(a/b) Single optical confocal slices of whole mount stained and tissue-cleared organoids showing the expression of the early cortical neuron marker CTIP2, precursor marker Pax6, and general neuronal marker Map2 at day 40. Expression of the different markers is generally confined to specific regions of the organoid. While Pax6 expression is highest in the neural rosette-like structures, CTIP2 is mostly found surrounding these regions (see (b) for an enlarged view of a rosette). The expression pattern of the general neuronal marker Map2 indicates that most neurons are located within the outer regions of the organoid with lower expression toward the center. (c) At day 30, staining for the forebrain precursor marker FoxG1 combined with Map2 further confirms the correct differentiation toward a cortical fate. (d/e) Consistently, the expression of the precursor markers Brn2 (d) and Sox2 (e) is mostly confined to the neural rosette-like structures, while the neural marker DCX (d) shows a distribution similar to that of Map2 in 30 days old organoids. (f/g) The precursor marker TBR2 is also mainly expressed in confined regions of the organoid surrounded by more mature cortical markers like TBR1 (f) and CTIP2 (g) at day 30 of differentiation. (h) Late-born SATB2+ neurons arise starting day 50. (i) At day 40, few synapsin-positive punctae on Map2-positive neurites indicate formation of first synapses. (j) Maximum intensity projection (MIP) showing the network of β-Tubulin III (TUBB3) positive neurons as well as Sox2-positive precursors throughout a 30 days old organoid. (k) While the mostly unguided nature of the differentiation protocol does allow for the emergence of other cell types, expression of TH is confined to a small area and limited to few cells after 40 days of differentiation. Organoids shown were at either day 30 (c, d, e, f, g, j), 40 (a, b, i, k), or 50 (h) of differentiation. Scale bars: 200 μm (a, c, d, e, f, g, h), 20 μm (b, g).