Tra 1 60

AP staining was performed according to the manufacturer's (Sidansai, China) instructions. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

Induced pluripotent stem cells (iPSCs) were generated as previously described (Holler et al., 2016 (link)). In brief, early passage fibroblast (Fig. S2C) using the following markers: Octamer-binding transcription factor 4 (Oct4; 1:400; Cell Signaling Technologies); Stage-Specific Embryonic Antigen 4 (SSEA; 1:500; Cell Signaling Technologies); Nanog homeobox (Nanog; 1:200; R&D Systems); Sex Determining Region Y-Box 2 (Sox2;1:200: ThermoScientific); Tra1–60 and Tra1–81 (1:150 each; Millipore). Correct chromosomal numbers were checked by karyotyping at WiCell (Madison, Wisconsin, USA; Fig. S2D).

Immunofluorescence (IF) staining, western blot analysis, and teratoma formation were performed as described previously [16 (link)]. Primary antibodies against the following proteins were used in this study: OCT4 (1:200), SSEA4 (1:200), TRA-1-60 (1:200), TG30 (1:200), HESCA-1 (1:200), HESCA-2 (1:200), MAP2 (1:500), CDX2 (1:200), and EOMES (1:200; Millipore, Temecula, CA, USA), PAX6 (1:50; DSHB, Iowa City, IA, USA), AFP (1:500; Dako, Glostrup, Denmark), SOX17 (1:200) and CG-α (1:500; R&D), GATA4 (1:200) and GATA2 (1:500; Santa Cruz, Dallas, TX, USA), a-SMA (1:500) and β-actin (1:800; Sigma), and CG-β (1:500) and NLRP2 (1:1,000; Abcam, Cambridge, UK). The following secondary antibodies were used: goat anti-mouse, rabbit Cy3 (1:500; Jackson ImmunoResearch laboratories, West Grove, PA, USA); and goat anti-mouse, rabbit 488 (1:200; Invitrogen).

Immunocytochemistry was performed as described previously13 (link). The primary antibodies used were P4HB (Acris Antibodies, USA), SSEA-4 (Millipore, USA), TRA-1-60 (Millipore, USA), and Nanog (Reprocell, Japan). Secondary antibodies were GFP anti-rabbit IgG (Molecular Probes, USA) to detect P4HB, Alexa Fluor 594 anti-mouse IgG (Molecular Probes) to detect SSEA-4 and TRA-1-60, and Alexa Fluor 488 anti-rabbit IgG (Molecular Probes) to detect Nanog.

Cells were fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature and incubated with primary antibodies against the following proteins: MeCP2 (1:200, Cell Signaling Technology), phalloidin (1:2000, Dyomics), NANOG (1:500, CosmoBio), OCT4 (1:200, Santa Cruz Biotechnology, Inc.), TRA-1-60 (1:1000, Millipore), TRA-1-81 (1:1000, Millipore), βIII-tubulin (1:2000, Sigma Chemical Co.), MAP2 (1:250, Sigma Chemical Co.), and GFAP (1:1000, Invitrogen). They were then washed with PBS and incubated with an Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated secondary antibody (1:500, Invitrogen), as appropriate. Images were obtained using an Axioplan 2 microscope (Carl Zeiss). The number of GFAP-positive cells was counted among 100 Hoechst-positive cells for each experiment (n = 5).

Cells were dissociated by treatment with 5 mM EDTA/DPBS for 3 min, followed by further treatment with a 0.05% trypsin/EDTA solution for 1 min. After two washes with DMEM/10% foetal bovine serum and one wash with staining buffer (0.1% bovine serum albumin/DPBS), 1 × 10⁵ cells were incubated for 30 min at 4 °C with primary antibodies diluted in staining buffer. The cells were then rinsed twice with staining buffer and incubated for 30 min at 4 °C with secondary antibodies diluted in staining buffer. Then, the cells were rinsed twice with staining buffer and counterstained with propidium iodide just prior to analysis. Fluorescence intensities were analysed on a FACSCanto flow cytometer (Becton Dickinson), and the FL-1 positive ratios in the propidium iodide-negative region were monitored using FACSDiva software. Primary antibodies against the following molecules were used: stage-specific embryonic antigen (SSEA)-3 [2 μg/ml; Developmental Studies Hybridoma Bank (DSHB)], SSEA-4 (1 μg/ml; DSHB), Tra-1-60 (1 μg/ml; Millipore), Tra-1-81 (1 μg/ml; Millipore), and SSEA-1 (2 μg/ml; DSHB). Anti-mouse Ig/FITC conjugated (10 μg/ml; Becton Dickinson) and anti-rat IgM/Alexa 488 conjugated (1 μg/ml; Molecular Probes) were used as secondary antibodies.