Extended Data Fig. 3: Selection of organoids for plating.

(a) Good-quality 1-month-old organoids with visible spatial arrangement of neural rosette structures. Scale bar, 1,000 μm. Mixed quality organoids, where there is a mixed population of fully differentiated organoids with visible rosette structures and incomplete differentiated organoids—select rosetted organoids only for downstream assays and discard incompletely differentiated organoids. Poor-quality spheroids that did not efficiently neuralize and differentiate into rosetted organoids—discard and start over. (b) Distinguish proper spatial organization and structural development in organoids; orange arrows indicate holes and green arrows indicate rosettes. Scale bar, 1,000 μm. (c) Use of immunohistochemistry to distinguish between good-quality organoids with spatial neural rosette arrangement (smaller circles within the organoid) and incompletely differentiated organoids that contain neurons but lack neural rosette structures and spatial organization. Immunostainings showing nuclei (DAPI), neuron microtubules (MAP2), and proliferating NPCs (Ki67 and Nestin). Scale bar, 50 μm.