Pluripotency Evaluation of PBMC-hiPSCs

To examine the pluripotency of the PBMC-hiPSCs, immunocytochemistry was performed as described previously (Yamasaki et al.2014 (link)). Briefly, the cells fixed with 4% paraformaldehyde were stained with antibodies against Oct4 (MAB4401, mouse monoclonal, 1/200, Millipore), Tra-1-60 (09-0010, mouse monoclonal, 1/200, Stemgent®, Cambridge, MA), and SSEA-4 (MC 813-70, mouse monoclonal, 1/100, R&D Systems Minneapolis, MN), and differentiated cells were stained with antibodies against βIII-tubulin (MAB3408/1637, mouse monoclonal, 1/300, Chemicon, Burlington, MA), α-smooth muscle actin (N1584, mouse monoclonal, pre-diluted, DAKO Cytomation, Glostrup, Denmark), and α-fetoprotein (MAB1368, mouse monoclonal, 1/100, R&D Systems) (Table 1). These primary antibodies were visualized with Alexa Fluor® 488-conjugated goat anti-mouse IgG (A11001, 1/300, Invitrogen, Carlsbad, CA). The cell nuclei and double-stranded DNA were stained with 4′, 6-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescence images were acquired using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Oberkochen, Germany).