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. 2020 Nov 10;9:e58178. doi: 10.7554/eLife.58178

Figure 3. hiPSCs differentiate into cortical neural lineages.

See also Supplementary file 2; Figure 4—figure supplement 1. (A) Schematic of neural differentiation of hiPSCs into cortical progenitor cells and neurons utilizing dual SMAD inhibition. Abbreviations: N3, basal neural differentiation medium, PDL-L, Poly-D-Lysine and Laminin coating; R-NPCs, radial NPCs. (B) Day 26 neural rosettes show the typical radially arrayed clusters of neural progenitor cells in brightfield micrographs. Rosettes are composed of PAX6-positive radial glia encircling a NCAD-positive, ZO-1-positive, and aPKCζ-positive apical adherens complex. Cells currently undergoing M-phase of mitosis, indicated by pHH3, are predominately localized around Pericentrin positive centrosomes at the apical end foot of radial glia (scale bars, 50 µm). Representative rosettes are shown from left to right for (WT_8343.2, WT_8343.2, WT_8343.4, and WT_2242.5). (C) Normalized transcript expression levels of neural regionalization candidate genes generated from RNA-Seq data, ordered from rostral to caudal cell fates, followed by general neuronal and non-neural cell fates, and housekeeping genes. Sex and Genotype status are indicated on the left.

Figure 3.

Figure 3—figure supplement 1. hiPSCs differentiate into NPCs and neurons.

Figure 3—figure supplement 1.

(A) Flow cytometric analysis of the transition between OCT4+ pluripotent hiPSCs and early PAX6+ telencephalic neural progenitor cells. Error bars represent the SEM. Replicates were as follows: WT (3 clones from two donors), DEL (6 clones from three donors). (B) Wild-type (WT), 16p11.2 microdeletion (DEL), and 16p11.2 microduplication (DUP) Day 26 neural rosettes show the typical radially arrayed clusters of neural progenitor cells in brightfield micrographs. Clone IDs for each image: DEL (DEL_5_9, DEL_1_2, DEL_5_8, DEL_9_984), DUP (DUP_3_1, DUP_1_9, DUP_3_3, DUP_1_8), WT (WT_8343.2, WT_8343.2, WT_8343.4, WT_2242.5). (C) Day 45 immature neurons are characterized by long neurites and expression of the neuronal markers TUJ1 and NEUN (scale bar, 50 µm). Clone WT_8343.5.