Extended Data Fig. 10: Characterization and data integration of MiSTR and MiSTR-like organoid cultures.

All experiments were performed with the hESC line H9. a, b, Expression patterns of key forebrain patterning genes across sections A-E in MiSTR tissue (a) and MiSTR-like organoids (b), showing a tendency towards higher ventralization in organoids, sampled at day 21. For panel a, n = 368 for A, n = 1902 for B, n = 404 for C, n = 2152 for D, n = 1562 for E. For panel b, n = 967 for Control, n = 483 for A, n = 955 for B, n = 669 for C, n = 1144 for D, n = 997 for E. For panels a-b, boxplots are shown with the median indicated, where boxes show the quartiles of the measurement and whiskers show the 1.5 IQRs of the lower and upper quartile. c, UMAP resulting from different methods showing the robustness of the integration, colored by sample source (top) and segment (bottom), sampled at day 21. d, Distribution of MiSTR (brown) and MiSTR-like organoid cells (orange) across the binned DV ranks, showing coverage of the same dorso-ventral domains at different frequencies, sampled at day 21. e, Dotplots showing cluster marker expression for the dataset at day 21. Annotations are kept consistent with Fig. 6c: 1-Cycling Telencephalic Progenitors, 2-Cortical-PSB-LGE Progenitors, 3-LGE-derived Interneurons, 4-MGE Progenitors, 5-MGE-derived Interneurons, 6-PoA / Hypothalamic Progenitors, 7-Hypothalamic Neurons, 8-Floor Plate, 9-Unknown. PSB, Pallial-Subpallial Boundary; LGE, Lateral Ganglionic Eminence; MGE, Medial Ganglionic Eminence; PoA, Preoptic Area. f, Fluorescence image showing NKX2.1-GFP expression in control MiSTR tissue cultured in the absence of PDMS microfluidic tree. This culture was performed under patterning conditions for segment D, and the image (representative of 3 cultures under D conditions) was taken on differentiation day 12. Induction patterns were similar across other 2D MiSTR cultures under different morphogen conditions. g, UMAP projection of Harmony-integrated single-cell RNA-seq data from MiSTR and organoid experiments, day 9, colored by cell type. h, UMAP resulting from different methods showing the robustness of the integration, colored by sample source (top) and segment (bottom). i,j, Cell type composition of each segment from MiSTR tissues (not h, but i) and separately patterned organoids (not i, but j), sampled at day 9. k, Dotplots showing cluster marker expression for the dataset at day 9. PSB, Pallial-Subpallial Boundary; LGE, Lateral Ganglionic Eminence; Prog., progenitors. l, Forebrain axis score ranking of cells from each experimental setup, representing shifts from anterodorsal identities (higher ranks) towards posterior-ventral identities (lower ranks). For each of them: left, ridge plot showing distribution of cells along FBaxis_rank values; middle, jitter plot showing the distribution of each progenitor type along the FBaxis ranking (most dorsal cells at the top, most ventral cells at the bottom); right, same jitter plot showing separately each MiSTR segment and organoid condition, sampled at day 9. m, Distribution of MiSTR (brown) and MiSTR-like organoid cells (orange) across the binned DV ranks, showing coverage of the same dorso-ventral domains at different frequencies, sampled at day 9. n, Boxplots showing distribution of percentage of progenitors, neurons and off-target cell types, split by tissue source at day 9 (left) and day 21 (right), with the median indicated, where the boxes show the quartiles of the measurement and the whiskers, the 1.5 IQRs of the lower and upper quartile. n = 5 for MiSTR and n = 6 for organoids category for each timepoint and cell class. NPC, neural progenitor cells; off, off-target cell types.