Cell culture and isogenic controls
RTT patient fibroblasts were derived from the Cell lines and DNA bank of Rett syndrome, X-linked mental retardation and other genetic diseases at the University Siena in Italy via the Network of Genetic Biobanks Telethon. We used fibroblast lines carrying MECP2 mutation showing a deletion in Exon 3 and 4 of the MECP2 gene (RTT Ex3-4), (RTT#2282C2). Fibroblasts were derived frozen, thawed and expanded in fibroblast medium (DMEM-F12, 20% FBS, 1%NEAA, 1%Pen/Strep, 50 µM β-Mercaptoethanol). To generate pure RTT, i.e. cells expressing affected X-chromosome, and isogenic control, i.e. cells expressing the healthy X-chromosome, fibroblasts were detached from cell culture plate and single fibroblasts were seeded in a 96-well plate. Cells were further expanded and characterized for their MeCP2 state by immunocytochemistry and PCR [24]. All of our experiments were exempt from the approval of the institutional review board.
Reprogramming
Reprogramming of fibroblasts was performed as described before [24]. In brief, fibroblasts were detached from cell culture plate and washed with PBS. 4 × 105 cells were resuspended in 400 µl Gene Pulser® Electroporation Buffer Reagent (BioRad) with 23,4 µg of each episomal plasmid (Addgene, Plasdmid #27078, #27080, #27076) containing the reprogramming factors OCT4, SOX2, KLF4 and C-MYC. Cell solution was carefully mixed and electroporated with three pulses of 1.6 kV, capacitance of 3 µF and a resistance of 400 Ω (Gene Pulser II (BioRad)). Fibroblasts were left for recovery in Fibroblast medium without antibiotics containing 10 µM Rock inhibitor (Y-27632). After cells reached a confluence of 60–70%, medium was changed to TeSR™-E7™ (STEMCELL). Colonies appeared after 21–28 days. These were picked manually and maintained in TeSR™-E8™ (STEMCELL). iPSC lines were characterized for pluripotency [24]. Six iPSC lines derived from one individual were selected and used in the present study; three iCTR clones and three RTT Ex3-4 clones.
Differentiation of neuronal stem cells
The 6 iPSC lines were differentiated towards neuronal stem cells as described before [21, 25]. As described in the paper by Shi et al, this protocol of cortical neurogenesis follows the same temporal order as occurs in vivo. iPSCs were plated in high-density on Geltrex®-coated wells of a 12-well plate in TeSR™-E8™ with 10 µM Rock inhibitor. Medium was changed daily for 2 days. Afterwards half of the medium was changed daily with Neuro-Maintenance-Medium (NMM) (1:1 DMEM/F12 + GlutaMAX:Neurobasal Medium, 1x B27, 1xN2, 2.5 µg/mL Insulin, 1.5 mM L-Glutamin, 100 µM NEAA, 50 µM 2-Mercaptoethanol, 1% penicillin/streptomycin) containing 1 µM Dorsomorphin and 10 µM SB431542 up to day 12. At day 10–12 rosette structures appeared, which were manually picked and further cultured on Poly-L-Ornithin (0.01%)/Laminin (20 µg/ml) coated cell culture plates in NMM medium containing EGF (20 ng/ml) and FGF-2 (20 ng/ml). Half of medium was changed daily and cells were cultured up to day 22.
Immunocytochemistry
To perform immunocytochemistry, cells were fixated with 4% Paraformaldehyde and blocked with blocking buffer containing 5% Normal Goat Serum (Gibco®), 0.1% bovine serum albumin (SigmaAldrich) and 0.3% Triton X-100 (SigmaAldrich). Primary antibody incubation for MeCP2 (D4F3, CellSignaling, 1:200, rabbit), OCT3/4 (C-10, Santa Cruz, 1:1000, mouse), SSEA4 (Developmental Studies Hybridoma Bank, 1:50, mouse), TRA1-60 (Santa Cruz, 1:200, mouse), TRA1-81 (Millipore, 1:250, mouse), SOX2 (Millipore, 1:1000, rabbit) was performed in blocking buffer over night at 4 °C. Next day cells were washed and secondary antibody Alexa Fluor® 488 (ThermoFisher, 1:1000, mouse or rabbit) and Alexa Fluor® 594 (ThermoFisher, 1:1000, mouse or rabbit) were applied in blocking buffer for 1 h at room temperature. To identify cell nuclei DAPI was used for 5 min before cells were mounted with Fluoromount™ (Sigma-Aldrich).
