Triple solution

Reprogrammed iPS cells were collected separately by digestion with TripLE solution (Invitrogen). We used 2% paraformaldehyde containing 0.05% Triton X-100 to fix and obtain permeabilized cells. After that, we stained the iPS cells with rabbit or mouse anti-human Oct4, Nanog, Sox2, SSEA-4 and TRA-1-60 antibodies (Stemgent, San Diego, CA) for analysis on FC500 flow cytometry (Beckman, Fullerton, CA) using CXP analysis software (Beckman). We assumed rabbit or mouse isotypic antibodies as negative controls respectively.

Individual cells were isolated from the contracticle cluster by digesting with the TripLE solution (Invitrogen). Then 2% formaldehyde with 0.05% Triton x-100 was used to fix and permeate cells. Cells were stained with rabbit or mouse anti-human Troponin T (Millipore), MLC-2a (Synaptic, Göttingen, Germany), and MLC-2v antibodies (Synaptic), and analyzed by CXP analysis software (Beckman Coulter). Rabbit or mouse isoforms were used as negative controls, respectively.

For embryoid body (EB) formation, after dissociating the putative human iPS colonies into individual cells by TripLE solution (Invitrogen), drops of 1000 cells were suspended in 20 μl culture medium containing no bFGF on petri dish lids for 48 h. The embryoid bodies were thereafter suspended in DMEM medium containing 10% FBS, 1 NEAA and 1 mM L-glutamine for 5 days, and then plated in a gelatin-coated 12-well plate in the same medium for another 7 days. The embryoid bodies were then settled and restained with a human embryonic germination characterization kit (Chemicon, Rolling Meadows, IL).