Fig. 3: Sensorimotor organoid cultures generate cells of an ectodermal lineage, including sensory and motor neurons and astrocytes.

a Dot plot of gene expression within neuronal clusters from single-cell RNA-seq data. Color intensity indicates expression level; dot size indicates the proportion of cells expressing each gene. b ISL+/TUJ1+ neurons in 4-week-old organoid cultures. c NCAM+ cells after FACS. d TUJ1 + NCAM + FACS purified cells (from (c)). e Staining (left) and quantification (right) of neuronal subtypes after FACS by expression of ISL (top), BRN3A (middle), and CHAT (bottom). For (c)–(e), one independent biological differentiation for each of control lines: 11a (red), FA0000011 (orange); and ALS lines: MGH5b (brown), and 19f (purple) at 4 weeks. f HB9, ChAT, and β-III tubulin labeling of motor neurons in 4-week-old cultures (three independent biological differentiation replicates of FA0000011). g BRN3A and PRPH staining of ganglia in 4-week-old cultures. h Ganglia connected by PRPH+/TUJ1+ axonal processes. i Sample calcium imaging traces of individual cells in ganglia-containing fields with (red) and without (gray) response to capsaicin at 6–7 weeks. Capsaicin sensitivity was confirmed in three independent biological differentiation replicates of two lines (11a, MGH5b). j Example whole-cell patch-clamp current traces recorded in the absence (gray) or presence of 300 nM TTX (cyan, cell without TTX-resistant sodium currents; magenta, the cell with TTX-resistant sodium currents). Quantification of peak sodium (middle) and potassium (right) current amplitudes in the absence (gray) or presence of TTX (cyan, cell without TTX-resistant currents; magenta, a cell with TTX-resistant currents). Recordings from four independent biological differentiation replicate of FA0000011 (sodium currents: n = 4 cells without TTX resistant currents, and n = 10 cells with TTX resistant currents, Mann–Whitney, U = 0; P = 0.002; potassium currents: n = 4 cells without TTX resistant currents, and n = 10 cells with TTX resistant currents, U = 19; P = 0.945). k GFAP + astrocytes in 4-week-old cultures. l Astrocyte morphology at 8 weeks. m GFAP+ cells in 6-week-old cultures. (n = 5–7 cultures from two independent biological differentiation replicates for each of five indicated lines; Kruskal–Wallis, P = 0.388). Scale bars 25 (f), (g), 50 (e), (k), (l), and 100 (b), (h) µm. Bars indicate mean and SEM.