Cell study

Materials used during this study included the following: D3 mESCs (ATCC); ES-qualified 0.1% gelatin (Embryomax, Millipore); D3 growth medium consisted of Dulbecco’s Modification of Eagle’s Medium (DMEM) (Corning) prepared with sodium bicarbonate (Sigma), supplemented with 10% ES-qualified fetal bovine serum (Millipore), 10⁻⁴ M β-mercaptoethanol (Millipore Embryomax), 4 mM L-glutamine (Life Technologies), 4.7 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hyclone GE Healthcare), and 1000 units/mL human recombinant leukemia inducing factor (GlobalStem); trypsin-EDTA (Sigma Aldrich); flow cytometry antibodies: anti-SSEA1 (BioLegend 125606) and isotype control (BioLegend 401611); Neural differentiation medium consisted of 70% DMEM/F-12 (Corning), 20% neurobasal medium (Life Technologies), 1X N2 supplement, 20% PluriQ serum replacement (GlobalStem), 10⁻³ M sodium pyruvate (Sigma), 2 mM L-glutamine (Life Technologies), and 2 μM retinoic acid (Sigma). TRIzol reagent (Life Technologies); cDNA Synthesis Kit (Quantabio), SYBR Green (Quantabio). Paraformaldehyde (Fisher Scientific); Triton-X (Sigma); sodium borohydride (MP Biomedicals); Primary antibodies: NES (Abcam 134017; 1:10,000), SSEA1 (DSHB MC480; 1:8), OCT4 (Abcam 198857; 1:1000), SOX1 (Cell Signaling Tech 4194; 1:200), GFAP (BioLegend 82401; 1:1000), and TUBB3 (Abcam 78078; 1:500) for early time points (days 1 and 3); GFAP, TUBB3, MAP2 (Abcam 11267; 1:500), GAP43 (Abcam 16053; 1:500), and OLIG1 (Abcam 53041; 1:500). Nuclei stain: {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 (Life Technologies H1399); Secondary antibodies were diluted at 1:400; goat anti-mouse IgM AF546 (Life Technologies A21045), donkey anti-rabbit IgG AF647 (Life Technologies {"type":"entrez-protein","attrs":{"text":"A31573","term_id":"87384","term_text":"pir||A31573"}}A31573), goat anti-chicken IgY AF488 (Life Technologies A11039), goat anti-mouse IgG2a AF546 (Life Technologies A21133), and goat anti-mouse IgG1 AF546 (Life Technologies A21123).