Adherent cortical organoid model.

A Schematic representation of the differentiation protocol. B/C/D Representative pictures of NPCs from three different iPSC lines with markers SOX2, Nestin and FOXG1 (scale bar B, 50 µm; C/D, 20 µm). E Live/Dead stain of a representative time course showing self-organization during differentiation, starting with radial organization between day 28 and day 42, seeding density 1500 NPCs per well; 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 Rudimentary separation of SOX2+ NPCs, CTIP2+ deep layer neurons and CUX1+ upper layer neurons (Day 67, scale bar 100 µm) E Full well overview of the CUX1 and CUX2 positive regions. F GAD67+ interneuron (white arrows) proportion of NeuN+ neurons (Day 67, 50 µm). G 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). H Quantification of GAD67+ proportion of NeuN+ neurons of cell line 1, at 3 different time points. The number of GAD67+ interneurons over the total NeuN+ population remains relatively stable although at day 114 there was a modest increase which disappeared at day 242. Data was collected from 4-6 organoids per time point. G/H The different symbols reflect from which organoid the data point was collected. I Astrocyte markers GFAP and S100β show the general radial pattern of astrocyte outgrowth (Day 66, 500 µm). J/K/L GFAP staining reveals the morphologies of different astrocyte subtypes (white arrows), including fibrous astrocytes (J, Day 65, 100 µm), protoplasmic astrocytes (K, Day 65, 50 µm) and interlaminar astrocytes (L, Day 65, 100 µm). M 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 a neural network with excitatory and inhibitory synapses.

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 Excitatory synapses marked by overlapping Synapsin+ and HOMER1+ puncta (scale bar: top 20 µm, bottom 5 µm). G Inhibitory synapses marked by overlapping Synapsin+ and Gephyrin+ puncta (scale bar: top 20 µm, bottom 5 µm). H Density of excitatory and inhibitory synapses per 100µm2. There are about twice as much excitatory synapses (10.9 / 100 µm2) as inhibitory synapses (5.0 / 100 µm2). I The proportion of Synapsin that co-localizes with HOMER1 is 0.51 and the proportion of Synapsin that co-localizes with Gephyrin is 0.21.

Activity in the Adherent Cortical Organoids

A Snapshot of radially organized neurons transduced with AAV1.Syn.GCaMP6s.WPRE.SV40 (Day 60, 100 µm). B Representative calcium traces from active neurons in two different Fields of View (FOVs) from cell line 1, showing both individual activity and network bursts where each line corresponds to a single cell. C Events per minute of all recorded cells in day 61 and 100 (day 61, 3.9 ± 0.5 events/min; day 100, 3.9 ± 0.6 events/min). D Network bursts per minute (D61, average of 1.4 ± 0.07 NB/min; D100 average of 1.6 ± 0.12 NB/min) which shows all active cells are participating with the network bursts to some degree. E Percentage of events that are part of network bursts, which indicates that some cells only have events that are part of a network burst while for other cells the network bursts are only a small part of their total calcium events. The data was collected from 2-3 organoids per time point from 2 different batches of differentiation.