Fig. 5: Long term perfused cortical organoids.

a Schematic representation of the experiment and differentiation protocol. Single hPSC cells were aggregated in E6 medium for 24 h, then a directed multistep neuronal differentiation was conducted for 62 days. b Bright field images of organoids in perfused (top row), non-perfused (middle row) grids and control organoids (bottom row) over the culture period. Scale bar 1 mm. c Immunofluorescent images of transverse sections of the three sample types. c1 Apoptotic marker cleaved Caspase 3 (green) indicates apoptotic cores in control organoids and non-perfused organoids, while healthy tissue shows neuronal identity (N-Cadherin, red, SOX2, white); non-perfused organoids have a characteristic mostly apoptotic dense core surrounded by sparse radially aligned cellular processes. Scale bars 1 mm and 70 µm on main and zoomed images respectively. c2 perfused organoids exclusively express myelin basic protein (MBP, yellow), marker of mature oligodendrocytes, while all three conditions express immature, migratory cortical neuron marker Doublecortin (DCX, blue). Scale bar 70 µm. c3 Staining for radial glia (BLBP (red) and DCX (green)) demonstrate a neuropil-like structure in perfused organoids formed by dense and intertwined networks of radial glia and neuronal processes, including a populations of mature neurons (NeuN, white), while in control organoids the cellular arborization is almost absent (also Supplementary Fig. 9b, c). Scale bar 70 µm. c4 SATB2 (red), marker of upper cortical layers and TBR1(red) and CTIP2 (gray) markers of deeper layers are expressed throughout the perfused organoids and within the non-apoptotic regions of non-perfused organoids, while in control organoids, these markers are localized in the proximity of the ventricle-like structures. Scale bar 70 µm. c5 Populations of glutamatergic (Glutaminase, green) and GABAergic (GAD67, white) neurons are expressed in all three conditions; while in perfused organoids a networked population is suggested by extended processes, in control organoids these neurons remain mainly segregated (also Supplementary Fig. 9d). Scale bar 70 µm. Representative images on (b) and (c) were acquired in three independent experiments; all experiments produced similar results.