Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Here, we have taken the approach of directly modeling early cortical development in probands with idiopathic ASD. We focused on individuals with increased head/brain size (macrocephaly), as this is one of the most consistently replicated ASD phenotypes () and confers poorer clinical outcomes among ASD patients (). Using induced pluripotent stem cells (iPSCs) obtained from affected families, we have produced telencephalic organoids that recapitulate transcriptional programs present in mid-fetal human cortical development. Transcriptome and cellular phenotype analyses in this model identified unexpected differences in cell-cycle time and synaptic growth, as well as an imbalance in GABA/glutamate neuronal differentiation in patients as compared to their unaffected family members.

The applicability of these pathogenetic mechanisms to the great majority of ASD cases remains unknown. Furthermore, the heterogeneity of phenotypes found when modeling these mutations in animals and the inherent difficulty in creating behavioral phenotypes of ASD in rodents have complicated the construction of credible animal models of ASD. It is possible that the heterogeneity of rare mutations found in ASD, as currently conceptualized, denies a unified understanding of the pathophysiology of the disorder. However, emerging evidence suggests that current genomic data, when considered in the framework of gene network analyses, point to a common pathophysiological substrate in ASD rooted in the embryonic development of the cerebral cortex ().

Rare penetrant mutations, common genetic variants, and environmental factors are known to contribute risk to ASD, yet, about 80% of the cases have no clear etiology and no pathogenetic model. A large number of rare mutations have been identified in the context of syndromic and non-syndromic ASD and have been modeled in various organisms. However, these mutations are extremely heterogeneous, each accounting for less than 1%–2% of cases. Furthermore, in no instance have they been shown to be sufficient to cause ASD; rather, they interact with other inherited and non-inherited risk factors. Some mutations involve synapse-associated molecules () and have led to the widespread notion that alterations in the assembly of synaptic connections are key in the pathophysiology of ASD. Others have formulated the hypothesis that there is an excitatory/inhibitory neuron imbalance in the disorder ().

Next, we correlated the patients’ HC and their autism symptom severity with gene expression indices in our organoid model in the four families used in this study. Despite the small sample size, we found a consistent positive correlation between HC and autism symptom severity with the upregulated modules as well as a negative correlation with the downregulated modules ( Figure 2 D). The HC Z scores of probands displayed particularly strong correlations with all module’s eigengene and levels of FOXG1 gene expression at TD31 ( Figure 2 D). However, the module’s eigengenes displayed no correlation with the HC of the unaffected fathers, despite the fathers presenting the same degree of macrocephaly as the probands (see Figures 2 E and 2F). Together, the observed patterns of correlations suggest that the upregulation in the magenta, blue, brown, and tan gene network is a maladaptive trait in the probands and that it may represent a pathophysiological antecedent of symptoms.

Previous studies suggest a strong association between increased head circumference (HC) in ASD and more severe autism symptoms and lower IQ (). Given the small sample size (n = 4) and limited range of severity scores in the probands, we were not able to evaluate these associations directly. However, in a supplementary analysis we obtained the correlations of interest after adding five participants who did not all meet the stringent criteria for macrocephaly that we required for deriving iPSCs (see “Participant” section in Supplemental Experimental Procedures ) and thus display a wider spectrum of head circumference sizes. Correlation analysis in this enriched sample (n = 9) indicated strong associations between HC and autism symptom severity (Spearman r = 0.79, p = 0.01) (data not shown).

To investigate the mechanism by which FOXG1 could affect the overproduction of GABAergic neurons, we compared cell proliferation in ASD- and control-derived organoids by BrdU incorporation with or without FOXG1 RNAi. Quantification of double-labeled BrdU/Ki67cells at TD11 revealed no general changes in proliferation between proband- and control-derived organoids ( Figures 7 A and 7B and Figure S6 A and S6B). However, there was a significant increase in the number of DLX2cells that incorporated BrdU in proband-derived organoids, as well as an increased proportion of BrdUcells that colocalized DLX1/2. Both effects were precluded by FOXG1-knockdown ( Figures 7 A–7C). Furthermore, at TD31, proband-derived organoids showed a greatly increased proportion of DLXand GAD1cells that had incorporated BrdU at TD11 ( Figures 7 D–7H), an effect that was also greatly attenuated by FOXG1 RNAi, which lowered the proportion of BrdU/DLXand BrdU/GAD1cells in ASD-derived organoids to levels comparable to those of the unaffected control ( Figures 7 D–7H). Moreover, GAD1cells at TD31 were not aberrantly entering the cell cycle, suggesting that GABAergic neurons were terminally differentiated in the organoids ( Figures S6 C and S6D). Taken together, these data suggest that the early increase in proliferation of GABAergic neuronal progenitor cells in proband-derived organoids gave rise to an increased proportion of mature GABAergic interneurons, and that FOXG1 RNAi restored both these early and late effects to levels comparable to those found in unaffected family members. Similar experiments revealed smaller or non-significant changes in the proliferation of PAX6and TBR1precursor cells after FOXG1 RNAi ( Figure S7 ), suggesting that upregulated FOXG1 expression in ASD neural cells was driving an early proliferative effect in neuronal precursor cells of the GABAergic lineage.

