Extended Data Figure 9. Derivation of TS hSS, migration and electroporation.

(a) Sequencing of PCR-amplified DNA showing the p.G406R mutation in exon 8a of CACNA1C in TS (subject: 8303). (b) Representative images of hiPSC colonies expressing pluripotency markers (OCT4, SSEA4) in one TS subject (c) Level of gene expression (RT-qPCR, normalized to GAPDH) for NKX2-1 showing no defects in ventral forebrain induction in TS (two-way ANOVA; interaction F(2,15)= 0.20, P= 0.81; TS versus Ctrl F(1,15)= 0.16, P= 0.68). (d–g) Representative immunostainings in cryosections of TS hSS (day 60) showing expression of NKX2-1, GABA, MAP2, GAD67, SST and CR. (h) Calcium imaging (Fura-2) in dissociated hCS derived from TS subjects and controls (Ctrl: n= 81 cells from 2 subjects; TS: n= 147 cells from 2 subjects). Quantification of residual intracellular calcium ([Ca²⁺]_i) following 67 mM KCl depolarization of Ctrl and TS cells in hCS cells. Residual [Ca²⁺]_i was calculated by dividing the plateau calcium (C–A) level by the peak calcium elevation (B−A); (t-test, ***P< 0.001). (i) Quantification of [Ca²⁺]_i following depolarization of Ctrl and TS cells in hSS (t-test, ***P< 0.001). (j) Representative image of fused TS hSS-hCS showing Dlxi1/2b::eGFP expression and migration. (k, l) Quantification of the number of saltations and saltation length of Dlx2i1/2b::eGFP cells in fused hSS-hCS across multiple Ctrl and TS lines (related to Fig. 3d, e). (m) Quantification of the speed when mobile of Dlxi1/2b::eGFP cells in fused hSS-hCS (Ctrl: n= 21 cells from 3 hiPSC lines derived from 3 subjects; TS: n= 29 cells from 3 hiPSC lines derived from 3 subjects; TS-Ctrl hybrid: n= 12 cells from 3 hiPSC line shown combinations; one-way ANOVA with Dunnett’s multiple comparison test; ***P< 0.001). (n) Electroporation of cDNA encoding the TS– and WT– Ca_V1.2 channels into slices of mouse E14 ganglionic eminences (GE). (o) Representative example of time-lapse live imaging depicting the saltatory migration of GFP⁺ cells in slices electroporated with CAG::GFP and either the WT–or the TS– CACNA1C. (p, q) Quantification of the number of saltations (t-test; **P< 0.01) and saltation length (t-test; ***P< 0.001) of GFP⁺ cells in electroporated mouse forebrain slices (WT: n= 33 cells; TS: n= 23 cells; from 3 litters). (r) Scheme illustrating pharmacological manipulation of LTCC during live imaging of fused hSS-hCS. (s) Quantification of speed when mobile following exposure to the LTCC blocker nimodipine (5 μM) (paired t-test; Ctrl: n= 13 cells from 3 hiPSC lines derived from 3 subjects, ***P< 0.001; TS: n= 12 cells from 2 hiPSC lines derived from 2 subjects, **P< 0.005). (t) Quantification of saltation length following exposure to roscovitine (15 μM) (paired t-test; Ctrl: n= 7 cells from 2 hiPSC lines derived from 2 subject, **P< 0.005; TS: n= 12 cells from 2 hiPSC lines derived from 2 subjects; ***P< 0.001). (u) Quantification of speed when mobile following exposure to roscovitine (15 μM) (paired t-tests; Ctrl: n= 9 cells from 2 hiPSC lines derived from 2 subjects, ***P< 0.001; TS: n= 12 cells from 2 hiPSC lines derived from 2 subjects; P= 0.05). Data are mean ± s.e.m.