Cerebral organoids express neural progenitor markers with ventricle-like, rosette, and dispersed morphologies

To generate cerebral organoids, we used a protocol similar to that published by the Knoblich lab6 with the following exceptions: we dissociated iPSCs from MEFs using Accutase instead of dispase/trypsin to obtain a single cell suspension, and we used AggreWells for initial embryoid body (EB) formation (3 × 106 cells per Aggrewell) instead of round-bottom plates. To characterize the cultures, organoids were pulsed with EdU during days 6 and 7 of differentiation, then continued in culture for 12 days without EdU. These aggregates were fixed, cryosectioned, and immunostained to examine the morphology of aggregates at this early stage of neurodevelopment. At day 19, wild-type organoids were composed of neural progenitor lineages that line fluid-filled cavities, similar to ventricles, along with regions of neural rosettes. There also were areas of “dispersed” cells that lacked discernible structure. Figure 1a shows a representative organoid stained for EdU and the NPC cytoskeletal marker Nestin (NES), with examples of each type of structural morphology. Interestingly, subsets of cells were found to express a marker of radial glia (PAX6) and a marker of intermediate progenitor cells (TBR2/EOMES) in areas with disparate morphologies including: cells lining the large ventricle-like structures, in rosettes, and in dispersed areas (Fig. 1b–i). This initial characterization revealed that organoids, at this time point, contain forebrain neural progenitors with heterogeneous structural organization.

Fig. 1: Cerebral organoids express neural progenitor markers with ventricle, rosette, and dispersed morphologies. a Organoids were differentiated to day 19 and pulsed with 2 μM EdU during days 6–7 before being fixed, sectioned, and stained for Nestin and EdU, showing areas of ventricle (asterisk), rosette (arrowhead), and dispersed (arrow) morphology. Day 19 organoids immunostained for b–e Tbr2 and Nestin, or f–i Pax6 and Nestin, showing areas of ventricle (asterisk), rosette (arrowhead), and dispersed (arrow) morphology. Scale bars: 100 μm (a), 50 μm (b–e, j–l), 20 μm (f–i) Full size image

DISC1 mutation disrupts cerebral organoid morphology in a manner that is phenocopied by WNT agonism

We previously described the generation of isogenic hiPSC lines with DISC1 disruption within exon 87, very near the site of a balanced translocation linked to mental illness in a large Scottish family8. Using TALEN targeting of exon 8, multiple clonal lines were derived that were wild-type or that harbored a premature stop codon within exon 8. Following differentiation to neural progenitor and neuronal fates of the cerebral cortex, we found that long splice variants of DISC1 were reduced by half in heterozygous lines, and eliminated in homozygous lines7. As the balanced translocation leads to elevated risk for mental illness in the heterozygous form (homozygous individuals have not been reported), we chose here to focus upon the model of DISC1 disruption most similar to the disease state, heterozygous exon 8 disruption (DISC1 ex8 wt/μ). To minimize the potential for clone and differentiation-specific effects, two clonal wild-type lines and two clonal DISC1 ex8 wt/μ lines were paired and analyzed over more than 10 differentiations. Unexpectedly, organoids derived from DISC1-mutant iPSCs displayed aberrant morphology (Fig. 2c, d). Mutant organoids lacked large and well-defined ventricle-like structures, and contained more small, disorganized rosette structures, and more areas of dispersed morphology when compared to wild-type organoids (Fig. 2a, b). These morphological changes were quantified for three differentiation rounds by counting the number of ventricles or rosettes present per organoid, average area of ventricles/rosettes per organoid, average length of the long axis of ventricles/rosettes present in each organoid, and total organoid area (Fig. 2o–r). As there are no clear criteria for differentiating rosettes from ventricle-like structures given the 3D structure of organoids, rosettes and ventricle-like structures were pooled for analysis, with example length/width measurements shown in Fig. 2m–n. Quantification revealed significant alterations with DISC1 mutation consistent with visible morphological changes, with increased numbers of rosettes per organoid (Fig. 2o), decreased ventricle/rosette area (Fig. 2p), and decreased length of the long axis of ventricles/rosettes (Fig. 2q). Notably, there was no clear difference in organoid area between wild-type and DISC1-mutant organoids (Fig. 2r).