Representative images (A) and stereological quantification (C and D) of PAX6 + /BrdU + double positive excitatory progenitor cells over total BrdU + cells in TD11 and TD31 organoids with or without RNAi-mediated FOXG1 knockdown. Representative images (B) and stereological quantification (E) of TBR1 + /BrdU + double positive excitatory neurons over total BrdU + neurons with or without RNAi-mediated FOXG1 knockdown. In all cases BrdU incorporation was at TD11 and analyses at TD11 or TD31. Data in (C–E) are presented as means ± SEM; ∗∗ p < 0.01, t test analysis. Scale bars, 10 μm (A), 10–20μm (B).

Not Significant or Transient Changes in the Proportion of Excitatory Progenitors after FOXG1-Knockdown, Related to Figure 7

(A and B) Proliferation in proband- and control-derived organoids with or without FOXG1 knockdown. Representative images and relative stereological quantification of BrdU + and Ki67 + cells in TD11 organoids derived from 07-F#2, 07-P#9 shRNA-C, and 07-P#9 shRNA-3. (C and D) Representative images of Ki67 and GAD1 double immunostaining and stereological quantification in organoids at TD31 in the indicated lines. Note that total proliferation as assessed by % Ki67 + cells is not changed at this time point and that there is no aberrant proliferation of GABAergic mature interneurons at TD31. Data in (B and D) are presented as means ± SEM; ∗∗∗ p < 0.001, t test analysis. Scale bars, 10 μm.

Data in (B, C, E, F, G, and H) are presented as means ± SEM;p < 0.05,p < 0.01,p < 0.001, t test analysis. Scale bars, 10 μm. See also Figures S6 and S7

(A–H) Representative images (A) and (D) and stereological quantification of BrdU + /DLX1-2 + proliferating cells (B), (C), (E), and (F) and BrdU + /GAD1 + neurons (G) and (H) in TD11 and TD31 organoids derived from 07-F#2 (father), 07-P#9 shRNA-C, and 07-P#9 shRNA-3 (proband’s iPSC lines transduced with shRNA-C or with shRNA-3). The selectively increased proportion of DLX 1-2 + /BrdU + double positive cells in patient-derived organoids at both TD11 (B) and (C) and TD31 (E) and (F), was restored to a physiological level after FOXG1-knockdown at both time points. The increased proportion of proliferating DLX1-2 GABAergic progenitors (A and D, upper panel) resulted in an overproduction of more mature GABAergic GAD1+/BrdU+ double positive interneurons (D, bottom panel) overproduction that was restored to physiological levels after FOXG1-knockdown (G and H).

As proof of principle, we generated four stable iPSCs lines (three of which stably expressed different shRNAs specifically targeting FOXG1 and one expressed a non-targeting shRNA control) from a proband-derived iPSC line (07-P#9). To confirm stable downregulation of FOXG1 expression, we performed qPCR analyses at TD11 ( Figure 6 A). Introduction of two FOXG1-targeting shRNAs (shRNA-2 and 3) downregulated FOXG1 mRNA expression to a level comparable to that of the unaffected family member ( Figure 6 A, compare bar 6 and 7 with bar 2). Immunostaining for FOXG1 confirmed that shRNA-2 and 3 were able to downregulate its expression also at the protein level ( Figures 6 B–6F). We next analyzed the expression of GABAergic markers after FOXG1-RNA interference (RNAi) at the transcript and protein level. At TD11, organoids derived from the iPSC lines stably expressing FOXG1 shRNA-2 and shRNA-3 (07-P#9 shRNA-2 and 3) showed downregulation of DLX1, DLX2, and GAD1 transcripts as compared to the same iPSC line expressing the shRNA control (07-P#9 shRNA-C) ( Figures 6 G–6I). Immunostaining and stereological quantification of DLX1-2 and GAD1 positive cells showed that FOXG1 RNAi restored the normal level of GABAergic neuronal differentiation in proband-derived organoids at both TD11 and TD31 ( Figures 6 K–6M). These results suggest that FOXG1 is involved, at least in part, in causing the overproduction of neurons of the GABAergic lineage found in ASD-derived organoids. FOXG1 RNAi had no or minor effects on the transcript/protein expression levels of dorsal forebrain markers (such as PAX6) ( Figures 6 B–6F,6J, 6L, 6M), or on TFs directing cortical excitatory neuron differentiation (such as TBR1) ( Figure 6 M).

(L and M) Stereological quantification of immunocytochemical (ICC) staining for GABAergic (DLX1-2, GAD1) and glutamatergic markers (PAX6, TBR1) at TD11 (L) and TD 31 (M). (Sample names: 07 = family name from which iPSCs were derived; F = Father; p = Proband; # = iPS clone number).

(G–J), qPCR for DLX1 (G), DLX2 (H), GAD1 (I), and PAX6 (J) in TD11 organoids from shRNA-C and shRNA-1/2/3. (K), DLX1-2 and GAD1 double immunostaining in organoids derived from the father or the proband transduced with shRNA-C or shRNA-3 at TD11 and TD 30.

(A) Relative expression levels of FOXG1 by qPCR among non-virally transduced undifferentiated iPSCs from proband #9 (i07-P#9), TD11 organoids from the proband (07-P#9), his father (lines 07-F#1), and proband’s organoids harboring a non-targeting shRNA-control (shRNA-C) or three different shRNAs targeting FOXG1 (shRNA-1, shRNA-2, and shRNA-3).

