Synaptic dysregulation in a human iPS cell model of mental disorders

Generation and characterization of patient iPS cells and isogenic iPS cell lines

Skin biopsy samples were obtained from four individuals in a previously characterized American family, pedigree H¹² (Fig. 1a). C1 fibroblasts were from ATCC (CRL-2097). All studies followed institutional IRB, ISCRO and animal protocols approved by Johns Hopkins University School of Medicine. Informed consents were obtained from individuals from pedigree H. Mouse embryonic fibroblasts (MEFs) were derived from E13.5 CF-1 mouse embryos as previously described¹⁶. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech) supplemented with 10% fetal bovine serum (FBS, HyClone) and 2 mM L-glutamine (Invitrogen).

iPS cells were generated with the EBV-based vectors as previously described¹⁶. Briefly, plasmids pEP4 EO2S ET2K (Addgene Plasmid 20927), pEP4 EO2S EN2L (Addgene Plasmid 20922), and pEP4 EO2S EM2K (Addgene Plasmid 20923) were transfected intohuman fibroblasts by Amaxa Nucleofector (Lonza; program U-023) at a concentration of 2 μg per 100 μl electroporation solution per 2 × 10⁶ cells. Colonies of iPS cells were manually picked after 3–6 weeks for further expansion and characterization. Lack of vector integration was confirmed by qRT–PCR analysis as previously described¹⁶. Two lines from each individual that passed stringent criteria were used for the current study (Supplementary Table 1a). iPS cells (passage ≤ 35) were cultured on irradiated MEFs in human iPS cell medium consisting of D-MEM/F12 (Invitrogen), 20% Knockout Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM MEM NEAA (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), and 10 ng ml⁻¹ human basic FGF (bFGF, PeproTech) as described^16,31. For feeder-free culture of iPS cells, cells were cultured on Matrigel (BD Biosciences) with mTeSR1 media (Stem Cell Technologies). Media were changed daily and iPS cell lines were passaged by collagenase (Invitrogen, 1 mg ml⁻¹ in D-MEM/F12 for 30 min at 37°C).

Karyotyping analysis by standard G-banding technique was carried out by the Cytogenetics Core Facility at the Johns Hopkins Hospital or Cell Line Genetics. Results were interpreted by clinical laboratory specialists of the Cytogenetics Core or Cell Line Genetics. Genotyping analysis was performed as described previously¹⁶. Genomic DNA of fibroblasts and derived iPS cells was extracted by DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s recommended protocol. Apair of specific primers was used to amplify the region around the 4-bp deletion (Supplementary Table 1c). PCR products were cloned by TA cloning and sequenced. Bisulphite genomic sequencing was carried out with the EZ DNA Methylation-Direct Kit (Zymo Research) as previously described³². After bisulphite conversion of genomic DNA from iPS cells, primers specific to human OCT4 and NANOG promoters (Supplementary Table 1c) were used to amplify genomic DNA sequences with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) for sequencing.

To assess the in vivo pluripotency of iPS cell lines, teratoma formation assays were performed¹⁶. iPS cells were injected subcutaneously into the dorsal flank of SCID mice. Animals were monitored and teratomas were dissected at 8 to 10 weeks post-injection. Tissues were fixed in 10% neutralized formalin solution (Sigma). Embedding, sectioning and haematoxylin and eosin staining were carried out by the Pathology Core Facility at the Johns Hopkins University Hospital.

TALEN designs and constructions were based on a Golden Gate Assembly protocol with modifications to the vector backbone³³. Donor DNA vectors with a loxP-flanked PGK-hygromycin cassette were cloned between 5′ and 3′ homology arms (Fig. 3a), which were amplified from genomic DNA of a healthy subject and a patient with the DISC14-bp mutation. For targeting, TALENs (4 μg DNA of each plasmid) and linearized donor vectors (10 μg DNA) were electroporated into individual iPS cells (1 × 10⁶ to 2 × 10⁶ cells pretreated with 5 μM ROCK inhibitor, Y-27632, Cellagentech) using Nucleofector 2b (Lonza; program A-023). Transfected cells were transferred onto a 6-well dish pre-plated with inactivated MEFs and supplemented with Y-27632 in standard iPS cell medium. Positive colonies were selected by 10mg ml⁻¹ hygromycin B (Invitrogen) after 5 days of culture or until small colonies appeared. Resistant colonies were sub-cloned and expanded in 48-well plates. Over 200 clonal lines were screened. The loxP-flanked PGK-hygromycin cassette was removed by electroporation of a Cre recombinase expression vector (4 μg DNA). Specific integration, correct genetic editing and efficient removal of PGK-hygromycin cassette at each stage were verified by Sanger sequencing.