Fig. 2: DISC1 disruption alters cerebral organoid morphology. a–l Wild-type and DISC1-disrupted organoids were pulsed with EdU for 2 days (culture days 6–7), then fixed, sectioned, and stained for EdU at day 19. Two representative organoids are shown for wild-type (a, b, e, f, i, j) and DISC1-disrupted (c, d, g, h, k, l) lines treated with either DMSO (vehicle, a–d), WNT agonist CHIR99021 (e–h), or WNT antagonist XAV939 (i–l). Well-defined ventricles/rosettes are labeled with asterisks, small poorly defined rosettes labeled with arrowheads, and areas of dispersed morphology labeled with arrows. Scale bars = 100 μm. Example measurements of ventricles/rosettes (labeled with red arrows) with blue lines labeling length and green lines labeling width, are shown in m (same image as b) and n (same image as d). Morphology of DMSO-treated wild-type or DISC1-disrupted organoids was quantified, blinded, in sections from the middle of the organoid, by counting number of ventricles or rosettes visible per organoid (o), average ventricle/rosette area per organoid (p), average length of the long axis of ventricles/rosettes per organoid (q), and organoid area (r). Data were derived from four independent differentiations. n = 4 (wt), 4 (DISC1 wt/μ). Statistics: two-tailed Student’s t-test. *p < 0.05, **p < 0.01. Variances were not significantly different in o, p, and r. Variances were significantly different in q, and t-test was performed with Welch’s correction. See also Supplementary Figure 1 for normalization per total area for each organoid Full size image

Multiple studies have previously linked DISC1 function to WNT signaling7,11,12. In our initial report describing DISC1-mutant neural cells in monolayer culture, we found that DISC1 disruption resulted in an elevation in baseline WNT signaling, which caused subtle alterations in cell fate at a neuronal time point (differentiation day 40)7. To examine whether elevated WNT signaling could induce the altered morphology observed in cerebral organoids with DISC1 disruption, organoids were treated with a WNT agonist (GSK3β inhibitor CHIR99021, 3 μM13) during days 6–19 of culture. Interestingly, incubation with the WNT agonist resulted in a structural change in wild-type organoids that phenocopied the effects of DISC1 disruption, including increased areas of dispersed cell morphology, and more small and disorganized rosette structures (Fig. 2e, f). WNT agonism of DISC1-mutant organoids did not have a marked effect on morphology (Fig. 2g, h). Furthermore, WNT antagonism (using tankyrase inhibitor XAV939, 2 μM14) similarly affected both wild-type and DISC1 ex8 wt/μ organoids to produce small, well-defined rosette/ventricle structures (Fig. 2i–l). Quantification showed that WNT antagonism with XAV939 rescued the phenotype of increased ventricle/rosette numbers with DISC1 disruption (normalized to organoid area, Supplemental Figure 1A).

DISC1 disruption reduces proliferation but does not affect apoptosis in early cerebral organoids

We compared expression of certain cell fate markers in wild-type and DISC1 exon 8-mutant organoids. Subventricular zone (SVZ) intermediate progenitor marker TBR2 and dorsal neocortical and ventricular zone (VZ) progenitor marker PAX615 were expressed similarly in WT and DISC1-disrupted organoids (Fig. 3a–d). The polarity of progenitors in neural rosettes was similar, with apical membranes directed interiorly, as identified by PKCλ and acetylated α-tubulin immunostaining (Fig. 3e–h). Markers of Cajal–Retzius cells, Reelin and p73, were identified in both WT and mutant organoids (Fig. 3i, j). Interestingly, expression of late neocortical progenitor and layer II–V neuronal marker BRN2/POU3F2, which has been shown to be critical for proper cortical lamination16, was markedly decreased with DISC1 mutation (Fig. 3k–l). Expression of neural progenitor markers PAX6 and TBR2 (EOMES), glutamatergic neuronal marker VGLUT1 (SLC17A7) and GABAergic neuronal marker VGAT (SLC32A1) were not significantly changed in d19 DISC1-mutant organoids, as measured by qPCR (Supplemental Figure 2).

