Human adherent cortical organoids in a multi-well format
- Department of Psychiatry, Erasmus MC, Netherlands
- Stavros Niarchos Foundation (SNF) Center for Precision Psychiatry & Mental Health, Columbia University, United States
- Department of Psychiatry, Columbia University Irving Medical Center, United States
- ENCORE Expertise Center for Neurodevelopmental Disorders, Erasmus MC, Netherlands
Figures
Figure 1 with 1 supplement
Adherent cortical organoid model.
(A) Schematic representation of the differentiation protocol. (B–D) Representative pictures of neural progenitor cells (NPCs) from three different induced pluripotent stem cell (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 days 28 and 42, seeding density 1500 NPCs per well; scale bars left to right 100, 150, 100, and 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 center of the structure, seeding density 1500 NPCs per well (scale bar, 500 µm).
Figure 1—figure supplement 1
Reproducibility of neural progenitor cells (NPCs) 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) Proportion of properly formed single structure adherent cortical organoids. Each dot represents the end point of a different batch of adherent cortical organoids where the size of the batch ranges from 10 to 40 organoids per plate. Total organoids line 1 n = 248, line 2 n = 70, line 3 n = 70. (D) 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.
Figure 2 with 4 supplements
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, 200 µm). (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+ neural progenitor cells (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 days 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 three 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 to 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). 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).
Figure 2—figure supplement 1
Neuronal maturation in cortical organoids is shown by a temporal shift in neural progenitor cell (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 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.
Figure 2—figure supplement 2
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).
Figure 2—figure supplement 3
Cortical layering in the adherent cortical organoids (ACOs).
Separation of CUX1 and CUX2 expression (A: day 70, scale bar, 100 µm; B: day 67, scale bar, 500 µm). (C) A SOX2+ band/layer is visible in the center of the ACO (day 63, scale bar, 500 µm).
Figure 2—figure supplement 4
Astrocyte distribution within adherent cortical organoids (ACOs).
The abundantly present GFAP+ and S100B+ astrocytes have a comparable distribution with the MAP2+ neurons in the ACOs. The astrocytes show radial patterns with many somas located in the center as well as a large presence outside the center (day 63, scale bars, 500 µm).
Figure 3
Adherent cortical organoids form oligodendrocyte lineage cells.
(A) Adherent cortical organoids show oligodendrocyte precursor cells (OPCs) as early as 44 days, indicated by OPC marker NG2 (day 44, 20 µm). 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 and 20 µm). After 5 months, MBP-positive oligodendrocytes show more mature morphology and initial wrapping of NF200+ axons (day 148; E, 50 and 20 µm, F, 5 µm). Oligodendrocyte distribution at day 161 where the MBP+ oligodendrocytes sit between axon bundles and co-localize with NF200+ axons (G–I, day 161, G 500 µm, H 100 µm, I 10 µm).
Figure 4
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 100 µm2. There are about twice as many 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.
Figure 5 with 1 supplement
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 days 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 to 3 organoids per time point from 2 different batches of differentiation.
Figure 5—video 1
Video showing 10 min of calcium imaging of an adherent cortical organoid (ACO) at day 61 using the genetically encoded calcium indicator AAV1.Syn.GCaMP6s.
WPRE.SV40 (rainbow filter indicates increasing calcium signal ranging from blue to red, speed increase indicated by time stamp in top left corner).
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Human adherent cortical organoids in a multi-well format
eLife 13:RP98340.
https://doi.org/10.7554/eLife.98340.3
