Figure 1. Phase contrast images of 2D smNPC culture at different stages of confluence and differentiation.

(a/b) Representative images illustrating the density of smNPCs 2 days after splitting at either 1:10 (a) or 1:20 (b). (c) Example image of a low-quality smNPC culture contaminated by large numbers of differentiated cells (very dark and large, located between colonies). Partially differentiated cultures may be rescued by maintaining high splitting ratios (at least 1:20) over several passages, as smNPCs usually proliferate faster than differentiated cells. These types of culture should not be used for generating organoids. (d) smNPCs at the optimal confluence for organoid generation or splitting.