Pluripotency Assessment of hiPSCs

We studied the pluripotency of hiPSCs by immunocytochemical analyses described previously (Yamasaki et al. 2014 (link)). Briefly, the cells were fixed with 4% paraformaldehyde (PFA) and stained with primary antibodies against OCT4 (diluted 1/200; MAB4401, mouse monoclonal, Millipore), Tra-1-60 (diluted 1/200; 09-0010, mouse monoclonal, Stemgent®, Cambridge, MA), and SSEA-4 (diluted 1/100; MC 813-70, mouse monoclonal, R&D Systems Minneapolis, MN), and the differentiated cells were stained with antibodies against β-III tubulin (diluted 1/300; MAB3408/1637, mouse monoclonal, Chemicon), α-smooth muscle actin (N1584, mouse monoclonal, pre-diluted, DAKO Cytomation, Glostrup, Denmark), and α-fetoprotein (diluted 1/100; MAB1368, goat polyclonal, R&D Systems) (Table 3). These primary antibodies were visualized with secondary antibodies conjugated with Alexa Fluor® 488 (diluted 1/300; 11,001, mouse monoclonal, Invitrogen, Carlsbad, CA). The cell nuclei and double-stranded DNA were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescence images were captured using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss GmbH, Jena, Germany).