Fig. 3: DISC1-mutant organoids exhibit decreased BRN2 expression and reduced proliferation that is rescued by WNT antagonism. Immunostaining was performed on WT and DISC1-mutant day 19 organoids for EdU incorporation and markers as shown. Expression of cell fate markers TBR2, PAX6, P73, Reelin and polarity markers PKC-λ, and acetylated-α-tubulin were grossly unchanged (a–j). However, immunostaining of BRN2 was markedly reduced in DISC1-mutant organoids (k, l). Scale bars: a–d 100 μm, e–h 20 μm, i–j 50 μm, k–l 20 μm. m, n WT and DISC1 exon 8 wt/μ organoids were immunostained at day 19 for markers of apoptosis (TUNEL and Cleaved Caspase 3). o, p Quantification of percentage of DAPI positive nuclei positive for TUNEL or cleaved caspase-3 shows no difference with DISC1 disruption. q–u WT and DISC1-mutant organoids with WNT agonism (CHIR) or WNT antagonism (XAV) were pulsed with EdU for 2 days (culture days 6–7), then fixed, sectioned, and stained for EdU and DAPI at day 19. One representative image for each condition is shown. s Percentage of EdU-positive nuclei were quantified. Data were derived from three independent differentiations. Statistics: o, p Variance was significantly different between conditions, Welch’s t-test. s Variance was significantly different between conditions, one-way ANOVA with Geiser–Greenhouse correction, Sidak’s multiple comparisons test. ***p < 0.001, ****p < 0.0001. Scale bars: m, n, q, p, t, u: 50 μm Full size image

DISC1 mutation does not alter apoptosis but results in a WNT-dependent decrease in EdU incorporation

We next investigated whether perturbations in proliferation or apoptosis were associated with the described morphologic alterations in DISC1-mutant cerebral organoids. To evaluate whether altered apoptosis also contributed to aberrant morphology, day 19 organoids were used for TUNEL immunohistochemistry and immunostaining for activated cleaved Caspase 3. There was no significant difference in the percentage of TUNEL+ or cleaved Caspase-3+ cells with DISC1 mutation, suggesting that altered apoptosis does not significantly contribute to the observed phenotype (Fig. 3m–p).

To examine potential changes in proliferation, cerebral organoids were pulsed with EdU during day 6–7 of differentiation, cultured in the presence of either DMSO, CHIR99021, or XAV939 during days 6–19, and then harvested and imaged at day 19 (Fig. 3q–u). As EdU was found to only incorporate into the outer areas of organoids, likely due to limited permeation of the 3D structure (Fig. 2a–l), EdU quantification was performed in these areas, defined in a blinded manner based on maximal penetration of EdU. Quantification showed that DISC1 disruption, as well as WNT agonism with CHIR99021, resulted in a decreased percentage of EdU-positive cells, whereas treatment of DISC1-mutant organoids with WNT antagonist XAV939 rescued this EdU incorporation phenotype (Fig. 3s). This implicates increased WNT activity as a mechanism resulting in decreased proliferation in DISC1-mutant organoids at this early developmental time point.

DISC1 disruption alters expression of genes implicated in neurodevelopment and migration and is mimicked by WNT agonism

To investigate factors contributing to the altered morphology of DISC1-mutant cerebral organoids, we assayed gene expression in day 19 organoids using a custom Nanostring panel of 150 genes related to neuronal development, maturity, and cell signaling. A heat map of expression of a subset of genes reveals the high organoid-to-organoid variability at the molecular level (Fig. 4a). Genes shown in the heat map mark particular subsets of cells in the central nervous system (CNS), revealing the variety in the cell types present in wild type and mutant organoids at day 19. These include markers of excitatory neurons (GRIA2, GRIK1, GRIN2A, VGLUT1, VGLUT2), inhibitory neurons (GAD1, GLRB), caudally located neurons of the hindbrain and spinal cord (HOXA1, HOXA2, HOXB1, HOXB4, IRX3, ISL1) and neural crest cells (HNK1), as well as cortical layer-specific markers (RELN, SATB1, CUX1, CTIP2, TP73, TBR1).

