Figure 1.

3D Neuronal Spheres from hPSC Lines Adopt a Predominantly Cortical Identity by Default

(A) Schematic illustration of the differentiation strategy used to obtain cortical neurons from 3D spheres. d, day; BDNF, brain-derived neurotrophic factor; GDNF, glial cell-derived neurotrophic factor.

(B) Representative bright-field image of HUES8-derived cortical spheres at day 15 of differentiation.

(C) Representative images of immunohistochemistry on sections of BJSiPS-derived cortical spheres at day 50 of differentiation, stained with antibodies specific for MAP2 (blue), TBR1 (red), and CTIP2 (green). Scale bar represents 200 μm.

(D–F) Representative results from a BJSiPS-derived cortical sphere cleared by SeeDB at day 50 of differentiation, stained with antibodies specific for TBR1 (blue), CTIP2 (green), and SATB2 (red), and imaged by LSFM. Photos in (D) show 3D images of the same sphere from two different angles. Scale bars in red and green represent 200 μm. Schematic images in (E) illustrate the virtual section of the sphere. The low-magnification 3D image in (E) shows the distribution of neurons inside the sphere expressing markers specific for distinct cortical subtypes. Scale bar represents 200 μm. (E′) is a higher-magnification image of the boxed area in (E). Schematic illustrations in (F) indicate how the sphere was virtually processed to generate a partially sectioned sphere.

Scale bars in green and red represent 100 μm. See also Figures S1 and S2; Movies S1A–S1C.