We therefore tested the hypothesis that abnormally high levels of FOXG1 and its downstream genes could be responsible for the phenotypic abnormalities identified in neuronal cells of macrocephalic ASD patients. To this end, using lentiviruses carrying short hairpin RNAs (shRNAs) targeting FOXG1, we tested whether an attenuation of the FOXG1 expression level in patients’ neural cells was able to revert some of the neurobiological alterations.

Our DGE results show that DLX6-AS1, TMEM132C, FOXG1, C14orf23, and KLHDC8A are consistently among the top 10 upregulated genes at both TD11 and TD31 ( Table S2 A). Among these genes, FOXG1, which is one of the top 100 hubs in the magenta module, with an 8.5- and 13-fold increase in expression at TD11 and TD31, respectively ( Table S2 A), is a transcription factor important for the development of the telencephalon (). Notably, loss-of-function mutations in FOXG1 have been found in patients with atypical Rett syndrome () and confer a small brain size (). As the probands under investigation have large brain size, it is possible that FOXG1 may be, at least in part, involved in the modulation of the brain size phenotype and possibly in the social disability component of the phenotype.

We also found evidence for increased expression of GABAergic phenotypes electrophysiologically. Although the size of sodium currents in control- and proband-derived neurons was similar ( Figure S3 ), there was substantial cell-to-cell variation in the voltage dependence of activation and inactivation, suggesting that different neurons express different proportions of brain sodium channel isoforms (Na1.1, Na1.2, Na1.3, and Na1.6). Figure 5 shows results for steady-state inactivation. Neurons from probands and their familial controls displayed substantial variation in the pre-test potential (V) at which the peak sodium current was reduced to half its maximal amplitude (E), and adequate fits to some of the data required two Hodgkin-Huxley components ( Figures 5 D and 5E). Interestingly, sodium currents in proband-derived neurons tended to inactivate at more hyperpolarized membrane potentials than the corresponding currents in control neurons ( Figure 5 C–5E; n = 7 ASD and 10 control neurons). The increased proportion of proband-derived neurons that gave Evalues in the range −72 to −65 mV ( Figure 5 F) is consistent with increased expression of the Na1.1 isoform in these cells. This isoform is preferentially expressed in GABAergic interneurons () and the increased proportion of cells with this sodium channel phenotype is consistent with a greater proportion of GABAergic neurons in cortical organoids from probands with ASD.

(F) Bar graph depicting the percentage of control and patient neurons (n = 10 and 7) with Evalues that fell within the indicated ranges. When two components were present, the fractional amplitudes of each were used in the calculation of mean percentages for each group. Cells that gave Evalues ≤ −65 mV gave half-activation voltages that ranged from −39.1 to −55.8 mV, a phenotype most consistent with the Na1.1 brain sodium channel isoform ().

(D and E) Steady-state inactivation data obtained from ten control neurons (D) and seven patient neurons (E). Data for individual cells are shown with different symbols and colors (normalized data in (C) are shown here with the same symbols: control, open black square; patient, filled black circle). For some neurons, the results were better fitted as the sum of two components (arrowheads point to E h1/2 values for each component).

(C) Peak inward current amplitudes as a function of pre-test potential from the data in (A and B). The results were normalized to Imax values obtained for each neuron from fits to the raw data (as in D and E).

(A and B) Inward sodium currents evoked by depolarizing test jumps to −20 mV from pre-test potentials of −90 mV to −25 mV (A) or −45 mV (B) in steps of 5 mV.

GABA precursors arose in a segregated fashion in an area of the organoids and did not colocalize with TBR1excitatory neuron precursors ( Figure S5 ). The number of cells immunoreactive for ASCL1/MASH1 and NKX2.1 (two TFs expressed by GABAergic progenitor cells) and the neurotransmitter GABA was also increased in ASD-derived organoids ( Figure S5 ). Moreover, western blot analyses further confirmed increased protein expression of GAD1/2 in ASD organoids ( Figures S5 Q and S5R). Along with the upregulation of GSX2, ASCL1, DLX1, DLX2, DLX5, DLX6, and DLX6-AS1, which is the top upregulated gene at TD11 ( Table S2 ) and the increase in GABA transporter immunostaining ( Figures 3 H and 3I), these cellular analyses strongly suggest an overproduction of progenitors and neurons of the GABAergic lineage as well as an altered balance between the number of excitatory and inhibitory neurons in organoids derived from probands ( Figure 4 K).

(A–R) GABAergic progenitors (DLX1-2 + ) and mature interneurons (GAD1 + ) in control (A, C) and probands (B, D, E-H) arise in distinct areas of the organoid devoid of excitatory neuronal progenitors expressing TBR1 (C, D) and Pax6/Tbr2 (not shown). (E–H) High magnifications of areas in boxed areas in (A–D). (I–P) GABAergic transcription factors ASCL1/MASH1 and NKX2.1, GAD1 and GABA show enhanced expression in ASD-derived organoids as compared to control-derived organoids. Representative images of control-derived and proband-derived organoids at TD11 are shown for ASCL1/MASH1 (I and J) and DLX1-2 and NKX2.1 (K and L). (M–P) Double-immunostaining of control-derived and proband-derived organoids at TD31 labeled with antibodies against GAD1 and GABA. (O, P) show high magnifications of areas in (M and N). Scale bars, 10-20 μm as indicated. (Q and R) Western blot images (Q) and relative quantification (R) of GAD1/2 expression at TD31 in cortical organoids derived form an independent set of neuronal differentiation experiment of family 07 and family 1120. C=controls (1120-F#6, 07-F#1), P=probands (1120-P#2, 07-P#7).

