. a mESCs in culture. b Karyotype analysis showing 40 chromosomes. c Expression of undifferentiated mESC transcription factors by RT-PCR: Oct3/4, Nanog and Sox-2. d–g Expression of pluripotency markers in mESCs by immunofluorescence: cells positive for d SSEA-1 (green) and e Oct3/4 (red); f nuclei labeled with Topro (blue); and g merged image. h, i Embryoid bodies (EBs) in suspension after 2 and 5 days of differentiation. j Adhered EBs after 8 days of differentiation with beating cells. k–n Flow cytometry analysis on day 14: representative graphs of k DAPI-positive differentiated cells used to isolate permeabilized cells only, l differentiated cells stained only with the secondary antibody and m cardiac troponin T-positive differentiated cells; and n histogram overlay of differentiated cells stained with secondary antibody (gray) and cells expressing cardiac troponin t (blue). o Representative trace of ventricular action potential. p Action potential duration at 30, 50, 70 and 90% of repolarization (APD30, APD50, APD70 and APD90, respectively; n = 8). Scale bars: a 50 μm, d–g 20 μm. mESC mouse embryonic stem cell, DAPI 4′,6-diamidino-2-phenylindole, FSC forward scatter, SSC side scatter