Electrophysiological signals were acquired using a patch clamp amplifier (Multiclamp 700B, Axon Instruments/ Molecular Devices) and digitizer (Digidata 1440A, Axon Instruments/ Molecular Devices), and recorded using pCLAMP 10 software (Axon Instruments/ Molecular Devices)

Electrophysiological signals were acquired using a patch clamp amplifier (Multiclamp 700B, Axon Instruments/ Molecular Devices) and digitizer (Digidata 1440A, Axon Instruments/ Molecular Devices), and recorded using pCLAMP 10 software (Axon Instruments/ Molecular Devices). myelination, and Chiari malformation7, 9, 14. There are many structural abnormalities in common between CFC and major psychiatric disorders: cortical atrophy is observed in schizophrenia15, 16, thinner corpus callosum in ASDs17, 18, and enlarged ventricles in both15, 16, 18, 19. Attempts to mechanistically model CFC in mice have led to mixed results. The ablation of in the developing mouse brain led to increased frequency of repetitive movements, seizures, hyperactivity, and impairments in sociability and learning20C22. Timapiprant sodium However, mouse models with gain-of-function mutations in failed to recapitulate the ART1 central nervous system (CNS) structural defects found in CFC subjects23, 24 and led to embryonic lethality or overall reduced viability. Human induced pluripotent stem cells (iPSCs) have proven to comprise a successful platform that allows for the direct examination of human neuronal development25C31. Our recent studies on another RASopathy, Costello syndrome32, 33, indicated that an activating mutation in led to an extended progenitor phase and subsequent increase in the number of cortical Timapiprant sodium neurons33 as well as excessive astrocyte-to-neuron signaling32, all of which correlated with the progressive postnatal brain overgrowth in Costello syndrome34. Because Ras/MAPK signaling controls differentiation in most tissues, we hypothesized that the gain-of-function p.Q257R mutation would affect neuronal maturation. We generated iPSC lines from four CFC subjects carrying mutation p.Q257R. Recruitment was described previously 6: all CFC subjects were recruited Timapiprant sodium at national RASopathy family meetings and two control participants at a UCSF Neurofibromatosis Symposium. The third control line used in our study, HS1-11, was kindly provided by Dr. Arnold Kriegsteins lab (UCSF). Control lines were sex- and age-matched to the subject lines (Supplementary Table 1; clinical features in Supplementary Table 2). Generation of pluripotent stem cell lines Dermal fibroblasts were isolated from skin biopsies as previously described35. When the dermal fibroblast culture reached passage 5, they were detached using 0.25% trypsin/EDTA, spun at 500xg for 5 minutes, resuspended in DMEM high-glucose media. We used 360,000 cells per individual per reprogramming. Before electroporation, one g of each of the plasmids pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXLE-EGFP (Addgene)36 was mixed with 100ul Resuspension Buffer R (Neon? Transfection System, Life Technologies), and added to the cells to be transformed. We pipetted 100l of the cell suspension into the Neon Transfection System chamber (Life Technologies) and electroporated the cell suspension with three 1,650V, 10ms pulses. Immediately after electroporation, the cell suspension was pipetted directly into a gelatin-coated well containing a warmed fibroblast medium (DMEM High Glucose supplemented with 10% fetal bovine serum) and incubated in a 37oC, 5% CO2 incubator. GFP-positive cells were examined during the first five days post-electroporation to monitor transfection efficiency. After day 5, transfected fibroblasts were detached and plated onto Mitomycin C treated Mouse Embryonic Fibroblasts (Millipore) at 10,000 cells/cm2. Until iPS-like colonies were ready to be picked and passaged, we fed the cells with hES-KSR Basal Medium – Knockout DMEM (Life Technologies), 20% Knockout Serum Replacement (Life Technologies), 5% L-glutamine (Life Technologies), 10% Non-essential amino acids (Life Technologies), and beta-mercaptoethanol and 10ng/ml bFGF (GlobalStem). Clonal colonies displaying iPSC morphology were manually selected and subsequently cultured on a Matrigel substrate (BD Biosciences) with mTeSR1 media (Stem Cell Technologies). All lines were shown to be pluripotent using a random differentiation protocol37. The presence of the Q257R point mutation in the CFC subjects was confirmed by Sanger sequencing of exon 6 of the gene for all the cell lines before and after reprogramming (forward primer: 5-GGGAGAGAAATACTGTCCATTCCA-3; reverse primer: 5-GCTTGAAATCAGTTGCCAGCC -3; annealing temperature= 58C). Both DNA fingerprinting analysis and genotyping using the Affymetrix Axiom EUR array38 were performed by the Genomics Core Facility at UCSF using standard protocols before reprogramming, after reprogramming and in one-week old neural differentiation cultures. Subclones utilized for each cell line and independent differentiations performed are listed in Supplementary Table 3. Immunofluorescence At the previously described time points, cells were fixed in 4% paraformaldehyde in PBS Timapiprant sodium for 10 min at room temperature and permeabilized with 90% ice cold methanol in PBS for 10 min. Nonspecific binding was blocked with IF blocking buffer [2% BSA (Sigma), 1% Fish Skin Gelatin (Sigma) and 0.2% Saponin (Sigma) in PBS] for 1h at room temperature. Cells were incubated with primary antibody (Supplementary Table 3) overnight at 4C, followed by three 5 minute PBS washes. Cells were incubated with appropriate secondary antibodies.