Generation of a human induced pluripotent stem cell line (UQACi001-A) from a severe epidermolysis bullosa simplex patient with the heterozygous mutation p.R125S in the KRT 14 gene

We have generated UQACi001-A, a new induced pluripotent stem cell (iPSC) line derived from skin ﬁ broblasts of a male patient with the generalized severe epidermolysis bullosa simplex phenotype (EBS-gen sev) and carrying the keratin 14 (K14) R125S mutation. Fibroblasts were reprogrammed using non-integrating Sendai virus vectors. The iPSC line displayed normal molecular karyotype, expressed pluripotency markers, is capable of di ﬀ erentiating into three embryonic germ layers and is genetically identical to the originating parental ﬁ broblasts. The established iPSC model provides a valuable resource for studying the rare disease of epidermolysis bullosa simplex and developing new therapies as DNA editing by CRISPR/Cas9 technology.


Resource utility
The iPSCs line was established from an EBS patient with the severe missense mutation p.R125S, not reported elsewhere (Bchetnia et al., 2012).This line might provide a cellular model to investigate the biological pathways altered by this mutation as well as to construct in vitro 3D skin models useful for novel personalized therapies.

Resource details
Epidermolysis bullosa simplex (EBS) is a rare skin disease characterized by skin fragility and blistering upon minor mechanical trauma.This disease is primarily caused by dominantly autosomal mutations in the keratin 5 (KRT5) or 14 genes (KRT14).These mutations lead to a collapse of the keratin cytoskeleton into cytoplasmic protein aggregates and the appearance of the EBS phenotype.close to 200 distinct pathogenic mutations have been identified (http:// www.interfil.org)and variants in the KRT14 gene that encodes keratin 14, are responsible for approximately 37% of all cases (Fine et al., 2014).A particular arginine codon within the helix initiation peptide in K14 (R125) is the most commonly mutated residue occurring in >30% of EBS-gen-sev cases, probably because it contains a hypermutable CpG dinucleotide.As a result, many studies in the literature reported severe affected patients with the cysteine (TGC) or histidine (CAC) in the place of the arginine codon (CGC) (Uitto et al., 2007).Our patient with the EBS-gen-sev phenotype is carrying a serine (AGC) at the 125 position (p.R125S) which is until now not reported elsewhere (Bchetnia et al., 2012).
Here we reprogrammed primary fibroblasts obtained from a skin biopsy of this patient (EBS21F) on iPSCs using non-integrative Sendai virus containing the human reprogramming factors, OCT4, SOX2, C-MYC and KLF4 (Takahashi et al., 2007) following instructions by manufacturer.Four weeks post transduction, colonies with a typical morphology of pluripotent stem cells appeared.These clones were subsequently manually picked and expanded to establish feeder-free iPSC cells (Fig. 1A).After two months' culture, the clearance of the virus and the exogenous reprogramming factor genes was confirmed in the resulting UQACi001-A cell line by PCR using specific primers (Fig. 1B).The presence of the heterozygous (K14 p.R125S) mutation was confirmed in the iPSC line by Sanger sequencing (Fig. 1C).Pluripotency was assessed by specific immunofluorescence staining for OCT4, NANOG, SSEA4, TRA-1-60 and TRA-1-81 (Fig. 1D) as well as by qRT-PCR for OCT4, NANOG, DNM3TB, hTERT, and TDGF (Fig. 1E).The iPSC line showed robust expression of all tested pluripotency markers.UQACi001-A cell line formed embryoid bodies that spontaneously differentiated into three germ layers.Using scorecard analysis, we observed expression of specific markers for ectoderm, mesoderm and endoderm (Fig. 1F).Examination of the genomic integrity of our iPSC line using array CGH, after six passages, showed a normal karyotype with no gain or loss that would be detected in a traditional karyotype (>5 MB) (Fig. 1G).STR analysis for 16 short tandem repeat markers showed identical profiles for iPSC line with the parental fibroblasts (available with the authors).Mycoplasma testing was negative proving that our iPSC line is free from mycoplasma contamination (Fig. S1).The current data proves that stable EBS-gen-sev patient specific iPSC line have been successfully generated.UQACi001-A cell line can provide a powerful tool for: 1) establishing an iPSC-derived skin equivalent; 2) identifying the biological pathways altered by the R125S mutation; 3) innovative drug screening and genome editing for EBS.

Mutation verification
Genomic DNA was extracted from primary fibroblasts and iPSCs cells using QuickExtract™ DNA Extraction Solution (Epicentre).Primers used for amplification and Sanger sequencing of K14p.R125S flanking region are described in Table 2.

Immunofluorescence analysis
The pluripotency status of UQACi001-A cell line was evaluated by immunostaining for NANOG, OCT4, SSEA4, TRA-1-60, and TRA-1-81.Briefly, the iPSCs were fixed with 4% para formaldehyde for 15 min at room temperature and washed with DPBS.They were permeabilized with 0.1% Triton™X-100, and blocked with 1%BSA, 0.3% Triton™X-100 in DPBS at room temperature.Cells were then stained with specific antibodies (Table 1).Images were captured under the fluorescent microscope (Zeiss Axio Observer Microscope).

PCR and qRT-PCR analysis
PCR was carried out on genomic DNA using HotStarTaq DNA polymerase kit (Qiagen) using specific primers to assess the presence of remaining Sendai virus vectors (Table 2).Total RNA was isolated from iPSC cells using Direct-zol™ RNA miniprep and reverse transcribed into cDNA using the Quantitect Reverse transcription kit.Pluripotency markers expression was performed by qRT-PCR using SYBR Green I Master hot start reaction mix.RPL13A and ACTIN were used as normalization controls.Markers characterizing the three germ layers were assessed by scorecard assay using the scorecard™ Kit 384w (Applied Biosystems) following manufacturer's protocol (Fergus et al., 2016).

In vitro differentiation
To evaluate the ability of UQACi001-A line to form three germ layers, spontaneous formation of embryoid body (EB) in vitro was assessed.iPSCs were harvested with accutase and plated in non-adherent dishes in EB medium consisting of DMEM/F12, 20% KnockOut™ Serum Replacement 1% non-essential amino acids and 1% GlutaMAX™ (ThermoFisher Scientific), 0.1 mM 2-mercaptomethanol and 50 µM rock inhibitor Y-27632.Forming EBs were transferred, after 8 days in suspension, onto gelatin coated plate and cultured for another 8 days.

Molecular karyotyping
Array comparative genomic hybridization (aCGH) at passage 6 was performed for UQACi001-A cell line at the Cell Line Genetics Inc laboratories (Madison, WI, USA).aCGH does not detect translocations or inversions, alterations in chromosome structure, mosaicism or polyploidy.

Mycoplasma contamination detection
The absence of mycoplasma contamination was detected using Venor®GeM Mycoplasma PCR Detection Kit (Cederlane).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table 1
Characterization and validation.