The generation of an induced pluripotent stem cell line (DCGi001-A) from an individual with FOXG1 syndrome carrying the c.460dupG (p.Glu154fs) variation in the FOXG1 gene

FOXG1 syndrome is a neurodevelopmental disorder caused by mutations in the FOXG1 gene. Here, an induced pluripotent stem cell (iPSC) line was generated from human dermal fibroblasts of an individual with the c.490dupG (p.Glu154fs) mutation in the FOXG1 gene. Fibroblasts were reprogrammed using non-integrating episomal plasmids and pluripotency marker expression was confirmed by both immunocytochemistry and quantitative PCR in the resultant iPSC line. There were no karyotypic abnormalities and the cell line successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin FOXG1 syndrome.


Resource utility
FOXG1 syndrome is a neurodevelopmental disorder caused by mutations in the FOXG1 gene. We generated iPSCs from fibroblasts of an individual with the c.460dupG FOXG1 mutation. This patient-derived cell line will be useful for modelling the pathogenic mechanisms that underpin FOXG1 syndrome.

Resource details
FOXG1 syndrome (originally called the congenital-onset variant of Rett syndrome) is a rare neurodevelopmental disorder associated with heterozygous mutations in the FOXG1 gene (located on chromosome 14q12). The range of variants associated with the disorder include chromosomal microdeletions, larger deletions and intragenic mutations (missense, nonsense and frameshift). Furthermore, a subset of cases are associated with structural variants occurring downstream of FOXG1 that may disrupt cis-regulation of FOXG1 expression. The clinical phenotype associated with FOXG1 syndrome includes severe intellectual disability, postnatal microcephaly, dyskinetic-hyperkinetic movement disorders, visual impairment, epilepsy, stereotypies, abnormal sleep patterns, and unexplained episodes of crying. Brain imaging of patients reveal structural abnormalities including hypoplasia of the corpus callosum and underdevelopment of the frontal cortex (Kortum et al., 2011;Vegas et al., 2018). The FOXG1 gene encodes the forkhead box G1 protein, a winged-helix transcriptional factor that is a master regulator of the development and regional specification of the ventral https://doi.org/10.1016/j.scr.2020.102018 Received 29 January 2020; Received in revised form 21 September 2020; Accepted 27 September 2020 telencephalon (Wong et al., 2019). Through its array of protein and DNA interactions FOXG1 has pleiotropic and non-redundant roles in brain development, reflected in the complex genotype-phenotype presentations observed (Vegas et al., 2018;Mitter et al., 2018).
Fibroblasts were isolated from a skin biopsy taken from an 18-year old FOXG1 syndrome patient with a previously reported heterozygous pathogenic variant, c.460dupG (p.Glu154fs) (Vegas et al., 2018). iPSCs were generated by a non-integrating episomal plasmid-based method A.J. Waite, et al. Stem Cell Research 49 (2020) 102018 through expression of the reprogramming factors OCT4, SOX2, KLF4, L-MYC, LIN28 and p53 shRNA. A panel of iPSC-like colonies were manually picked after 20 days post-electroporation and expanded in culture over several passages for establishment as iPSC clones for further characterisation. Amongst the panel of clones was FS_E154#3 that is described herein. Polymerase chain reaction (PCR) analysis performed at the 10th passage using validated primers against the EBNA1 backbone, common to all the episomal plasmids, confirmed their elimination from the iPSC line ( Supplementary Fig. 1A). The established FS_E154#3 iPSC line showed typical human embryonic stem celllike morphology as judged by brightfield microscopy (Fig. 1A, scale bar is 100 μm). The line expressed the pluripotency markers OCT4 and SSEA4 as shown by immunocytochemistry (Fig. 1A). Quantitative realtime PCR analysis (qPCR) demonstrated the expression of pluripotency markers NANOG, OCT4 and SOX2 in the FS_E154#3 line comparable to a control iPSC line (sex-matched) previously generated in house (Fig. 1B). FS_E154#3 displayed a normal diploid 46 XX karyotype (passage 9, Fig. 1C), and sequencing analysis confirmed the presence of the heterozygous FOXG1 variant found in the parental fibroblast line (Fig. 1D). Short tandem repeat analysis of genomic DNA extracted from FS_E154#3 and parental fibroblasts confirmed matching genetic identity at the 16 loci tested. in vitro differentiation potential was verified using an embryoid body (EB) assay, followed by qPCR analysis of gene expression relative to undifferentiated cells (Fig. 1E, 12 germ layer markers). This detected increased expression of marker genes indicating the presence of cells derived from endoderm, ectoderm and mesoderm lineages. Furthermore, epifluorescence microscopy detected cells immunoreactive for ectoderm (nestin, PAX6), endoderm (AFP) and mesoderm (αSMA) specific markers (Fig. 1F). The absence of mycoplasma contamination was confirmed by a PCR-based assay ( Supplementary  Fig. 1B).

Reagents
Reagents were purchased from Thermo Fisher Scientific unless otherwise stated.

In vitro differentiation
EBs were formed as previously described (Ungrin et al., 2008) with modifications. iPSCs were dissociated using Accutase, seeded into vbottom plates (Greiner; 2000 cells/well) and cultured for 24 h in StemFlex medium with 10 μM Y-27632 (Hello Bio) and 0.4% (w/v) PVA (Sigma). EBs were washed in D-PBS, resuspended in E6 media and transferred to low-attachment 96-well plates (Greiner). EBs were cultured for a further 7 days before seeding onto Matrigel-coated 24-well plates and cultured in E6 media for an additional 7 days prior to analysis.

RNA isolation, PCR and qPCR
RNA was isolated using the RNeasy Mini Kit (QIAGEN) followed by DNase treatment (DNAfree kit). Complementary DNA was reverse transcribed from 250 ng total RNA using SuperScript IV. qPCRs were performed using Power SYBR Green PCR master mix and an ABI 7300 Real Time PCR System, with data analysed by the ΔΔCt method using ACTNB for normalisation. PCR and qPCR parameters are available on request and Table 1.

Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 20 mins at room temperature (RT), permeabilised in 0.1% (v/v) Triton X-100 for 15 mins at 4 °C and incubated in blocking solution (10% FBS in PBS) for 30 mins at RT. Fixed cells were incubated with primary antibodies (Table 2) in blocking solution (1-3 h at RT). Following three PBS washes, cells were incubated with Alexa Fluor-conjugated secondary antibodies (Table 2) in blocking solution (1 h at RT), washed three times Expression of OCT4 and SSEA4. Fig. 1

Sequencing
The FOXG1 c.460dupG variant was confirmed in iPSCs and parental fibroblasts by Sanger Sequencing of PCR-amplified sequence from genomic DNA using specific primers (Table 2). DNA was isolated using the QIAamp DNA mini kit (QIAGEN). Sequencing reactions were performed using Big Dye v3.1 chemistry and analysed by the All Wales Medical Genomics Service (Cardiff, UK).

STR analysis
STR analysis of 16 loci was performed using the Promega Powerplex 16HS kit with amplicon detection using an Applied Biosystems 3730xl (undertaken by Source BioScience, Nottingham, UK).

Mycoplasma test
Mycoplasma contamination was tested using the PCR-based Venor®GeM Classic detection kit (Minerva Biolabs) according to manufacturer's instructions.