Collection and characterization of hiPS cell lines
Control hiPS cell lines in this study were reported and have been extensively characterized using standardized methods44,50,51. hiPS cell lines from 11 idiopathic ASD individuals were obtained from Coriell. The 22q11.2 deletion44, 22q13.3 deletion108 and Timothy syndrome45 hiPS cell lines were reported and characterized before. To confirm the various genetic mutations, whole-genome sequencing was performed. Cultures were regularly tested for mycoplasma and maintained mycoplasma free. Approval for reprogramming and differentiation of these cell lines was obtained from the Stanford Institutional Review Board panel, and informed consent was obtained from all individuals.
Reprogramming and hiPS cell culture
Human fibroblast cells were reprogrammed using the Sendai virus-based CytoTune iPS 2.0 kit (CytoTune 2.0, Invitrogen, A16517). Fibroblasts were plated in six-well plates 2 days before transduction to achieve a density of 2 × 105 to 3 × 105 cells per well. Cells were then transduced with the indicated set of Sendai vectors at a multiplicity of infection of 5–5–3 (KOS–Myc–Klf4) as we previously described in ref. 44. Virus was removed after 24 h and cells were cultured in fibroblast medium with 10% FBS (Gibco, 16000-044) in Dulbecco’s modified Eagle medium (DMEM) (Gibco, 10569-010) for a total of 7 days, with media changes every other day. On day 7 after transduction, cells were collected with TrypLE (Gibco, 16563-029) and plated in the fibroblast medium onto truncated recombinant human vitronectin (VTN-N, Gibco, A14700)-coated plates. The following day, fibroblast medium was changed to Essential 8 medium (Gibco, A1517001) and cells were fed daily until days 20–22. For further culture, individual colonies were manually dissected and transferred to fresh vitronectin-coated dishes. Once hiPS cell clones were established, they were routinely cultured on vitronectin-coated dishes in Essential 8 medium, and cells were split every 4–5 days using 0.5 mM EDTA (Invitrogen, 15575-020). hiPS cell clones and lines were also generated using retroviruses or non-integrating episomal vectors from fibroblasts109. We did not observe any systematic differences between cells reprogrammed by the two methods. hiPS cells were cultured as previously described in ref. 51. Briefly, hiPS cells were cultured on six-well plates coated with recombinant human vitronectin (VTN-N, Gibco, A14700) in Essential 8 medium (Gibco, A1517001) and passaged with 0.5 mM EDTA (Gibco, 15575). To coat the six-well plates, 1 ml of vitronectin (diluted at a 1:100 ratio with Dulbecco’s phosphate-buffered saline (DPBS); Gibco, 14190) was added per well and then incubated at room temperature for 1 h. To passage hiPS cells with 80–90% confluency, cells were rinsed with 3–4 ml of DPBS per well, and then 1 ml of 0.5 mM EDTA (Gibco, 15575) was added and incubated for 7 min at room temperature. After the EDTA was removed, 2 ml of prewarmed complete Essential 8 medium was added to collect cells. The cell suspension was then diluted in Essential 8 medium (1:6–1:20 depending on the hiPS cell line) and distributed on vitronectin-coated wells.
Generation of hCOs from hiPS cells
For the generation of 3D spheroids, hiPS cells were incubated with Accutase (Innovate Cell Technologies, AT-104) at 37 °C for 7 min and dissociated into single cells. To obtain uniformly sized spheroids, we used AggreWell 800 plates (STEMCELL Technologies, 34811) containing 300 microwells. There were roughly 3 × 106 single cells per well in Essential 8 medium supplemented with the ROCK inhibitor Y-27632 (10 μM, Selleckchem, S1049), centrifuged at 100g for 3 min to capture the cells in the microwells and incubated at 37 °C with 5% CO 2 . After 24 h, day 0 of differentiation, spheroids were collected from each microwell by firmly pipetting (with a cut end of a P1000 tip) medium in the well up and down, and transferring it into ultra-low-attachment plastic dishes (Corning, 3262) in Essential 6 medium (Gibco, A1516401) supplemented with two SMAD pathway inhibitors: dorsomorphin (2.5 μM, Sigma-Aldrich, P5499) and SB431542 (10 μM, Tocris, 1614) together with Wnt pathway inhibitor XAV939 (1.25 μM, Tocris, 3748). Media changes were performed daily, except for day 1. On the sixth day in suspension, neural spheroids were transferred to neural medium containing Neurobasal-A (Gibco, 10888), B-27 supplement without vitamin A (Gibco, 12587), GlutaMax (1:100, Gibco, 35050) and penicillin and streptomycin (1:100, Gibco, 15140). The neural medium was supplemented with epidermal growth factor (20 ng ml−1, R&D Systems, 236-EG) and basic fibroblast growth factor (20 ng ml−1, R&D Systems, 233-FB) until day 24. From days 25 to 42, to promote differentiation of the neural progenitors into neurons, the neural medium was supplemented with brain-derived neurotrophic factor (20 ng ml−1, Peprotech, 450-02) and neurotrophin 3 (20 ng ml−1, Peprotech, 450-03), with medium changes every other day. From day 43 onwards, only neural medium without growth factors was used for medium changes every 4–5 days.
RNA-seq processing
Sequencing libraries were prepared using TruSeq stranded RNA RiboZero Gold (Illumina) on ribosomal RNA-depleted (RiboZero Gold, Illumina) RNA. The libraries were sequenced with 100-base-pair paired-end reads on an Illumina HiSeq 4000. The reads were mapped to the human genome (hg38) with Gencode v.25 annotations using STAR (v.2.5.2b)110 and gene expression was quantified using RSEM (v.1.3.0)111. Genes that were expressed at very low levels (fewer than 10 reads in 30% of samples from a given day) were removed from the analysis. Samples with standardized sample network connectivity Z scores below −2 in each mutation were defined as outliers and removed112. To control for technical variation due to the sequencing and library prep we calculated the principal components of the Picard sequencing metrics (http://broadinstitute.github.io/picard/) using the CollectAlignmentSummaryMetrics, CollectRnaSeqMetrics and MarkDuplicates modules, and included them in our model. To infer genetic ancestry, we called single-nucleotide polymorphisms (SNPs) from the aligned reads using the GATK (v.3.3) Haplotype caller113. Sites with more than 5% missing samples, with rare minor allele frequency (less than 0.05) and Hardy–Weinberg disequilibrium (less than 1 × 10−6) were removed using plink (v.1.09)114. We then used the remaining high-quality SNPs to run MDS together with HapMap3.3 (hg38)115. The first two MDS values, referred to as genetic ancestry principal components 1 and 2 (PC1, PC2), were then included in our model. Sample identity was verified using the identity by descent algorithm from PLINK (v.1.09)114. Identity by descent was calculated for each pair of samples based on genotypes derived from RNA-seq analysis as well as for all pairs of RNA-seq and DNA sequencing samples. Samples with a \(\hat{\pi }\) < 0.8 from other samples derived from the same individual were removed. Sex was verified for each sample by detection of genes expressed by the Y chromosome.
Sample with high levels of duplication (Picard tools, PERCENT_DUPLICATION > 65), high levels of intergenic mapping (Picard tools, PCT_INTERGENIC_BASES > 60) or with low levels of messenger RNA (RNA) (Picard tools, PCT_MRNA_BASES < 0.5) were removed. All RNA-seq samples passing quality control are listed in Supplementary Table 1. Variance partitioning was performed using the variancePartition package (v.1.20.0) with default parameters. Gene-expression reproducibility was measured using Spearman’s correlation between every pair of samples in a given time point and mutation both within and between individuals.
Whole-genome sequencing
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