Paraformaldehyde (pfa)

Total cell number and the fraction of cells undergoing S-phase were determined using Click-iT Edu (Life Technologies) followed by quantitative image analysis using Imaris (Bitplane). Briefly, after 3 days of culture, tumor-ECM construct medium was refreshed with medium containing 10 μM 5-ethynyl-2’-deoxyuridine (Edu) and cultured for another 24 hours. Constructs were then fixed with 3% paraformaldehyde (Mallinckrodt), permeabilized using 0.1% Triton X-100 (Sigma Aldrich), and incubated with Click-iT reaction cocktail prepared following manufacturer’s instructions. Constructs were subsequently counterstained with Draq5 and z-stack images of 50 μm thickness were collected using laser scanning confocal microscopy on an Olympus IX81 inverted microscope with an Olympus Fluoview FV1000 system (Olympus) and a 20X air objective. The Imaris spot detection algorithm was used to independently detect nuclei stained with Click-iT Edu and Draq5 for quantification of the number of cells undergoing S-phase and total cell number, respectively. S-phase fraction was then calculated by dividing the number of S-phase cells by the total number of cells. Images (2 per well) from two separate experiments (N = 2) performed in triplicate (n = 3) for each matrix stiffness were used for final calculations and statistics.

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).