Depletion of furin in our in vivo zebrafish model had the largest impact on head size. Thus we further tested the impact of FURIN knockdown in human neural progenitor cells (NPCs) capable of differentiating into mixed populations of post-mitotic neurons and astrocytes37,38. Neurosphere outgrowth is a well-established neural migration assay measuring the distance NPCs migrate away from the neurosphere. NPCs were differentiated from human induced pluripotent stem cells (hiPSCs) reprogrammed from human fibroblasts using sendai viral vectors39,40. Pairwise isogenic comparisons were conducted in 307 neurospheres from three independent unaffected controls. We measured migration of DAPI-positive nuclei from pLKO.1 non-hairpin-PURO control neurospheres (n = 147) and LV-FURIN shRNA-PURO (shRNA-FURIN) knockdown neurospheres (n = 160). FURIN knockdown in the hiPSC NPCs resulted in significantly decreased total radial migration for all three individuals (C1: 1.16-fold decrease, P < 0.0017; C2: 1.23-fold decrease, P < 3 × 10−6; C3: 1.22-fold decrease, P < 2 × 10−6) (Fig. 4).
