Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders, such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome, we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs, both parental alleles are present, distinguishable by the mutation, and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation, in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein.


Cell culture
All media, PBS and supplements were obtained from Life Technologies unless otherwise stated. Cells were split once a week in a ratio of 1:2 to 1:6 (feeder-dependent cells) or 1:6 to 1:12 (feeder-independent cells). For propagation, colonies were dissociated either using collagenase (feeder-dependent), or cell dissociation reagent (feeder-independent; STEMCELL Technologies), according to the manufacturer's recommendations. To yield single cell suspensions, cells were treated with Accutase for 10 minutes at 37°C and triturated. To support survival of single cells, 10 µM ROCK inhibitor (Y-27632, Calbiochem) was added to the medium until the first medium change. in culture medium (DMEM high glucose, 10 % FCS, 50 U / ml Penicillin / Streptomycin, 1 % sodium pyruvate, 1 % glutamine, 1 % non-essential amino acids and 0.2 % βmercaptoethanol). Transduction was performed by adding vector supernatant according to a multiplicity of infection of ten. Medium was exchanged 24 hours later. After flow cytometric cell sorting, cells were and plated at a density of 1,000 cells / cm 2 in a 6-well plate on proliferation-arrested CF-1 feeder cells in KSR medium containing 10 µM ROCK inhibitor).

Reprogramming of human dermal fibroblasts
Colonies were picked approximately four weeks post transduction and expanded on irradiated feeder cells in KSR medium.

Virus excision
Virus excision was mediated by direct protein transduction of Flp recombinase into iPSCs. Vitronectin-coated 6-well plates and cultured in mTesR1 containing 10 µM ROCK inhibitor.
50 µl of vector-containing supernatant was added with daily medium changes until colonies appeared (about 5 days). Single clones were isolated and expanded. Genomic DNA was isolated using the QIAamp DNA mini kit (Qiagen) and 100 ng were used as template for PCR amplification. PCR amplification was conducted as a three-primer PCR which amplified a product specific for the intact integrated virus genome (290 bp) and an internal control (170 bp). The internal control is a fragment of the ∆U3-region of the virus that is present before and after excision. Successful excision was confirmed by absence of the 290 bp amplification product.

Immortalization of fibroblasts
Primary fibroblasts of the patient's skin biopsy showed insufficient proliferation and early onset of senescence. In order to maintain cells and prolong proliferation, fibroblasts were immortalized by lentiviral transduction of a SV40 large T / small T antigen fusion protein encoded by the plasmid pCL12SVT (a kind gift of Helmut Hanenberg, Indiana University, USA). Since selection of transduced cells is not possible with this construct, different volumes (2, 5, 10, 15, 20 and 40 µl) of virus supernatant were added to 2x10 5 cells per well in a 6-well plate. Transduced cells were selected by proliferation rate and survival.
Immortalized patient fibroblasts were solely used for analysis of DNA methylation.

HLA typing
Isolated genomic DNA of iPSCs was used for high resolution HLA-A, -B and -C typing, using the Luminex Multiplex Technology together with commercially available sequence-specific oligonucleotides (High Definition LABType) 1 . Briefly, exons 2 and 3 of HLA class I alleles were amplified, biotinylated and subsequently hybridized to the sequence-specific, microbead-bound oligonucleotides. Bound oligonucleotides can be identified by the Luminex flow analyzer. HLA class I alleles with identical nucleotide sequences were grouped.

Alkaline phosphatase staining
iPSC colonies were stained for alkaline phosphatase activity using the Alkaline Phosphatase Detection Kit from Millipore as recommended by the manufacturer. The staining reaction was stopped after 15 minutes by washing with PBS and cell colonies were evaluated by phase contrast microscopy.

Immunofluorescence
For antibody stainings, cells were cultivated on feeder cells in standard 24-well cell culture dishes or in 24-well Lumox plates (Sarstedt). Cell colonies were fixed using 4 % paraformaldehyde for 15 min at room temperature. For staining of nuclear proteins, cells were permeabilized by incubation in 0.3 % (v / v) Triton X-100 for 5 min at room temperature.
After washing and blocking in PBS with 3 % normal goat serum (serum of secondary antibody host species; Cell Signaling Technology), incubation with the primary antibody was conducted at 4 °C overnight. After washing with PBS, fluorescently labelled secondary antibody was applied for 2 hours at room temperature. Cells were counterstained with DAPI (200 µg / ml; diluted 1:1000 in PBS) and imaged on a Zeiss Axio Observer.D1 fluorescence microscope using the Axiovision acquisition software from Zeiss. Antibodies and dilutions used are listed in Supplementary Table S7.