We next investigated determinants of GABAergic inhibitory neuronal fates using DLX1-2 (two TFs which are among the earliest determinants of GABAergic fate in telencephalic precursors cells and upregulated members of the magenta module) and GAD1/GAD67 (the GABA synthetic enzyme, an upregulated member of the tan module). The expression of these GABAergic markers was increased significantly in organoids derived from ASD individuals compared to those from unaffected family members. This increase was strong at TD31 and was already detectable at TD11 ( Figures 4 G–4J). The increase in DLX1/2 and GAD1/GAD67 was consistent in probands irrespectively of iPSC line, or individuals within different families ( Figures S4 G–S4L). Also, the increases in DLX1/2 and GAD1 in probands with respect to their fathers were reproducible across independent differentiation experiments ( Figure S4 M).

Next, we directly tested whether there was any bias for differentiation into specific neuronal subtypes. In this analysis, we used as markers TFs which control fate choice, cell proliferation, and neuronal differentiation during normal telencephalic development, many of which were members of the magenta and blue modules ( Figure 2 C; Tables S5 and S6 ). We found that the proportions of cortical excitatory neuron precursors of the subventricular zone expressing EOMES/TBR2, of layer 6 neurons expressing TBR1, and of early-born layer 5 CTIP2 neurons (also known as BCL11B) were not significantly different in ASD and control organoids at TD31 ( Figures 4 A–4F), although some of these cortical excitatory neuron markers (such as TBR1, TBR2, CTIP2 and SOX5) were upregulated in proband-derived organoids at the mRNA level.

C = Controls, p = Probands.p < 0.01,p < 0.001, t test analysis. Scale bars, 10 μm (A), (C), (E), (G), and (K), 20 μm (I) and (J). See also Figure S4 and Figure S5

(K) Plots showing percentages of inhibitory (GAD1 + ) and excitatory (TBR1 + , CITIP2 + ) neurons in ASD-derived and control-derived organoids at TD31. Total cells are estimated by counting DAPI + nuclei.

(A–J) Representative images of control-derived and ASD proband-derived organoids: (A–C) Immunostaining of SOX1 + and PAX6 + proliferating radial glia progenitors, cortical excitatory TBR2 + intermediate progenitors, and more mature TBR1 + and CTIP2 + excitatory neurons at TD31. (G–I) Immunostaining of GABAergic inhibitory progenitor cells (DLX1-2 + ) and mature GABAergic interneurons (GAD1 + ) at TD11 and TD31. (D, E, F, and J) Box plots showing the stereological quantification of the immunostaining. The upper and lower error bars in each box plot represent the top whisker (maximum value-quartile3) and bottom whisker (quartile1-minimum value), respectively.

We then assessed neuronal maturation and synaptic formation in organoids at TD31. Quantification of microtubule-associated protein 2 (MAP2) showed a significant increase in its density in ASD-derived neurons ( Figures 3 E and 3F). Moreover, synapse number quantification revealed a significant increase in Synapsin I-immunoreactive (SynI)puncta in ASD-derived neurons, suggesting increased neuronal maturation and synaptic overgrowth ( Figures 3 E and 3G). This is in agreement with the upregulated expression of the blue and brown modules ( Figure 2 A), whose signatures suggested accelerated or increased neuronal differentiation and synaptic connections in probands. Furthermore, quantification of the inhibitory VGAT (vesicular GABA transporter) and excitatory VGLUT1 (vesicular glutamate transpoter-1) immunoreactivity revealed a significant increase in VGAT-immunoreactive puncta ( Figures 3 H and 3I) and no significant changes in VGLUT1 puncta ( Figure 3 J), suggesting an increase in the number of inhibitory synapses in ASD-derived neurons.

In summary, the signatures that emerged from our transcriptome analyses as significantly perturbed in probands were transcriptional regulation of cell proliferation/cell fate, neuronal differentiation/process outgrowth, and synaptic transmission. To functionally validate these signatures, we performed morphometric cellular analyses and immunostaining for cell fate markers. We first studied the dynamics of the cell cycle in undifferentiated iPSCs and neuronal progenitors (TD11) by BrdU incorporation ( Figures 3 A and 3B ). These experiments revealed a significant decrease in cell-cycle length in ASD-derived iPSCs ( Figure 3 A). We saw a similarly strong trend in early neuronal progenitors cultured as monolayers ( Figure 3 B). However, when we estimated the proportion of proliferating cells in more mature organoids (TD31), there was no significant difference in proliferation between ASD- and control-derived organoids ( Figures 3 C and 3D). Taken together, these results suggest that a decrease in cell-cycle length could be an early event that is present at the iPSC undifferentiated stage and during the early stages of neuronal differentiation, which is consistent with the transient increase in organoid size at TD11 ( Figure S3 B).

The data in (A, B, D, F, G, I, and J) are presented as means ± SEM; ∗∗ p < 0.01, ∗∗∗ p < 0.001; t test analysis. Scale bars, 10 μm (A), 10–20 μm (B), as indicated, 10 μm (C), 5 μm (E) and (H).

(E–J) Increased neuronal maturation and synaptic formation in ASD-derived neurons (TD 31). Representative images of controls and probands organoids at TD 31 labeled with MAP2 and SynI (E), or MAP2 and VGAT (H), and relative quantification of MAP2 density (F), number of SynI + puncta (G), number of VGAT + puncta (I), and number of VGLUT1 + puncta (J).