Differentiation of iPS cells into forebrain-specific neural progenitors and cortical neurons

The protocol is illustrated in Extended Data Fig. 2a. Specifically, iPS cell colonies were detached from the feeder layer with 1 mg ml⁻¹ collagenase treatment for 1h and suspended in embryoid body (EB) medium, consisting of FGF-2-free iPS cell medium supplemented with 2 μM dorsomorphin and 2 μM A-83, in non-treated polystyrene plates for 4 days with a daily medium change. After 4 days, EB medium was replaced by neural induction medium (hNPC medium) consisting of DMEM/F12, N2 supplement, NEAA, 2mg ml⁻¹ heparin and 2 μM cyclopamine. The floating EBs were then transferred to Matrigel-coated 6-well plates at day 7 to form neural tube-like rosettes. The attached rosettes were kept for 15days with hNPC medium change every other day. On day 22, the rosettes were picked mechanically and transferred to low attachment plates (Corning) in hNPC medium containing B27. For neuronal differentiation, resuspended neural progenitor spheres were dissociated with Accutase at 37°C for 10 min and placed onto poly-D-lysine/laminin-coated coverslips in the neuronal culture medium, consisting of Neurobasal medium supplemented with 2 mM L-glutamine, B27, 10 ng ml⁻¹ BDNF and 10 ng ml⁻¹ GDNF. Half of the medium was replaced once a week during continuous culturing. For electrophysiological recordings, neural progenitors were plated on a confluent layer of rodent astrocytes as previously described^21,34. These cultures exhibited similar neuronal densities and parallel cultures were used for recordings of different iPS cell lines in a blind fashion.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min at room temperature. Samples were permeabilized and blocked with 0.25% Triton X-100 (Sigma) and 10% donkey serum in PBS for 20 min as previously described¹⁶. Samples were then incubated with primary antibodies (Supplementary Table 1b) at 4°C overnight, followed by incubation with secondary antibodies for 1 h at room temperature. Antibodies were prepared in PBS containing 0.25% Triton X-100 and 10% donkey serum. Images were taken by Zeiss LSM 710 confocal microscope, or Zeiss Axiovert 200M microscope, and analysed with ImageJ (NIH). Images were acquired with identical settings for parallel cultures. For analysis of synaptic bouton density, total SV2⁺ puncta in a given image were counted by ImageJ Analyze Particles, and the total dendritic length were measured by ImageJ plugin NeurphologyJ³⁵. The numbers of SYN1/PSD95 pairs were manually counted. The synaptic density was determined by D (D = total SV2⁺ puncta or SYN1/PSD95 pair per 100 μm total dendritic length).

Electrophysiological and FM1-43 imaging analyses

Whole-cell patch-clamp recordings were performed using Multiclamp 700A patch-clamp amplifier (Molecular Devices, Palo Alto, CA) as previously described²¹. Briefly, the recording chamber was constantly perfused with a bath solution consisting of 128 mM NaCl, 30 mM glucose, 25 mM HEPES, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂ (pH7.3; 315–325 mOsm per litre). Patch pipettes were pulled from borosilicate glass (3–5 MΩ) and filled with an internal solution consisting of 135 mM KGluconate, 10 mM Trisphosphocreatine, 10 mM HEPES, 5mM EGTA, 4 mM MgATP, and 0.5 mM Na₂GTP (pH 7.3). The series resistance was typically 10–30 MΩ. For SSC recording, the membrane potential was typically held at −70 mV. Drugs were applied through a gravitydriven drug delivery system (VC-6, Warner Hamden, CT). Data were acquired using pClamp 9 software (Molecular Devices, Palo Alto, CA), sampled at 10 kHz and filtered at 1 kHz. Spontaneous synaptic events were analysed using MiniAnalysis software (Synaptosoft, Decatur, GA). All experiments were conducted at room temperature.

For monitoring synaptic vesicle release, human iPS cell derived neurons were loaded with 10 μM FM1-43 for 2 min in FM image buffer (FMIB, consisting of 170 mM NaCl, 3.5 mM KCl, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 1.2 mM Na₂SO₄, 1.2 mM MgCl₂, 1.3 mM CaCl₂, 5 mM glucose, 20 mM N-tris (hydroxymethyl)-methyl-2-aminoethane-sulfonic acid, pH7.4, ~360 mOsmol) supplemented with 60 mM KCl, followed by a wash with 10 mM FM1-43 in FMIB for 2min. Subsequent washing was with FMIB for 5 min, followed by FMIB supplemented with 1 mM ADVASEP-7. FM1-43 imaging was performed on a Nikon TE2000 imaging system with a 20× objective. Neurons were perfused with FMIB for1 min as the baseline, followed by stimulation with 60 mM KCl for 4 min. Cells were excited at FITC spectra, and the green fluorescence was collected as FM1-43 signal. Images were acquired every 5 s and analysed using NIH ImageJ software. The FM1-43 signal was determined by F [F = (F₁−B₁)/(F₀−B₀)], which was normalized to the mean fluorescence intensity measured at the baseline condition (set as 1).

qPCR and RNA-seq analyses

Human forebrain neurons without astrocyte co-culture were used for gene expression analyses. Total RNA was isolated using mir Vana kit (Invitrogen) according to the manufacturer’s instructions. For qPCR, a total of 1 μg RNA was used to synthesize cDNA with the SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative RT–PCR was then performed using SYBR green (Applied Biosystems) and the StepOnePlus Real-Time PCR System (Applied Biosystems). Quantitative levels for all genes were normalized to the housekeeping gene GAPDH and expressed relative to the relevant control samples. All primer sequences are listed in Supplementary Table 1c.