RNA collection, Sequencing and PCR analysis
To isolate RNA samples, standard TRIzol®-Chloroform isolation was done. RNA was stored at -80 °C until further processing. For PCR analysis RT-PCR was performed. cDNA was synthesized by using SuperScriptIV-Kit (ThermoFisher) following manufacturer’s recommendations and could be stored until further processing at -20 °C. To perform PCR different primer sets were used (Table 1) and PCR was executed with Phire Hot Start II DNA Polymerase (ThermoFisher).
Table 1
Primers used for iPSC characterization.
OCT3/4 | Fwd: | GAC AGG GGG AGG GGA GGA GCT AGG |
Rev: | CTT CCC TCC AAC CAG TTG CCC CAA AC |
SOX2 | Fwd: | GGG AAA TGG GAG GGG TGC AAA AGA GG |
Rev: | TTG CGT GAG TGT GGA TGG GAT TGG TG |
NANOG | Fwd: | CAG CCC CGA TTC TTC CAC CAG TCC C |
Rev: | CGG AAG ATT CCC AGT CGG GTT CAC C |
C-MYC | Fwd: | GCG TCC TGG GAA GGG AGA TCC GGA GC |
Rev: | TTG AGG GGC ATC GTC GCG GGA GGC TG |
TDGF1 | Fwd: | TGC TGC TCA CAG GGC CCG ATA CTT C |
Rev: | TCC TTT CGA GCT CAG TGC ACC ACA AAA C |
UTF1 | Fwd: | CAG ATC CTA AAC AGC TCG CAG AAT |
Rev: | GCG TAC GCA AAT TAA AGT CCA GA |
DNMT3B | Fwd: | CAG GAG ACC TAC CCT CCA CA |
Rev: | TGT CTG AAT TCC CGT TCT CC |
MECP2 (Set 1) | Fwd: | GGA GAA AAG TCC TGG AAG C |
Rev: | CTT CAC GGC TTT CTT TTT GG |
MECP2 (Set 2) | Fwd: | CACGGAAGCTTAAGCAAAGG |
Rev: | CTGGAGCTTTGGGAGATTTG |
EIF4G2 | Rev: | CTT CAC GGC TTT CTT TTT GG |
Rev: | AGT TGT TTG CTG CGG AGT TGT CAT CTC GTC |
Western blotting
Frozen cell pellets were lysed by adding WB-Lysate buffer (50 mM Hepes ph 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% TritonX-100, 10% Glycerol, 1 mM DTT). To determine protein concentration Bradford-Test was performed and 30 µg of sample were used. For SDS-PAGE pre-casted Gels were used (Biorad) and ran in 10x Tris/Glycine Buffer for Western Blots and Native Gels (Biorad #1610734). Gels were blotted in tank-blotter (Biorad) on PVDF membranes (Biorad) according to manufactures protocol. After protein transfer blots were blocked in 5% BSA/TBS for 1 h and stained for SOX2 (1:100, Millipore AB5603), SOX9 (1:250; CellSignalling 82630) and ß-actin (1:1000; Chemicon, C4 MAB 1501) in 5% BSA/TBS over night at 4 °C. Next day blots were washed and stained with secondary antibodies in 5% BSA/TBS for 1 h at room temperature. After another 3 TBS washes blots were stained with SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher) and analysed with LiCor analyser.
Sample collection
Samples were collected at different days throughout the differentiation. First samples were taken at day 3 (D3) of protocol, one day after medium change towards NMM with Dorsomorphin and SB431542. Second samples were taken at day 9 (D9), before rosette structures were cut, reminiscent to the early stage of secondary neurulation [26] followed by third sample collection at day 15 (D15), after rosettes were manually picked, comparable to complete neural tube formation state. Finally, fourth samples were taken at day 22 (D22), after first passage was performed and cells were recovered. To collect, cells were washed once with PBS and then scraped off the cell culture plate. Solution was collected in an Eppendorf Microtube and centrifuged at maximum speed for 5 min. Supernatant was discarded and pellet was frozen at -80 °C until further processing for mass spectrometry.
Cell lysis and protein digestion
Samples were lysed, reduced and alkylated in lysis buffer (1% sodiumdeoxycholate (SDC), 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 40 mM chloroacetamide (CAA) and 100 mM TRIS, pH 8.0 supplemented with phosphatase inhibitor (PhosSTOP, Roche) and protease inhibitor (Complete mini EDTA-free, Roche). After sonication, samples were centifugated at 20,000 x g for 20 min. Protein concentration was estimated by a BCA protein assay. Reduction was done with 5 mM Ammonium bicarbonate and dithiothreitol (DTT) at 55 °C for 30 min followed by alkylation with 10 mM Iodoacetamide for 30 min in dark. Proteins were then digested into peptides by LysC (Protein-enzyme ratio 1:50) at 37 °C for 4 h and trypsin (Protein-enzyme ratio 1:50) at 37 °C for 16 h. Peptides were then desalted using C18 solid phase extraction cartridges (Waters).
