Fig. 5

Spontaneous differentiation of mES cells cultured on flat glass substrates. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without either a feeder layer or LIF from days 4 to 21. a Immunofluorescence for OCT4 (red), GATA4 (green), SOX-17 (green), NKX2.5 (green), α-SMA (red), collagen type I (red), osteocalcin (red), βIII-tubulin (green) and DAPI (blue) at days 3 and 21. The scale bar is 50 μm. b Phase contrast image of differentiated cells at day 19, showing a morphology resembling pacemaker-like cells (arrows). The scale bar is 100 μm. c Phase contrast image of beating cells (black arrows) surrounding two embryoid bodies at day 19 (cell beating is shown in the Additional files 2: Video 1 and Additional file 3: Video 2). The scale bar is 100 μm. d Quantification of the beating frequency by image processing