Fig. 4: DISC1 disruption alters BRN2 levels in organoids and monolayer cultures. Wild-type and DISC1 ex8 wt/μ organoids were harvested at day 19 and RNA was used for Nanostring analyses. a Heat map of cell type markers for wild type (wt) and DISC1 ex8 wt/μ organoids. b Volcano plot of gene expression changes in DISC1 ex8 wt/μ vs. wild-type organoids are shown. Statistics: Student’s t-test, unadjusted p-values plotted; n = 7 for wild-type, 8 for DISC1 wt/μ, from four independent differentiations. See also Supplementary Table 1. c, d qPCR was performed for BRN2 and CALB1 on RNA derived from day 19 organoids. Data were derived from three independent differentiations. For each differentiation, over five organoids were pooled for analysis. e Wild-type and DISC1 ex8 w/μ iPSCs were differentiated to NPCs using an EB protocol7 and subsequently dissociated and plated as monolayer culture. Day 40 neuronal RNA was harvested and used for Nanostring. Data were derived from three to seven independent differentiations. Statistics: c One-way ANOVA with Sidak multiple comparisons test, d,e Student’s t-test, *p < 0.05, **p < 0.01, ****p < 0.0001. f Table summarizing gene expression changes across both differentiation methods: organoid day 19 qPCR (from c, d and other data not shown) and day 40 monolayer Nanostring data. Asterisks indicate significance by two-tailed Student’s t-test; #indicates data published in ref. 7 Full size image

Due in part to inherent variability of cerebral organoids at the molecular level, no genes achieved statistical significance with correction for multiple comparisons across the 150 genes examined. Using unadjusted p-values, DISC1 expression was one of the more significant findings, which is consistent with our previous findings of decreased DISC1 expression with this genomic mutation due to nonsense-mediated decay (as previously observed in neural cultures derived from these cells7). Among the other gene changes highlighted by this discovery set were decreased expression of BRN2/POU3F2, Calretinin (CALB2), and EAAT2/SLC1A2, and increased expression of Pancortin (OLFM1), Calbindin (CALB1), FEZF2, and NRG1 (Fig. 4a, b).

To test the validity of observed gene expression changes, a set of replication experiments were performed to measure a selection of genes using qPCR. This confirmed decreased BRN2 and increased CALB1 expression (Fig. 4c, d). Although some other genes showed trends for altered expression in agreement with Nanostring results (decreased EAAT2 and CALB2; increased FEZF2 and OLFM1), these did not reach significance.

Given the variability of organoid cultures, we complemented this assay with a study of neuronal cells differentiated via a similar protocol but with plating of neuroepithelial cells in a monolayer9. Previous reports have shown that non-neural cells can arise using the protocol on which our organoid protocol is based, which could be contributing to our observed variability6. An important difference between our organoid and monolayer protocol is the addition of a purification step to separate the neural from non-neural cells via adherence to Matrigel followed by rosette selection. Indeed, this reduced the heterogeneity in the cell types present between individual cultures, therefore reducing variability at the gene expression level (Fig. 5e, Supplementary Table 2). Interestingly, of the genes highlighted in Fig. 5b, BRN2, EEAT2, and NRG1 expression all were significantly reduced with DISC1 mutation in this monolayer system (Fig. 4e and ref. 7). A summary of organoid qPCR data (Fig. 4c, d and data not shown), as well as monolayer day 40 Nanostring data (Fig. 4e) is shown in Fig. 4f.

