a. Representative images of SMI312 staining of day 105 whole organoids compared with ALI-COs (20 days ALI, 85 days total) used for OrientationJ analysis shown in Fig. 1d. Shown are representative images out of six samples from two independent batches each. b. Comparison of more extended cultures of 169 days total whole organoid compared with ALI-CO (100 days ALI, 169 days total). Note the more abundant neuronal staining (TUBB3) and extensive tracts in the ALI-CO at this time point compared with whole organoid. GFAP staining marks glia including astrocytes. c. Higher magnification imaging reveals a dense TUBB3+ neuropil containing axons (SMI312), dendrites (MAP2) and astrocytes (GFAP) in ALI-COs, while whole organoids exhibit disrupted morphology with decreased neuronal processes. b-c are representative images of three ALI-COs compared with three whole organoids from the same batch, with similar results. Scale bars, 500 μm (a, b) and 100 μm (c).