Neonatal human epidermal keratinocytes were transduced and cultured with two factor mixtures of lentiviruses encoding human Oct4, mouse Klf4 and Sox2 Ganetespib distributor as previously described. Lentiviral vectors were obtained from Addgene. A day later, 1 105 transduced NHEKs were seeded on the irradiated x ray inactivated CF1 MEF feeder cells in a 100 mm dish by keratinocyte method. Seven days after, the medium was altered to human ES cell medium: DMEM/F12, 2006-2007 Knock-out serum alternative, 1% GlutaMAX, 1% non-essential amino acids, 1% penicillin/streptomycin, 0. 1 mM w mercaptoethanol, and 100 ng/ml basic fibroblast growth factor and treated with GSK 3 inhibitor CHIR99021 alone or mixed with SB431542, BIX 01294, RG108, Parnate, PD0325901, and valproic acid. The media containing the above little molecule combinations were changed each day. A couple of weeks after-treatment, the cells were subcultured on new feeder cells. After still another 2 weeks, the little molecules were removed and the cells were stained with Alexa Fluor 555 conjugated mouse anti Cholangiocarcinoma human TRA 1 81 antibody. The colonies were marked and picked up for expansion on feeder cells in human ES cell medium about 7 days after transduction. The human iPS cells were subcultured regularly by Accutase. All cell culture products were from Invitrogen/Gibco BRL except where mentioned. Cytochemistry and Immunofluorescence Assay Alkaline phosphatase staining was performed based on the manufacturers protocol using the Alkaline Phosphatase Detection Kit. For immunofluorescence analysis, cells were fixed in 4% paraformaldehyde for 10 minutes and washed 3 times with phosphate buffered saline containing 0. One of the Triton X 100. The fixed cells were then incubated in blocking buffer, 0. 1000 Triton X ten percent and 100 regular donkey serum in PBS, for half an hour at room temperature. PFT The cells were then incubated with primary antibody over night at 4 C in blocking buffer. The day after, cells were washed with PBS and incubated with secondary antibody in PBS containing 0. Hands down the Triton X 100 for 1 hour at room temperature. Mouse anti Oct4 antibody, rabbit anti Sox2 antibody, mouse anti SSEA1 antibody, rabbit anti Nanog antibody, rat anti SSEA3 antibody, mouse anti SSEA4 antibody, mouse anti TRA 1 81 antibody, goat anti Sox17, mouse antibIII Tubulin antibody, and rabbit anti Brachyury antibody were employed as primary antibodies. Secondary antibodies were Alexa Fluor 486/ 555 donkey anti mouse, anti rat, anti goat, or anti rabbit IgG. Nuclei were visualized by 40,6 diamidino 2 phenylindole staining. Pictures were taken using a Nikon Eclipse TE2000 U microscope. Differentiation of iPS Cells In Vitro The in vitro differentiation of miPSCs OK and hiPSCs OK was carried out by the conventional embryoid body differentiation technique. The iPS cells were dissociated by both 0.