8 well glass chamber

Cells plated on 8-well glass chambers (Thermo Fisher) were fixed with 4% paraformaldehyde (Mallinckrodt Baker) for 15 minutes, permeabilized with 0.1% Triton X100 (Mallinckrodt Baker) for 10 minutes, and blocked with blocking buffer (5% BSA in PBS) for 1 hour at room temperature (RT). Cells were incubated overnight at 4°C with primary antibodies (Supplementary data, Table S2). On the next day, cells were incubated for 1 h at RT with secondary antibodies. Both primary and secondary antibodies were diluted in blocking buffer. Slides were mounted with a ProLong® Gold Antifade reagent with DAPI (Life Technologies), and confocal images were captured by using a LSM 510 Meta microscope (40x objective). Images were acquired using ZEN Black software. Counting of NKX2.1⁺ and PAX 6⁺ cells was performed by two independent raters in a blinded fashion. For each condition, at least three images with at least 100 cells per image were counted. The NKX2.1/PAX6 ratio was calculated based on cell counts for each condition. The experiments were conducted in triplicates. Purity of the hESC-derived MGE progenitor population was assessed by NKX2.1⁺ and PAX6⁺ cell count in ICC images. It was determined by the ratio of NKX2.1⁺ cells divided by the total number of generated NPC (NKX2.1⁺ and PAX6⁺ cells).