Generation of TARDBP knock-in iPSC lines and differentiation into MNs
(A) Schematic representation of CRISPR-Cas9-mediated genome editing via homology-directed repair.
(B) iPSC lines genotyping using Sanger sequencing.
(C) Schematic representation of the protocol for sequential differentiation of iPSCs into neuroepithelial progenitors (NEPs), MNPCs, and MNs with representative phase-contrast images of cells along differentiation. Scale bar, 250 μm. For time-lapse movie depicting maturation of MNPCs into MNs, see Video S1.
(E–I) Representative images (E) and quantification (F–I) of MNs differentiated for 2 weeks (D14) and 4 weeks (D28) subjected to immunocytochemistry for the common MN markers HB9, ISL1/2, ChAT and VAChT. Scale bar, 50 μm. Data shown as mean ± SEM. Two-way ANOVA. n = 5 independent experiments. See also Figures S1–S4.