Adherent cortical organoid model.

A Schematic representation of the differentiation protocol. B/C/D Representative NPCs from three different iPSC lines with markers SOX2, Nestin and FOXG1 (scale bar B, 50 µm; C/D, 20 µm). E Representative time course showing self-organization during differentiation, starting with radial organization between day 28 and day 42, seeding density 1500 NPCs per well (green is Live-stain from Viability/Cytotoxicity kit; scale bars left to right 100 µm, 150 µm, 100 µm, 100 µm). F Full well showing radial organization at day 42 in culture were only a few dead cells are visible in red in the dense centre of the structure, seeding density 1500 NPCs per well (scale bar, 500 µm).

Adherent cortical organoids show an organized network of neuronal and astrocyte subtypes.

A MAP2+ somas and dendrites alongside Tau+/MAP2– axons show segregation of dendritic and axonal compartments, with SOX2+ progenitors concentrated in the center of the organoid (Day 75, 200µm). B MAP2+ and NeuN+ cells indicate mature neurons (Day 72, 50 µm). C Deep-layer cortical marker CTIP2 and upper layer marker CUX1 show rudimentary segregation of cortical layers in expected inside-out pattern (Day 64, 50 µm). D A subset of the MAP2-positive neurons are GAD67+ (white arrows), indicating the presence of an interneuron population in the cortical organoids (Day 60, 50 µm). E Astrocyte markers GFAP and S100β show the general radial pattern of astrocyte outgrowth (Day 66, 500 µm). F/G/H GFAP staining reveals the morphologies of different astrocyte subtypes (white arrows), including fibrous astrocytes (F, Day 65, 100 µm), protoplasmic astrocytes (G, Day 65, 50 µm) and interlaminar astrocytes (H, Day 65, 100 µm). I Co-localization of astrocyte marker GFAP and PAX6 marks radial glia (white arrows) (Day 65, 50 µm).

Adherent cortical organoids form oligodendrocyte lineage cells.

A Adherent cortical organoids show OPCs as early as 44 days, indicated by OPC marker NG2 (Day 44, 20 µm). B/C The NG2+ OPCs are still present in the cortical organoids after 4 months (Day 119, B 50 µm, C 20 µm). D Young oligodendrocytes start to emerge after 4 months indicated by rudimentary MBP staining (Day 119 100 µm and 20 µm). E/F After 5 months, MBP-positive oligodendrocytes show more mature morphology and initial wrapping of NF200+ axons (Day 148; E, 50 µm and 20 µm, F 5 µm). G/H/I Oligodendrocyte distribution at day 161 where the MBP+ oligodendrocytes sit between axons bundles and co-localize with NF200+ axons. (G/H/I Day 161, G 500 µm, H 100µm, I 10 µm)

Adherent cortical organoids show synaptic connectivity and network bursts.

A Synapsin staining shows synapse formation along MAP2+ dendrites (Day 70, 20 µm). B Co-localization of pre-synaptic marker Synapsin and post-synaptic marker PSD-95 (Day 205, 20 µm). C MAP2+ dendrites and soma are decorated with Synapsin+ synapses (Day 251, 100 µm) D Overview of entire well, showing alignment of Synapsin staining with MAP2 (Day 251, 500 µm). E Sparse labeling of neurons with AAV9.CamKII.eGFP allows detailed imaging of glutamatergic dendritic spines with mature mushroom morphology, contacting pre-synaptic Synapsin puncta (Day 310, 5 µm). F Snapshot of radially organized neurons transduced with AAV1.Syn.GCaMP6s.WPRE.SV40 (Day 60, 100 µm). G Calcium events per minute from 36 cells from 7 recordings (each showing a cluster of neurons) from 2 different cortical organoids at day 61. H Network bursts per minute from 37 cells from 6 recordings from 2 different cortical organoids. I Percentage of events that are part of network bursts indicates that the majority of cells are involved in network burst activity. J Representative calcium traces from 2 different clusters in which cells are showing individual activity as well as network bursts where multiple cells are active simultaneously.

Reproducibility of Neural Progenitor Cells and Adherent Cortical Organoids.

A NPCs are positive for SOX2, Nestin, FOXG1, PAX6 and TBR2 (scale bars 20 μm) B LIVE/DEAD staining of adherent organoids (N=4) showing reproducibility of the structure formation. Seeding density line 1; 1250 NPCs, line 2; 750 NPCs, line 3; 1000 NPCs (scale bars 500 μm). C Function of the necessary NPC seeding density to form adherent cortical organoids in relation to the doubling time as a measure of the proliferation rate of the NPCs. The doubling time of the NPCs explains more than half the variation (r2 = 0.67) of the required seeding density showing that fewer cells need to be seeded for NPC lines with a higher proliferation rate. D Proportion of successful, single structure adherent cortical organoids. Each dot represents a different batch of adherent cortical organoids where the size of the batch ranges from 10-40 organoids.

Neuronal maturation in cortical organoids is shown by a temporal shift in NPC and neuronal markers.

A Representative images exhibiting change in expression of NPC and neuronal markers over time. Number of cells positive for NPC markers SOX2 and PAX6 decreases over time. Deep-layer CTIP2+ neurons start appearing at day 28, while upper-layer CUX1+ positive neurons appear in higher numbers around day 56. (Scale bars 20 μm). B The percentages of DAPI+ cells expressing each of the markers were quantified at four time points. Significant differences between week 2 and week 8 expression were seen for SOX2+ (down by 39.6 %), CTIP2+ (up by 13.5 %) and CUX1+ cells (up by 23.1 %). *** p < 0.0001, ANOVA one-way followed by Tukey-Kramer’s multiple correction test. Error bars indicate SEM, n=3-6 images taken over two wells, for each time point.

Distribution of axons and dendrites in adherent cortical organoids.

Radially organized MAP2+/Tau+ dendrites along with both radially and circumferentially organized NF200+/Tau+ axons in duplicate for each cell line (Day 63, scale bars 500 µm)

Cortical layering in the adherent cortical organoids.

A Rudimentary separation of SOX2+ NPCs, CTIP2+ deep layer neurons and CUX1+ upper layer neurons (Day 67, scale bar 100 µm) B-E Separation of CUX1 and CUX2 expression (B day 70, scale bar 100 µm; C day 70, scale bar 500 µm; D day 67, scale bar 500 µm; E day 67, scale bar 100 µm)

GAD67+ interneurons make up a small proportion of NeuN+ neurons in the adherent cortical organoids.

A Example image of GAD67+ interneuron proportion of NeuN+ neurons (Day 67, 50 µm). B Quantification of GAD67+ proportion of NeuN+ neurons. Every point is the percentage of GAD67+ interneurons out of all NeuN+ neurons in a spatially randomized selected image with on average 130.6 (± 16.1) NeuN+ nuclei per frame. (Line 1: N= 26 images of 6 different cortical organoids at day 65-67; Line 2/3: 6 images of 2 different organoids at day 65).

Astrocyte distribution within Adherent Cortical Organoids.

The abundantly present GFAP+ and S100B+ astrocytes have a comparable distribution with the MAP2+ neurons in the adherent cortical organoids. The astrocytes show radial patterns with many somas located in the centre as well as a large presence outside the centre (Day 63, scale bars 500 µm).