Extended Data Figure 5. Migration of Dlxi1/2::eGFP⁺ cells in fused hSS-hCS.

(a, b) Representative immunostaining in cryosections of hSS showing co-expression of Dlxi1/2::eGFP and GAD67 and GABA. (c) Quantification by immunostaining of the proportion of Dlxi1/2::eGFP⁺ cells that co-express GAD67 in hSS derived using the ISA or ISRA condition (n= 3 hiPSC lines; t-test, P= 0.35). (d) Proportion of Dlxi1/2::eGFP and GAD67 positive cells in hSS derived using the ISA or ISRA condition that co-express SST (t-test, P= 0.48), CR (t-test, *P= 0.04) or CB (t-test, P= 0.43); n = 3 hiPSC lines. (e) Representative images of hSS-hCS at day 60 showing migration of Dlxi1/2b::eGFP⁺ cells (from fluorescently labeled hSS) in fused hSS-hCS but not in hSS-hSS over time. (f) The number of Dlxi1/2b::eGFP⁺ (hSS-derived) or hSYN1::mCherry cells (hCS-derived) that moved in hSS-hCS or hSS-hSS was quantified in snapshots of live, intact spheroids at different time points (from day 3 to 25). The values shown are absolute number of cells that migrated to the other side (approximately the same area and thickness was imaged in each session); one-way ANOVA for cells at 17 days after assembly; F(2, 32)= 8.24, P= 0.001. (g) Representative images of fused hSS-hCS at day 91 showing migration of Dlxi1/2b::eGFP⁺ cells (from fluorescently labeled hSS) into fused hSS-hCS. (h) Representative image of an hSS that was plated on a glass coverslip and cultured for ~7 days. (i) Percentage of Dlxi1/2::eGFP inside the coverslip-plated hSS, in the rim (0–200 μm) or beyond this region (> 200 μm). (j) Quantification of the number of saltations of Dlxi1/2b::eGFP⁺ cells (n= 32 cells) inside the one-week coverslip-plated hSS, in the rim and outside this region (one-way ANOVA, interaction F (2, 30)= 22.12, P< 0.001; Bonferroni post-hoc ***P< 0.0001). (k) Diagram showing the angle of movement of migrating Dlxi1/2b::eGFP⁺ cells at 8–9 days after assembly of hSS-hCS. The angle was calculated between the leading process of Dlxi1/2b::eGFP⁺ cells that have moved into hCS and the fusion interface (n= 92 cells from 5 hiPSC lines). (l) Histogram showing the distribution of the distance of migrated Dlxi1/2b::eGFP⁺ cells relative to the edge of hCS in hSS-hCS at 30–50 days after assembly. The distance was measured in fixed 18 μm cryosections (n= 73 cells from 2 hiPSC lines). (m, n, o) Representative examples of Dlxi1/2b::eGFP⁺ cells migrated in the hCS that moved within a VZ-like region. The VZ-like region contains GFAP-expressing cells, is surrounded by TBR1⁺ cells and the migrated cells show GABA expression. Supplementary Video 2 shows movement of Dlxi1/2b::eGFP⁺ cells that is reminiscent of the ventricular-directed migration described in rodents.