For deep RNA sequencing, libraries for three biological replicates of 4-week-old forebrain neurons derived from iPS cells of three individuals within the pedigree H (C3-1, D2-1 and D3-2) without astrocyte co-culture were prepared and sequenced on an Ion Proton Torrent. Libraries were prepared using the Ion Total RNA-Seq Kit v2 for Whole Transcriptome sequencing following the protocol provided by the manufacturer (Life Technologies, Carlsbad, CA). Briefly, poly (A)-enriched mRNA samples were fragmented, Ion Adaptors were hybridized, and cDNA generated through reverse transcription. Barcodes were added and the libraries were amplified for sequencing. From the poly(A)⁺ RNA-seq libraries, a total of 123 million reads were generated comprising between 9 and 34 million reads from each of the 9 samples (Supplementary Table 2a). Sequencing generated strand-specific singleend reads of variable length between 8–24 bp. Reads were mapped to the UCSC Human Reference Genome (hg19) using TopHat³⁶ (v2.0.10) and Bowtie³⁷ (v2.1.0). Resulting sequence alignment files were analysed using RSeQC package for quality control³⁸. Reads covering gene coding regions were counted with BED Tools and count data were analysed for differential expression using edgeR³⁹ (v2.15.0). For Gene Ontology analysis, gene lists were obtained for disease enrichment from PharmGKB, KEGG pathways from the Encyclopedia of Genes and Genomes and gene ontology (GO) from AmiGO. IDs for each gene list are provided in Supplementary Table 2. P values were calculated from a cumulative hypergeometric distribution, calculated at (http://www.geneprof.org/GeneProf/tools/hypergeometric.jsp). The total population size was set to 20,687. Additional gene ontology analysis was performed with WebGestalt⁴⁰.

DNA constructs and biochemical analyses

Full-length human DISC1 cDNA was amplified by PCR and subcloned with HA-tag through AgeI and EcoRV into the pFUGW vector. The 4-bp deletion mutation was introduced through a synthesized long length PCR primer and cloned with Flag-tag through AgeI and EcoRV into the pFUGW vector. All expression plasmids were confirmed by DNA sequencing.

HEK293cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 10%FBS (Gibco). Once reaching 70% confluence, the cells were transiently transfected with cDNA constructs using Lipofectamine 2000 (Invitrogen). Cells were harvested 48h after transfection for biochemical analyses. Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS; 50 mM Tris, pH8.0) containing Complete Protease Inhibitor Cocktail (Roche). Samples were left on ice for 30 min and sonicated briefly. The insoluble fraction was removed by centrifugation at 15,000 r.p.m. for 15 min at 4°C. Protein concentration was determined by BCA protein assay kit (Bio-Rad). 2× SDS sample buffer (Bio-Rad) containing5% β-mercaptoethanol (Sigma) was added to equal amounts of protein. Proteins were then separated by 4–15% SDS PAGE (Bio-Rad) and transferred to nitrocellulose membrane (0.45 μm). 5% dried milk in TBST (Tris buffered saline with 0.1% Tween 20) was incubated for blocking, and membranes were applied with specific antibodies as listed in Supplementary Table 1b. After washing with TBST and incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology), the antigen-antibody was detected by chemiluminescence (ECL; Pierce) and X-ray film (GE Healthcare).

For immunoprecipitation experiments, cells were lysed using an IP lysis buffer containing 25 mM HEPES, pH7.4, 1 mM EDTA, 10 mM NaCl, 0.5% Triton X-100, protease inhibitors cocktail (Roche) and 1 mM PMSF, except for the ubiquitination assay in which the IP lysis buffer contains 0.2% SDS (Extended Data Fig. 4e). Equal amount of protein was incubated overnight with Fast Flow Protein G agarose beads (Millipore) and mouse IgG or specific antibody in IP lysis buffer. After pull-down, protein-G beads were washed five times with IP washing buffer (25 mM HEPES, pH 7.4, 1 mM EDTA, 100 mM NaCl, 0.5% Triton X-100 and protease inhibitors cocktail (Roche) and boiled with 2×SDS sample buffer (Bio-Rad) containing 5% β-mercaptoethanol (Sigma). Western blotting was then carried out with primary antibodies listed in Supplementary Table 1b.

Data collection and statistics

All experiments were replicated at least three times using iPS cell lines indicatedin Supplementary Table 1a and data from parallel cultures were acquired. The sample size and description of the sample collection are reported in each figure legend. Quantification of synaptic puncta density, FM-imaging and electrophysiological analyses were performed in a blind fashion. Statistical analyses used for comparison are reported in each figure legends.