L

L. in the generation and maintenance of PSCs. differentiation of ES cells grown under different culture conditions was performed by embryoid body formation. ES cell serum-free neural differentiation (5% knockout serum replacement medium) and unbiased differentiation in serum-containing medium (10% FBS) were performed as described previously (29,C31). Briefly, ES cells were dissociated into single cells after treatment with 0.05% trypsin-EDTA at 37 C for 2 min. Single cells were aggregated in Petri dishes at a density of 1 1 105 cells/ml in differentiation medium. Differentiation day 0 indicates the day on which the ES cells were seeded to differentiate. Embryoid bodies were collected every other day and analyzed by quantitative reverse-transcription PCR (qRT-PCR). AP Staining and Immunofluorescence Staining AP staining was performed as described previously (30). Briefly, cells were washed with PBS three times and stained with BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) (Promega) for 10 min. For immunofluorescence staining, samples were washed once with PBS and fixed with a 4% pure formaldehyde solution (Sigma-Aldrich) in PBS for 20 min, followed by two washes with PBS. Blocking and permeabilization were done with a 3% BSA and 0.3% Triton X-100 (Sigma-Aldrich) solution in PBS for 1 h at room temperature. All primary antibodies were diluted in 0.3% Triton X-100 and incubated overnight at 4 C. After 1 h of washing with PBS, samples were incubated with Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen) for 2 h at room temperature. The following primary antibodies were used. Mouse monoclonal antibodies included anti-Oct4 (1:200, Santa Cruz Biotechnology), anti-Cytokeratin18 (Ck18) (1:200, Abcam), anti-SSEA-1 (1:200, Santa Cruz Biotechnology), and anti-Tuj1 (1:500, Covance). The rabbit polyclonal antibody was anti-Nanog (1:400, Cell Signaling Technology). The goat polyclonal antibodies were anti-T (1:200, Santa Cruz Biotechnology) and anti-Gata6 (10 g/ml, R&D Systems). Western Blotting Western blotting was performed as described previously (32, 33). Briefly, cells were lysed in cell lysis buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.5% NaDOC, 0.1% SDS, 1% Nonidet P-40, 5 mm EDTA, 0.25 mm phenylmethanesulfonyl fluoride, and a mixture of protease inhibitors. Lysate samples were boiled for 10 min, separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) ELN-441958 BSA in PBS at room temperature for 1 h and incubated with primary antibodies at room temperature for a further 1 h. The following primary antibodies were used. Anti-phosphorylated Smad (pSmad)1/5/8 (1:2000, Cell Signaling Technology), anti-Smad1 (1:2000, Cell Signaling Technology), anti-pERK1/2 (1:1000, Cell Signaling Technology), anti-ERK1/2 (1:3000, Santa Cruz Biotechnology), anti-pSmad2 (1:2000, Cell Signaling Technology), anti-Smad2 (1:2000, Cell Signaling Technology), anti-Tubulin (1:10,000, KangChen Bio-Tech), and anti-GADPH (1:10,000, KangChen Bio-Tech). After three washes with 0.1% Tween in PBS (PBST), the membrane was incubated with HRP-conjugated IgG for 1 h at room temperature. The membrane was washed again with PBST. Antibody-reacted proteins were visualized using ECL detection reagents (Tanon). All images were taken using a Tanon-5500 ELN-441958 chemiluminescence imaging system (Tanon). Gene Knockdown in MEFs Lentiviral vector pSilencer-expressing shRNA was used for knockdown in MEFs. The shRNA target sequences were as follows: shRNA-1, 5-AAAGTAACTGTAGGACGAA-3; shRNA-2, 5-GTTTGGAGAAGTTTGGCGA-3. Lentiviral packaging and lentiviral ELN-441958 transfection were performed as described above. Briefly, 293T cells were transfected with pSilencer shRNAs with the packaging plasmids VSVG and 8.9. Viruses were collected by ultracentrifugation 72 h after transfection. 2 105 MEFs were plated on a Matrigel-coated dish and infected with viral supernatants (multiplicity of infection = 4). RNA Preparation and qRT-PCR Analysis Total RNA was extracted from cultured cells using TRIzol (Invitrogen) reagent. One microgram of RNA was reverse-transcribed using SuperScript reverse transcriptase (Invitrogen). qRT-PCR was performed with JumpStart Taq ReadyMix (Sigma-Aldrich). Reactions were carried out in triplicate using 1/40 concentration of the cDNA obtained as described above. The expression level of the transcript in each sample was normalized to test. RESULTS Inhibition of TGF- Signaling Promotes the Generation of iPSCs To obtain iPSCs from differentiated cells under serum-free conditions, dox-inducible Oct4, Sox2, Klf4, and c-Myc (OSKM) were transduced into MEFs by lentiviral vectors. Three days after transduction, one group of cells was cultured in serum-free N2B27 medium containing PD03, CHIR, and VPA (VPA/PD03/CHIR, VPC) (34). The other group of cells was grown in serum-containing medium supplemented with VPA (serum/VPA) as the control. 24 days after induction, many AP-positive colonies were observed in the control serum/VPA Mmp10 medium, but AP-positive colonies were barely detectable under VPC conditions (Fig. 1, and < 0.05; Student's ELN-441958 test. < 0.05; Student's test. < 0.05; Student's ELN-441958 test. = 100.