(C and D) Representative images (C) and stereological quantification (D) of the proportion of proliferating Ki67 + neuronal progenitor cells in both control and ASD-derived organoids at TD31.

(A and B) Cell-cycle time determined by the formula Tc = Ts/ (%BrdU/Ki67), where Tc = cell-cycle time and Ts = S phase time. (A) Representative image of double immunostaining for BrdU (red) and Ki67 (green) of undifferentiated iPSCs from a control individual and (B) early neuronal progenitors in monolayer cultures at TD11. ∗ p < 0.05, ANOVA with family as covariant. To prepare monolayer cultures for neuronal progenitors the neural rosettes were dissociated into single cells and cultured in adhesion as monolayers until TD11.

For the brown and the tan modules, the GO and top pathways annotation were mostly related to synaptic functions, ion channels, and ligand-receptor interactions ( Tables S5 and S6 ), which is in harmony with their more significant eigengene upregulation in probands at the later time point ( Figure 2 A). The brown module displayed the strongest enrichment with respect to the SFARI autism gene dataset, a collection of genetic information that includes data from linkage and association studies, cytogenetic abnormalities, and specific mutations associated with ASD ( https://gene.sfari.org ) (SFARI category S to 4) () (p value = 6.51E-5). Among the top 100 hub genes of the brown module, 64 were upregulated at TD31, including molecules involved in synaptic assembly, ion transport, and post-synaptic signaling (i.e., NRXN1, NRXN2, SLITRK1, CAMK2B, CAMK1D, NRSN1, SYT13, GRIN1, and SCN2A) ( Table S7 . Among the hub genes, NRXN1, SCN2A, and TSPAN7 were significantly upregulated as well as overlapping with genes in the SFARI autism database ( Figure 2 C). The tan module was characterized by an upregulation at TD31 of transcripts for several potassium channels and for key components of GABAergic neurons, including the GABA synthetic enzyme GAD1 and three GABA receptor subunits, most of which were in the tan module’s 100 hub genes ( Table S7 ).

For the blue “neuronal differentiation” module, canonical pathway annotation was enriched in axon guidance and generic transcription pathways ( Tables S5 and S6 ). The axon guidance genes, all upregulated in probands, included members of the neural cell adhesion family (NCAM1, NRCAM, L1CAM, and NFASC), semaphorin (PLXNC1 and PLXNB3), netrin (DCC and UNC5A), Rho GTPases (PAK3 and PAK7), CDK5R1/P35, and DCX. Many of these molecules were among the top 100 hub genes, i.e., CDK5R1, DPYSL5, DCX, DPF1, and APC2 ( Figure 2 C; Table S7 ). Hence, genes in the blue module are involved in cytoskeletal regulation of various cellular functions, including neurite outgrowth, axon guidance, cell proliferation, migration, and survival.

We further refined our understanding of the functional annotations of the upregulated magenta, blue, brown, and tan “neuronal” modules by investigating their canonical pathways annotations. The magenta module was significantly enriched in transcription- and cell-cycle-related canonical pathways ( Tables S5 and S6 ). Indeed, among the significantly upregulated genes in this module are a number of transcription factors (TFs) crucial for acquisition of neural cell fates and precursor cell proliferation in the telencephalon, including DLX6-AS1, FOXG1, EOMES, POU3F3/BRN1, SOX3, SOX5, GSX2, ETV1, DLX1, DLX6, E2F2, and SYNE2 ( Tables S4 S5 , and S6 ). Many of these TFs are also hub genes ( Figure 2 C; Table S7 ). Overall, genes in the magenta module function in the transcriptional regulation of cell fate and cell proliferation in the forebrain.

We then asked whether, at a system level, the DEGs underline coherent alterations in groups of co-expressed genes. Using weighted gene co-expression network analysis, WGCNA (), we identified 24 modules of co-expressed genes across probands and controls at TD11 and TD31, all of which survived permutation analysis ( Table S3 ). We estimated the modules’ eigengenes (i.e., the first principal component of the module’s genes expression profiles) and assessed their changes over time and across diagnosis (see Figure 2 A). The yellow and green modules (annotated by “vascular development” and “lipid metabolism” gene ontology [GO] categories, respectively) were enriched in downregulated DEGs ( Table S4 ), and their eigengenes were consistently downregulated across time points ( Figure 2 A). The blue and magenta modules (annotated by “neuronal differentiation” and “regulation of transcription” GO terms, respectively) were enriched in upregulated genes at both TD11 and TD31 ( Table S4 ), and their eigengenes were consistently upregulated across time points, in keeping with their developmental annotation. The brown and tan modules (annotated by “synaptic transmission” and “gated channel activity” GO terms, respectively), however, were enriched in upregulated genes only at TD31 ( Table S4 ), and consistently, their eigengenes were upregulated more at TD31 than at TD11, in line with their synaptic functional annotation ( Figure 2 A). Hierarchical clustering of module eigengenes also showed a tighter correlation among the green and yellow, the blue and magenta, and the brown and tan modules ( Figure 2 B), suggesting similar functions and/or tighter regulatory interactions. In sum, probands were characterized on the transcriptome level by a downregulation of non-neuronal and a corresponding upregulation of neuronal transcript modules.

