Figure 1.

Active Neurons Generated from PGPC iPSCs Display Similar Dendrite Morphology and Network Circuitry

(A) Differentiation scheme to generate excitatory cortical neurons by induction of Ngn2. Transduced iPSCs were dissociated to single cells and plated in the presence of ROCK inhibitor. At D1, medium was changed to CM1 and Ngn2 was induced by incubating with doxycycline until D7. Puromycin was added from D2 to D4 to remove any non-transduced cells. Culture medium was changed to CM2 on D3. Ara-C was added from D6-8 to remove any remaining dividing cells. On D8, neurons were re-seeded for downstream assays in BrainPhys medium.

(B) Representative immunocytochemistry image of iPSC-derived neurons after 6 weeks in culture labeled with DAPI and MAP2 and sparsely labeled with GFP (independent experiments = 2; technical replicates = 20). Scale bar represents 100 μm. Color channels were independently altered to adjust contrast for publication.

(C–E) Plots of (C) soma area, (D) total dendrite length, and (E) number of intersections (Sholl analysis) from 6-week-old neurons (independent experiments n = 2; technical replicates per batch = 20). (C and D) Boxplots indicate median values. (E) Mean values were plotted with error bars indicating SE. Statistically significant pairwise comparisons indicated by ^∗ are inset.

(F and G) (F) Representative raster plots of PGPC3 neurons from a single-well of recordings collected by micro-electrode arrays at different time points (two independent experiments each with eight technical replicates). Each spike is indicated by a black line, blue lines represent bursts defined by at least five spikes each separated by an inter-spike interval of no more than 100 ms. (G) Bursts are absent after treatment of week 7 neurons with CNQX (left). Bursts begin to return after compound removal and replacement with fresh basal medium (right).