Fig. 5: DISC1 disruption alters expression of genes implicated in neurodevelopment and migration and is phenocopied by WNT agonism. Wild-type and DISC1 ex8 wt/μ iPSCs were differentiated to NPCs and treated with vehicle (DMSO) or WNT agonist CHIR99021 (CHIR) during days 7–17, followed by withdrawal of small molecules and subsequent monolayer culture. Day 40 neuronal RNA was harvested and used for Nanostring. a Venn diagram showing genes with significantly altered expression with shared direction of fold-change compared to wild-type. Number of genes per category is shown within the diagram. Statistics: Student’s t-test with multiple comparisons correction using 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. b–d Nanostring data for select genes are shown, including genes associated with cell fate (b), genes associated with glutamatergic neurotransmission (c), and genes associated with interneuron development (d). Statistics: Holm–Sidak; WT n = 15, WT CHIR n = 18, DISC1 ex8 wt/μ n = 7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Table 2 Full size image

As WNT agonism phenocopied the organoid morphology of DISC1 disruption, we sought to investigate the shared effects of DISC1 mutation and WNT agonism on gene expression. Neuroepithelial aggregates were cultured with WNT agonist CHIR99021 during days 7–17 of differentiation, followed by withdrawal of the WNT agonist and neuronal culture in traditional differentiation media until day 407. Changes observed at day 40 therefore represent long-lasting changes in cell state that persist more than 20 days after cessation of WNT agonism. Day 40 RNA was harvested and used for the Nanostring assay. A selection of gene expression changes observed under these conditions was published previously7, but here we sought to more globally evaluate changes in gene expression that were shared between DISC1 disruption and WNT agonist treatment. Expression of a subset of general markers of neuronal and astrocyte fates were unchanged with DISC1 mutation or CHIR99021 treatment (Supplementary Table 2, ref. 7). DISC1 exon 8 mutation altered expression of 74 genes, whereas early CHIR99021 treatment of wild-type cells changed expression of 115 genes (Fig. 5a). Strikingly, 51 shared genes were significantly altered with the same directional fold-change from wild-type in each condition. This represents 69% of all genes altered with DISC1 disruption. The marked overlap of gene expression changes with WNT agonism and DISC1 mutation further support a model in which DISC1 disruption leads to downstream changes in neural cells via augmented WNT signaling.

Gene expression changes shared between DISC1 ex8 wt/μ and CHIR99021-treated wild-type cells included many genes important for neurodevelopment and cell fate. A handful of these were published previously (including BRN2 (POU3F2), DAB2, FOXG1, GSX2, HES1, IRX3, SIX3, TBR2, and WNT3A)7. Of those genes that showed a suggestion of alteration in day 19 organoids, BRN2 and EAAT2 were also decreased in neurons with DISC1 mutation and in wild-type neurons exposed to CHIR99021 (EAAT2 shown in Fig. 5c; BRN2 in ref. 7, normalized data in Supplementary Tables 1 and 2). DISC1 mutation and CHIR99021 treatment also led to decreased expression of lower-layer cortical neuronal marker FOXP2 and increased expression of upper-layer marker CUX1 (Fig. 5b). Decreased expression of the immature neuronal marker DCX and increased expression of pro-proliferative protein CCND2 and neural progenitor marker Vimentin (VIM) further pointed to alteration in neural progenitor fates with DISC1 mutation at this later developmental stage (Fig. 5b). We also observed increased expression of DIXDC1, a regulator of neurogenesis (Fig. 5b). Expression of glutamatergic cell markers showed decreased GRIN1 and GRIN2B expression, but increased VGLUT1 (SLC17A7) expression (Fig. 5c). Interestingly, expression of ventral progenitor markers GSX27 and GSX1, and GABAergic cell markers GAD1, GAD2, and VGAT (SLC32A1) were decreased, suggesting perturbed interneuron development (Fig. 5d). Recapitulation of DISC1 ex8 wt/μ expression changes with WNT agonism in wild-type cells was consistent with increased WNT signaling in DISC1-mutant lines causing cell fate and morphologic phenotypes. Overall, these data show that DISC1 disruption alters expression of select genes related to neural development and migration in cerebral organoids and monolayer neurons, many of which are induced by WNT agonism in wild-type cells.