(C–F) Top 200 hub genes networks for three representative modules (C). Circles: genes; diamonds: genes overlapping with genes in the SFARI database classified as associated to ASD; red: genes overexpressed at either TD11 or TD31; gray: no changes in gene expression; larger font: genes differentially expressed at both TD11 and TD31; (D) Pearson’s correlation between modules’ eigengenes with log transformed HC Z scores and ADOS severity scores at TD11 and TD31; ∗ nominal p value < 0.1; (E and F) Pearson’s correlation between HC Z scores and modules’ eigengenes or FOXG1 expression levels for the unaffected fathers (E) and the affected probands (F) at TD31.

(A) Relationship between the modules’ eigengenes with diagnosis and time in culture. TD11: 11 days of terminal differentiation; TD31: 31 days of terminal differentiation; F: fathers; P: probands.

Next, we compared the transcriptomes of the four probands to those of the unaffected family members (two to three iPSC clones per person) at two time points, TD11 and TD31. Differential gene expression (DGE) between the probands and the respective fathers, used as sex-matched normal controls, identified 1,062 differentially expressed genes (DEGs) at TD11 and 2,203 DEGs at TD31 ( Table S2 A), hinting at a possibly divergent developmental trajectory between controls and probands. Validation by qPCR of a subset of the DEGs identified by RNA-seq revealed a 0.98 correlation coefficient between logfold changes from the two techniques and 100% concordance in direction of change ( Table S2 B). The individual-to-individual (biological) variability, as modeled in RNA-seq DGE analyses, is clearly narrow ( Table S2 ). The iPSC line-to-line variability is also quite low, as shown by the boxplots of correlation coefficients within each individual (intra) and across individuals (inter) ( Figure S4 ). In fact, the variability between lines from the same individual is lower than the variability between lines across individuals ( Figures S4 A–S4C). This reproducibility is likely due to the robustness of our telencephalic organoid preparation, which selects for forebrain progenitors while allowing spontaneous 3-D organization.

(D–M) Variability in stereological cell counts when comparing organoids of different iPSC lines of the same individual, organoids of different individuals within a family, and individuals of different families. F= fathers, M= mothers, P= probands; # indicate different iPSC lines; family and day of terminal differentiation are shown above each plot. Note the consistent increase in DLX1/2 (G and I) and GAD1/GAD67 (H and J) in probands irrespectively of iPSC lines, or individuals within different families. Also note that general neuronal markers TUBB3 and SOX1 show no change across lines, individuals and families. (M) Variability across experiments. Note that the increases in DLX1/2 and GAD1 in probands with respect to their fathers are reproducible in 3 independent differentiation experiments. Data in (D–M) are presented as means ± SEM.

(A–C) Variability in global gene expression between iPSC lines of the same individual (intra) and across individuals (inter). The box plots are made by computing the Spearman correlation coefficients between RNA-seq data (in RPKM) of organoids from different iPSC lines within or across different individuals, at the 2 different time points. TD= day of terminal differentiation. Note that the variability between lines is much lower than the variability between individuals.

To assess both the region specificity and maturity of our organoid preparation in a comprehensive fashion, we analyzed the global transcriptome of organoids by RNA-seq at each of three time points (rosette stage, TD11, and TD31, two to three iPSC lines per individual, total 45 samples). The organoid’s transcriptomes were compared with BrainSpan, the largest dataset of post-mortem human brain transcriptomes from embryonic age to adulthood (). This comparison indicated that our preparation best reflected the transcriptome of the human brain during early fetal human development (9 weeks post-conception), with TD31 cells displaying significant correlations also with second trimester human brain samples, up to 13–16 weeks post-conception ( Figure 1 G). With regards to regional specification, the transcriptome of iPSC-derived organoids was most similar to the human dorsal telencephalon (cerebral cortex and hippocampus), particularly to dorsolateral, medial, and orbitofrontal cortical areas, with smaller but significant homology to the cerebellar anlage and the amygdala ( Figures 1 H and 1I).

To compare the electrical excitability of iPSC-derived neurons from probands with those from familial controls, we made whole-cell patch-clamp recordings from neurons in dissociated cultures or at the edge of organoids. All neurons examined (n = 99) expressed voltage-activated sodium and potassium currents that were of similar amplitude in control and proband neurons from two different families ( Figures S3 E–S3G). Because we observed that iPSCs differentiated using dissociated monolayers were not able to robustly generate ventral telencephalic neurons and express virtually no GAD1cells, we limited our experiments to organoid preparations. In recordings from neurons in the organoids, the voltage-activated currents supported action potential firing in 18/18 control neurons and 12/14 proband neurons, with thresholds of −42.5 ± 0.8 mV and −37.9 ± 2.1 mV (controls, probands; p = 0.06) and action potential overshoots of 30 to 65 mV. Most neurons fired only a single action potential, but some fired multiple spikes ( Figure 1 C). In total, the electrophysiological data indicate that the cells studied here display voltage-gated channels similar to those in central neurons. In addition to these signatures of electrical excitability, we also recorded spontaneous synaptic currents in some neurons ( Figures 1 D–1F). In three control neurons (of 20 tested), the fast rise (0.5–0.7 ms) and decay (1.8–2.4 ms) of these currents and their reversal near 0 mV strongly suggested that they were AMPA-receptor EPSCs ( Figures 1 D–1F). In three other neurons (2 of 17 from patients, 1 additional control), we saw occasional synaptic currents that were larger (40 to 325 pA, at −70 mV) and which decayed with time constants of 7 to 10 ms. Although the percentage of neurons showing synaptic currents was low, the results clearly demonstrate that neurons in the organoids form functional synaptic connections.

