For single-cell self-renewal assays the experimental setup was modified as follows: undifferentiated (sorted or unsorted) iNPCs were dissociated and plated into matrigel-coated 96-well plates as single cells as described52. Each condition was done in 24 technical replicates with a minimum of three biological duplicates (n⩾62). One day after plating, all wells were treated with the respective compounds as indicated. After 21 days, the colony number was evaluated by microscopy analysis using an upright microscope (Olympus). For single-cell self-renewal assays in the presence of PI3K (LY294002; 5 μM) and MAPK (PD98059; 5 μM) inhibitors, individual cells were plated as described above and the media supplemented with the respective chemicals or the solvent control (DMSO) at the indicated concentration and colonies counted after 21 days. Biological triplicates with technical triplicates (n=9) were performed for each tested cell line and condition.
