Fig 1: Murine brain organoids show progressive cellular organization and maturation into discrete regions(A) Graph showing Relative gene expression (normalized to GAPDH and TBP) levels of genes related to bona fide NSCs (i.e., SOX5, Pax6, Sox1, Vim, Dcx), SGZ-NSCs (Sox2, Msi1, Slc1a3, Nes, Dach1) and to ESCs (i.e., Nanog, Oct3/4, Klf4, Stat3, Cdh1) evaluated in n = 4 different SGZ-derived NSCs batches. Data are expressed as mean ± SEM.(B) Representative confocal immunofluorescence images of SGZ-derived NSCs neurospheres at 3d in culture, showing the presence of the specific NSCs markers SOX1 and Vimentin, the SGZ-NSCs markers Musashi, SOX2 and Nestin, and little or no expression of Nanog, Oct3/4 and E-cadherin ESCs markers.(C) Representative confocal immunofluorescence images of sliced murine brain organoids at different protocol stages, showing the stemness (Vimentin) marker. At day 7 and day 14, the distribution of Vimentin+ cells is widespread, and no evident cellular organization is observed. At a later stage of maturation (day 21 ad 32), few Vimentin+ cells were still detected.(D) Representative confocal immunofluorescence images of sliced NSCs-derived organoids at different protocol stages, showing the stemness (SOX2) and the neuronal progenitors (DCX) markers. At day 7, all the cell populations inside the organoids do not show any type of organization but are scattered and strongly staminal. Starting from the induction phase (day 14), the immature neuronal cells spatially rearrange to cluster on the external region, while the middle layer is mainly staminal. At later time points (days 21 and 32), few SOX2+ cells were detected while DCX+ cells still populated the edge of the organoid.(E) Representative confocal immunofluorescence images of sliced murine brain organoids at different protocol stages, showing the immature neurons (TUBB3) marker. At day 7, few immature neurons were detected within the organoid structure and progressive organization to the external layer was observed along the developmental stages. Starting from the induction phase (day 14), the immature neurons mainly cluster on the external region with a progressive thickening of the TUBB3+ cells layer.(F) Representative confocal immunofluorescence images of sliced murine brain organoids at different protocol phases, illustrating the mature neuronal cells (MAP2) in close association with astrocytes (GFAP) cells. At later stages of culture, organoids show an ongoing neuronal maturation, mainly restricted to the outer organoid edge. A progressive thickening of the neuronal cells layers and the ability to form a neuronal network is observed from day 21 to day 32. All the pictures in B-F are maximum intensity projections of z stack images.(G) Fluorescence intensity analysis showing expression of SOX2 (red), DCX (green), TUBB3 (blue) and MAP2 (orange) plotted versus the distance from the center (left) to the edge (right) of brain organoids at day 7, 14, 21, 32. Dashed lines delineate different zones of the organoid slice, from the core to the edge. A progressive accumulation of neuronal cells population from the core to the edge is observed during the later time point.