After 11 days of terminal differentiation (TD11), the organoids were composed of polarized, proliferating progenitors expressing the radial glial cell markers BLBP, NESTIN, PAX6, BRN2, and SOX1/2. The radial glial cells underwent mitoses on the apical (luminal) side, whereas neuronal precursor cells expressing the immature neuronal proteins DCX and TUBB3, and more mature NeuNneurons after 31 days of terminal differentiation (TD31), accumulated on the basal side of the layer of radial glial cells ( Figure 1 A; Figures S3 C and S3D). Both ASD-derived and unaffected family member-derived iPSCs had an equivalent potential to generate neuronal cells ( Figures 1 A and 1B; Figures S3 C and S3D). We observed no significant change in either perimeter or area of the organoids between control and affected groups, except for a transient increase in ASD-derived organoid size at day 11 ( Figure S3 B). This is most likely due to intrinsic factors, since the starting number of iPS cells seeded per aggregate was the same in all experiments and the size of EBs at the beginning of the differentiation was not different between patients and controls (see Supplemental Experimental Procedures ).

For the four families, we differentiated two to three iPSC lines per person into organoids using a modification of our free-floating tridimensional (3D) culture method () (see schematic outline in Figure S3 A). Similarly to our previously published preparation () and to other protocols described in the literature () we obtained organoids characterized by autonomously organized layers of radial glia, intermediate progenitors, and neurons ( Figure 1 Figures S3 C and S3D).

(G–I) Transcriptome correlation between organoids at terminal differentiation day 0 (d0) (rosette stage), day 11 and 31 (d11 and d31) (n = 45 samples, see Table S1 ) and post-mortem human brain samples from the BRAINSPAN project (n = 524 samples). The x axis shows the post-conceptional age in weeks or the brain region of the BrainSpan post-mortem brain samples. The y axis is the number of times each iPSC-derived neuron sample was classified. Classification was based on the maximum correlation coefficient and on the 95% confidence interval of the maximum correlation (see Supplemental Experimental Procedures ). AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral frontal cortex; HIP, hippocampus; ITC, inferolateral temporal cortex; MFC, medial prefrontal cortex; OFC, orbital frontal cortex; STC, superior temporal cortex; STR, striatum. In (I) x axis shows BRAINSPAN agglomerated brain regions (i.e., more brain regions were merged into a single “larger” region): NCX (i.e., FC, PC, TC, OC), neocortex; HIP, hippocampus; AMY, amygdala; VF (i.e., VF, MGE, LGE, CGE, STR), ventral forebrain; URL (i.e., CBC, CB, URL), upper rhombic lip.

(F) Histogram of the amplitude of spontaneous inward EPSCs recorded from the same neuron (604 events). The overall frequency of events detected in this neuron was 0.81 per second.

(E) average EPSC obtained from 30 spontaneous events. The decay of the current was fitted with a single exponential that gave a time constant of 1.85 ms.

(D) Examples of spontaneous excitatory post-synaptic currents (EPSCs) recorded at a holding potential of −70 mV in a neuron from a parental control that was maintained in vitro for 52 days.

(C) Action potentials elicited by depolarizing current injections in iPSC-derived neurons from a patient and the corresponding parental control. The dashed lines indicate a membrane potential of 0 mV.

(B) Stereological quantification of SOX2 + proliferating radial glia progenitors and TUBB3 + neuronal cells at TD31. Data are presented as means ± SEM. Scale bars, 10 μm or 20 μm as indicated.

(A) Both control-derived and ASD-derived organoids express markers for proliferating neural progenitors (SOX2) and the neuronal markers TUBB3. The organoids have apico-basal polarity with N-CADHERIN + apical end feet of radial glial cells and pH 3 + cells undergoing mitosis at the apical side of the neuroepithelium.

(G) Histogram of the amplitude of maximum inward currents recorded from neurons along the edge of organoids (33 control and 28 patient neurons). The distribution of current sizes was log-normal (see Experimental Procedures) and the control and patient data gave mean peak inward currents of 2.46 nA (2.37-3.22 nA) and 2.09 nA (1.72-2.54 nA), respectively (means and ranges are the anti-logs of mean ± SEM values from the log-transformed data). The size of the currents did not change significantly from 32 to 72 days in vitro.

(F) Current-voltage data for the fast sodium currents in (E). The inward currents reverse direction near +40 mV (results normalized to the maximum inward current recorded from each neuron). The mean reversal potential for similar currents was 53.5 ± 3.8 mV for control neurons (n=12) and 58.7 ± 7.1 mV for patient neurons (n=8).

(E) Currents evoked in neurons from a control and a patient by 20-ms test depolarizations from -60 mV to +50 mV (in 10 mV steps). The pre-pulse potential was -120 mV and the currents were leak-subtracted. Both rapidly activating sodium currents and more slowly activating outward currents are present. Similar currents were seen in all neurons examined in dissociated cultures (n=38) or organoids (n=61). The inward and outward currents were blocked by 1 μM tetrotoxin (n=3) and 10 mM tetraethylammonium (n=4), respectively. The voltage dependence and kinetics of activation and inactivation of the inward currents are typical of sodium currents in central neurons.

