Tra 1 60

Cells were washed with PBS and then immersed in 4% paraformaldehyde PBS and fixed for 10 min. Permeabilization Buffer (10×) from eBioscience (Vienna, Austria) was used instead of 0.1% Triton X-100 in PBS. Liquid 1% FBS added to PBS was used as an antibody diluent.
Immunofluorescence staining was performed using specific antibodies for Troponin T, Cardiac Isoform Ab-1 (Clone 13-11; Thermo Fisher Scientific K.K.), Anti-β-actin (C4; Santa Cruz Biotechnology) and Anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology). Cellstain^®-Hoechst 33342 solution (DOJINDO LABORATORIES, Kumamoto, Japan) was used for nuclear staining of the cells. Images were recorded using a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Since SeV is carried on the SeV vector, SeV-positive cells can be positively identified with a GFP light source without the need for fluorescently labelled antibodies. Tra-1-60 was then purchased from R&D Systems, Inc. and used for live-staining. After fluorescent staining, cells were detached with trypsin according to the usual cell detachment method and then counted after making cell suspensions with StemFit AK03 containing ROCK inhibitor (10 μM).

For AP staining, colonies were fixed with 90% alcohol for 2 min, washed three times with Tween-BST solution (PBS with 1% bovine serum albumin and 0.2% Tween-20) and then stained with BCIP/NBT (alkaline phosphatase substrate solution, Maxim Biotech) for 30 min in the dark. They were then washed with PBS twice and observed under the microscope.
For immunostaining, cells were fixed in 4.0% paraformaldehyde for 20 min, permeabilized with 0.5% Tween-20 in PBS, incubated with primary antibody overnight and then incubated with secondary antibodies (Invitrogen) for 1 h. Imaging was performed using an inverted confocal microscope. Primary antibodies used in this study were SSEA-4 (1:100, R&D), TRA-1-60 (1:200, R&D), OCT4 (1:500, R&D), SOX2 (1:500, R&D), AFP (1:500, R&D), nestin (1:100, R&D), SMA (1:500, R&D) and TNNT2 (1:500, R&D). 4, 6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining.