Extended Data Fig. 2: NSC-Chimeroids created by aggregating different numbers of cells display variable development.

a. Brightfield images of single-donor H1 NSC-Chimeroids seeded with 9,000 (upper) and 12,000 (lower) cells/well at day 3 and day 16 after reaggregation (left panels), and brightfield images of single donor PGP1 NSC-Chimeroids seeded with 9,000 (top left), 12,000 (bottom left), and 20,000 (top right) cells/well at day 3 and day 16 after reaggregation (right panels). Scale bar: 500 μm. Barplot showing growth over time of PGP1 NSC-Chimeroids compared across all different cell counts at aggregation (bottom right). Bars show median values, whiskers show upper and lower quartiles (n = 170 Chimeroids across all 4 timepoints). Significance was calculated using ANOVA followed by two-sided Tukey post-hoc pairwise tests. P-values: *** < − 0.001; **** - <0.0001. b. Immunolabelling of PGP1 single-donor NSC-Chimeroids made by aggregating the indicated number of cells, at DIV35. Upper images display whole organoids; lower images, enlargement of indicated portions. Lefthand panel, immunolabelling for SOX2 (progenitors), MAP2 (neuronal dendrites), and TBR1 (deep layer neurons). Righthand panel, immunolabelling for SOX2, ZO-1 (tight gap junctions), and EMX1 (dorsal cortical progenitors). Scale bar: 500 μm (upper) and 125 μm (lower). Arrowheads indicate non-cortical (grey) and cortical (white) regions. c. Left panel: whole-organoid brightfield images of H1 single-donor NPC-Chimeroids seeded with 100,000 cells at aggregation, at 18 and 35 days after mixing. Scale bar: 500 μm. Right panel: immunolabelling of H1 single-donor NPC-Chimeroids showing SOX2, ZO-1, and EMX1. Scale bar: 500 μm and 125 μm (zoom-in).