Flow cytometry
For FACS analysis of pluripotent cells cultured on feeder layer, feeder cell depletion was Data analysis was done using Kaluza® software (Beckman Coulter). Antibodies are listed in Supplementary Table S7.

Karyotyping
Cells were cultured in 6-well plates. At 50 to 60 % confluency cells were treated with 0.1 µg / ml colcemid (Roche) for 3-5 hours in a humidified incubator at 37 °C. Cells were harvested using cell dissociation buffer and collected by centrifugation at 200 x g for 7 min. Hypotonic treatment in 0.4 % Hepes-KCl was performed for 20 min at 37 °C in a water bath. At the end of hypotonic treatment, 100 µl of freshly prepared fixative (3:1 methanol: acetic acid) were added and the suspension was mixed by inversion. Cells were collected by centrifugation at 200 x g for 7 min. 5 ml of fixative were added to the cell pellet and the suspension was incubated for 20 min at room temperature. After centrifugation, the pellet was resuspended in using a microscope. For karyotype analysis, slides were baked overnight at 60 °C, digested with 0.25 % trypsin solution for a maximum of 5 min and stained with Giemsa (MerckMillipore) for 3.5 min. Chromosome analysis was done using a Zeiss Axioskop microscope and the Ikaros software (MetaSystems). Per sample eleven metaphases were counted and analyzed. To determine the origin of the marker chromosome in patient #H, XCyting chromosome paints (Metasystems) for chromosomes 12 and 17 were applied for fluorescence in situ hybridization according to manufacturer's instructions.

Neuronal differentiation
For positive selection of neural progenitor cells at day 13 magentic PSA-NCAM MicroBeads (Miltenyi Biotec) were used. In brief, a single cell suspension was passed through a 40 µm cell strainer (Biologix) and a maximum of 1x10 7 cells was blocked in 60 µl blocking buffer (1% BSA in PBS) for 5 minutes at room temperature. Next, 20 µl of the magnetic beads were added and cells were incubated at 4 °C for 15 minutes. After addition of 2 ml blocking buffer and centrifugation, the pellet was resuspended in 500 µl induction medium and loaded onto an LS column placed into a magnetic separator (Miltenyi Biotec). The column was washed three times with expansion medium. After removal of the column from the magnetic separator, cells were resuspended in 5 ml expansion medium and seeded at a density of 4 x 10 5 / cm 2 on Matrigel-coated culture vessels. Culture was continued in expansion medium.
Cells were passaged every three to four days as single-cell suspension and seeded at densities of 4 x 10 5 / cm 2 at the first four passages and afterwards at 1 x 10 5 / cm 2 on Matrigel-coated culture vessels. Terminal differentiation into neurons followed.

qRT-PCR and TaqMan human stem cell pluripotency arrays
RNA was prepared by cell lysis in Qiazol (Qiagen), chloroform extraction and subsequent processing of the aqeous phase through columns from the RNAeasy kit (Qiagen) according to the protocol. 500 ng DNaseI-treated RNA was reverse transcribed into cDNA using either

Deep bisulfite sequencing
Genomic DNA of cultured cells was prepared using standard alkaline lysis protocols. Bisulfite

Southern blot analysis
Genomic DNA was prepared by alkaline lysis and 20 µg of genomic DNA were digested either with BamHI or EcoRV overnight and run on 0.8 % (w / v) agarose gels at 60 V for 5 to 6 h. Gels were denatured in 0.5 M NaOH / 1.5 M NaCl solution, twice for 30 min. Wet blotting to positive-charged nylon-membranes (Amersham Hybond-XL, GE Healthcare) was performed for at least 16 h in denaturing solution. Blots were hybridized with a wPRE fragment isolated from the reprogramming vector by EcoRV digestion. The probe was labeled with α-32 P-dCTP using the Megaprime kit (GE Healthcare) and purified using the QIAquick Nucleotide Removal Kit (Qiagen). Hybridization was performed at 65 °C overnight in Church buffer (7 % SDS, 1 mM EDTA, 0.5 M Na 2 HPO 4 / NaH 2 PO 4, pH 7.2). After stringent washing in Church buffer at 65 °C for at least 30 min twice, blots were exposed to film at -80 °C and developed after one to ten days.

Western blot analysis
Protein lysates of 1 x 10 6 were prepared in 100 µl NP-40-lysis buffer (500 mM    Note that images of #D are also shown in Fig. 2 of the main paper. Positive staining indicates pluripotency. The hESC H1 line was used for reference. The scale bar represents 100 µm.