(C and D) Representative images of control-derived and ASD-derived organoids at TD11 (C) and TD31 (D). Both control-derived and ASD-derived organoids express markers for proliferating neural progenitors (BLBP, NESTIN, SOX1, PAX6, BRN2, Ki67, pH3), and the neuronal markers TUBB3, DCX and NeuN. Scale bars, 10 μm. (E–G) Voltage-activated currents in iPSC-derived neurons.

(B) Representative bright field (BF) images of control- and ASD-derived cortical organoids at TD11, TD31, and TD50. The bar graph shows quantification of the organoids perimeter at the 3 different time points of terminal differentiation. The data are presented as means ± SEM; ∗∗ p < 0.01; t test analysis. Scale bars, 200 μm.

(A) For the neuronal differentiation we modified our previously published 3D organoid culture (Mariani et al., 2012) by combining it with a monolayer protocol (Kim et al., 2011). Our previous protocol showed that neural progenitors kept in a 3D conformation were able to differentiate and recapitulate early aspects of forebrain development (Mariani et al., 2012). To improve its efficiency we added a neural rosette step (Kim et al., 2011) to enrich for neural progenitor cells (see Supplemental Experimental Procedures for details). The neural rosettes were cultured in suspension for five days in EGF and FGF and kept as free-floating undissociated organoids during terminal differentiation (TD), unless noted.

Neuronal Differentiation Potential and Electrophysiological Properties of Organoids Do Not Differ between Patients and Controls, Related to Figure 1

(E) Quantitative real time PCR assay for expression of the exogenous reprogramming genes OCT4, SOX2, c-MYC and KLF4 in retroviral-derived iPSC clones. Expression levels were calculated as average expression and normalized to the housekeeping gene GAPDH (2 -ddCTmean ). Parental fibroblasts four days after viral infection were used as positive controls.

(C and D) Expression of pluripotent cell markers by RT-PCR analysis in iPSC clones for families 1120 (C) and 07 (D) generated by the standard retrovirus approach. OCT4 en and SOX2 en indicate expression of the endogenous reprogramming genes. The iPSC line PGP-1-1 (pgp) and hES line H1 were used as positive controls. Fibroblast (Fib) and PCR Negative control (Neg) were used as negative controls.

(A) RT-PCR analyses for endogenous and exogenous (total) reprogramming genes and exogenous episomal genes (Epi) in iPSC clones from family 1120 generated by a viral-free episomal reprogramming method(Okita et al., 2011) (see methods). PGP-1-1 (pgp) is a control retrovirus-derived iPSC line used as a comparison. Fibroblast (Fib) and PCR Negative control (Neg) were used as negative controls; Plas indicates plasmid positive control.

Whole-genome sequencing data were obtained on all fibroblasts and iPSCs for members of all families in this study. Data were analyzed with CNVnator () for copy-number variation (CNV) discovery and with a GATK-based pipeline for single nucleotide variation (SNV) discovery (see Supplemental Experimental Procedures ). For three families with both parents participating in the study (03, 07, and 1123), we predicted de novo CNVs and SNVs. Of the putative de novo CNVs, only one 4.8 kb deletion (chr14:39987476-39992327) found in the 1123 proband could be validated by qPCR. For this event, qPCR validation showed roughly 30% copy-number decrease in the proband relative to either of the parent, suggesting that the deletion is possibly a somatic mosaic variant. This deletion did not overlap any known genes. Proband 07-03 carries a previously uncharacterized deletion involving exon 2 in the PTEN gene (chr10:89,641,498-89,658,394), which was also found in his unaffected father. Proband 1120 and 03 did not carry any deleterious CNVs that could potentially be pathogenic. On average, each proband had ∼112,000 rare SNVs not previously detected by the 1000 Genomes Project. This is also likely to include most of de novo SNVs. By intersecting rare SNVs in probands with lists of genes whose disruption has been previously linked to ASD () as well as the SFARI syndromic genes dataset ( https://gene.sfari.org/autdb/Welcome.do ), we found no rare SNVs that cause a known deleterious loss of function of an ASD gene. In summary, we found no de novo CNVs and/or no rare SNVs in probands that cause a known deleterious loss of function in the protein coding sequence of a gene previously involved in rare cases of syndromic or non-syndromic ASD.

To analyze neurodevelopmental aspects of idiopathic ASD, we generated iPSC lines from members of four families that each included an ASD proband with increased head circumference (HC) and one to three unaffected, first-degree family members (see Figure S1 A for family structure and Table S1 for data collection and participants details).

(C) Correlation plot of RNA-seq [log2 (RPKM+1)] data from iPSCs lines and fibroblasts in our dataset, as well as hESCs (H1 line). Red squares show clustering of iPSC with each other and H1 but not with fibroblasts.

(A) Schematic view of the family structure of the subjects included in the neurobiological analyses and their families. Square symbols indicate male subjects, circles indicate females, and shaded symbols indicate individuals affected by ASD.

Discussion

Our study provides a framework for studying normal human brain development and its disorders. Using iPSC-derived cortical organoids that recapitulate human first trimester telencephalic development, we performed genome-wide transcriptome analysis in four families affected by idiopathic ASD. The affected individuals do not share any obvious underlying genomic alterations, but all express a phenotypic trait that confers increased symptom severity, macrocephaly. Despite the heterogeneity in genotypes, we were able to identify perturbations in coherent programs of gene expression and associated features of altered neurodevelopment, namely upregulation of cell proliferation, unbalanced inhibitory neuron differentiation, and exuberant synaptic development.

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