Tandem Mass Tag (TMT) 10 plex labelling
Aliquots of ~ 100 µg of each sample were chemically labeled with TMT reagents (Thermo Fisher) according to Fig. 1. In total three TMT mixtures were created for each biological replicate. Peptides were resuspended in 80 µl resuspension buffer containing 50 mM HEPES buffer and 12.5% acetonitrile (ACN, pH 8.5). TMT reagents (0.8 mg) were dissolved in 80 µl anhydrous ACN of which 20 µl was added to the peptides. Following incubation at room temperature for 1 hour, the reaction was then quenched using 5% hydroxylamine in HEPES buffer for 15 min at room temperature. The TMT-labeled samples were pooled at 1:1 ratios followed by vacuum centrifuge to near dryness and desalting using Sep-Pak C18 cartridges.
Off-line basic pH fractionation
Before the mass spectrometry analysis, the TMT mixture was fractionated and pooled using basic pH Reverse Phase HPLC. Samples were solubilized in buffer A (5% ACN, 10 mM ammonium bicarbonate, pH 8.0) and subjected to a 50 min linear gradient from 18% to 45% ACN in 10 mM ammonium bicarbonate pH 8 at flow rate of 0.8 ml/min. We used an Agilent 1100 pump equipped with a degasser and a photodiode array (PDA) detector and Agilent 300 Extend C18 column (5 µm particles, 4.6 mm i.d., and 20 cm in length). The peptide mixture was fractionated into 54 fractions and consolidated into 20. Samples were acidified with 10% formic acid and vacuum-dried followed by re-dissolving in 5% formic acid/5% ACN for LC-MS/MS processing.
Mass spectrometry analysis
We used nanoflow LC-MS/MS using Orbitrap Lumos (Thermo Fisher Scientific) coupled to an Agilent 1290 HPLC system (Agilent Technologies). Trap column of 20 mm x 100 µm inner diameter (ReproSil C18, Dr Maisch GmbH, Ammerbuch, Germany) was used followed by a 40 cm x 50 µm inner diameter analytical column (ReproSil Pur C18-AQ (Dr Maisch GmbH, Ammerbuch, Germany). Both columns were packed in-house. Trapping was done at 5 µl/min in 0.1 M acetic acid in H2O for 10 min and the analytical separation was done at 300 nl/min for 2 h by increasing the concentration of 0.1 M acetic acid in 80% acetonitrile (v/v). The mass spectrometer was operated in a data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra were acquired in the Orbitrap from m/z 350–1500 with a resolution of 60,000 FHMW, automatic gain control (AGC) target of 200,000 and maximum injection time of 50 ms. Ten most intense precursors at a threshold above 5,000 were selected with an isolation window of 1.2 Da after accumulation to a target value of 30,000 (maximum injection time was 115 ms). Fragmentation was carried out using higher-energy collisional dissociation (HCD) with collision energy of 38% and activation time of 0.1 ms. Fragment ion analysis was performed on Orbitrap with resolution of 60,000 FHMW and a low mass cut-off setting of 120 m/z.
Data processing
Mass spectra were processed using Proteome Discover (version 2.1, Thermo Scientific). Peak list was searched using Swissprot database (version 2014_08) with the search engine Sequest HT. The following parameters were used. Trypsin was specified as enzyme and up to two missed cleavages were allowed. Taxonomy was set for Homo sapiens and precursor mass tolerance was set to 50 p.p.m. with 0.05 Da fragment ion tolerance. TMT tags on lysine residues and peptide N termini and oxidation of methionine residues were set as dynamic modifications, and carbamidomethylation on cysteine residues was set as static modification. For the reporter ion quantification, integration tolerance was set to 20 ppm with the most confident centroid method. Results were filtered to a false discovery rate (FDR) below 1%. Finally, peptides lower than 6 amino-acid residues were discarded. Within each TMT experiment, reporter intensity values were normalized by summing the values across all peptides in each channel and then corrected for each channel by having the same summed value. After that the normalized S/N values were summed for all peptides. Finally proteins were Log2 transformed and normalized by median subtraction. Proteins were included that are identified in 2 out of 3 replicates.
Data visualization
The software Perseus was used for data analysis and to generate the plots. Volcano plots for each time point was generated and up- or down-regulated proteins were considered significant with a fold change cut-off = 1.3. Functional analysis to enrich to GO terms were done on David Database and pathway enrichment analysis was done on Reactome Functional Interaction (http://www.reactome.org/). Furthermore, protein interaction network was performed using Cytoscape, Gnenmania plugin.