Nestin

Cells were seeded on 35 mm glass bottom plates (Cellvis). Cells were fixed with 4% paraformaldehyde for 30 min at 4°C and permeabilized during blocking in 2% BSA containing 0.3% Triton X-100 for 1 hr at room temperature. After blocking, cells were treated with primary and secondary antibodies using standard methods. The following primary antibodies were used: OCT4 (Cell Signaling Technology, Cat. 75463S), NANOG (Cell Signaling Technology, Cat. 4903S), PAX6 (Cell Signaling Technology Cat. 60433), NESTIN (STEMCELL Technologies, Cat. 60091), TUBB3 (Cell Signaling Technology, Cat. 4466S), MAP2 (Thermo Fisher Scientific, Cat. 131500), Cytochrome c (BD Pharmingen, Cat. 556433). All secondary antibodies were conjugated to Alexa fluorophore derivatives (Thermo). See detailed information about antibodies in S7 Table. Nuclei were stained with Hoechst 3342 (Thermo). Fixed and stained cells were mounted with Fluoromount-G slide mounting medium (Electron Microscopy Sciences). Images were acquired using an Andor DU-897 camera mounted on a Nikon Spinning Disk. The software used for image acquisition and producing representative images was Nikon Elements.

Day 5 EBs were plated onto Matrigel-coated chambered microscope slides (Falcon, VWR, USA), and were then subjected to the differentiation procedure described above. At the indicated timepoints, the cells on the slides were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The samples were blocked with 5% bovine serum albumin and stained with primary antibodies overnight at 4 °C followed by secondary antibodies and incubated at room temperature for 60 min. EBs were stained with the following antibodies: OCT4 (Stem Cell Technologies, 1:250), nestin (Stem Cell Technologies, 1:1000), and PAX6 (Invitrogen, Waltham, MA, USA, PA1-801, 1:50). Neural rosettes were stained with PAX6 and nestin antibodies. Neural progenitors were stained with $\beta$ -tubulin III
 antibodies (Stem Cell Technologies, 1:125) and PAX6 antibodies. We also used 53BP1 Polyclonal Antibody (PA1-16565, Thermo Fisher Scientific). Primary antibodies were followed with their appropriate secondary antibodies Alexa Fluor^® 488 Goat anti-mouse IgG (Invitrogen, A-11011, 1:100) and Alexa Fluor^® 594 Goat anti-rabbit IgG (Invitrogen, A-11012, 1:100). All samples were imaged with the Zeiss Axiovert 200 fluorescence microscope.