Induced pluripotent stem cell line from an atopic dermatitis patient heterozygous for c.2282del4 mutation in filaggrin: KCLi001-A

We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-of-function mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.


A B S T R A C T
We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-offunction mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.
Resource Resource utility Generation of a library of human iPSC lines with the most common variants in the FLG gene can be efficiently used to construct highly specific in vitro 3D skin models (Petrova et al., 2014) for drug discovery towards novel personalized therapies in AD.

Resource details
AD or eczema is an incurable, non-contagious, extensive inflammatory and extremely pruritic chronical cutaneous disorder. AD is one of the most common skin diseases which affects up to 20% of children and approximately 3% of adults worldwide, while its (caption on next page) prevalence is continuously increasing, particularly in underdeveloped countries (Asher et al., 2006). Several loss-of-function mutations within FLG exon 3, including c.2282del4 variant, are considered to be the most significant risk factors for atopic dermatitis in the European population (Palmer et al., 2006). The epidermal keratinocytes derived from a female AD patient who is heterozygous for c.2282del4, were reprogrammed into iPSCs following previously established protocol with genome non-integrating Sendai virus (SeV) vectors (Miere et al., 2016a). Three weeks post-transduction colonies with a typical morphology of pluripotent stem cells appeared and were selected to establish feeder-free iPSC clones (Fig. 1A). After ten passages, the elimination of the SeV vectors was confirmed in the KCLi001-A cell line by RT-PCR using specific primers (Fig. 1B). The clones were screened with restriction enzyme digestion and we have verified that the AD-related mutation (NM_002016.1:c.2282del4) was retained in the iPSCs. This finding was also confirmed independently by Sanger sequencing (Fig. 1C). Endogenous expression of pluripotency-related molecular markers (TRA-1-60, TRA-1-81, OCT4, NANOG) in the iPSCs was assessed by double immunofluorescence technique (Fig. 1D). Furthermore, undifferentiated colonies were also positive for alkaline phosphatase (AP) (Fig. 1D). Differentiation capacity of the KCLi001-A cells into three germ layers was determined by specific immunofluorescence staining of AFP (liver, endoderm), ACTA2 (cardiac muscle, mesoderm), and TUBB3 (neurons, ectoderm) in vitro ( Fig. 1E), as well as in vivo through a teratoma formation assay. All three germ layers, ectoderm, mesoderm, and endoderm, were present in the teratoma, as demonstrated by immunohistochemical analysis (Fig. 1F).
Since our aim is to use the line for modeling AD in vitro, we tested differentiation of the KCLi001-A iPSCs into epidermal keratinocytes (Petrova et al., 2014). The cells expressed keratinocyte-specific markers -keratins 14 and 18 (KRT14, KRT18), and isoform of TP63 known as ΔNp63 within three weeks in culture as expected (Fig. 1G).
Examination of the genomic integrity of our AD-iPSC line using array CGH after more than twenty passages showed a normal female karyotype (46, XX), whereas smaller imbalances have not been excluded (submitted in archive with journal). Taken together, these results prove that we have successfully produced a stable AD patient specific iPSC line which can provide a powerful tool for: 1) developing the first iPSC-derived 3D in vitro AD-human skin equivalents (HSE); 2) deciphering the molecular mechanisms of the disease; 3) innovative drug screening platform in atopic dermatitis. (Table 1). Fig. 1. Characterization of KCLi001-A line. A, The colonies display typical morphology of pluripotent stem cells under feeder-dependent and feeder-free conditions. B, The absence of 181 bp positive SeV band at 45 and 60 days of culture indicates that the cells are SeV free. β-actin 455 bp band serves as an internal control. C, The line is heterozygous for c.2282del4 mutation in FLG as indicated with restriction enzyme digestion and Sanger sequencing of PCR product spanning the mutation site. Mutation generates a site for DraIII that is not present in wildtype allele. The enzyme digestion cuts 811 bp product in mutated allele into 667 bp and 134 bp, whereas 811 bp remains intact in wildtype. hESC line KCL038 is used as a negative control. D, Pluripotency markers. Alkaline phosphatase activity (AP) was restricted to the iPSC colony (green) growing on AP-negative feeder cells. Both iPSC colony and feeders are positive for actin (phalloidin) staining (red). iPSC colonies are positive for POU5F1 (red), TRA-1-81 (green), NANOG (red) and TRA-1-60 (green) pluripotency markers. E, Spontaneous differentiation in vitro. The cells can differentiate into all three germ layers as demonstrated with markers specific for ectoderm (TUBB3), endoderm (AFP) and mesoderm (ACTA2). Nuclei are visualized with Hoechst 33342. F, Spontaneous differentiation in vivo. Gross anatomy and staining for human-specific MTCO2 marker iPSC indicated that the teratoma is incapsulated and did not invade surrounding tissues. Teratoma contained cells from all three germ layers as demonstrated with markers specific for ectoderm (TUBB3, GFAP), endoderm (AFP, GATA4) and mesoderm (AB-PAS, DESMIN). G, Directed differentiation into keratinocytes. The cells expressed keratinocyte-specific markers KRT14, KRT18, TP63 (ΔNp63) in time-dependent manner.

Epidermal keratinocytes reprogramming
Patient keratinocytes of passage 3 were transduced with genome integration-free SeV virus kit (CytoTune 2.0, Life Technologies) as described (Miere et al., 2016a). Clonal selection of fully reprogrammed cells was performed manually by picking individual clones with hESClike appearance ( Table 1). The iPSCs under feeder-free culture conditions were maintained on Matrigel (BD Biosciences) in TeSR2 medium (STEMCELL Technologies).

FLG mutation verification
Genomic DNA was extracted from KCLi001-A cells using DNeasy Blood & Tissue Kits (Qiagen) and samples were verified independently by restriction enzyme digestion and Sanger sequencing using the primers described in Table 2. A 311 bp sequence in exon 3 of FLG was amplified and Eurofins Genomics provided DNA sequencing service. Mutation c. 2282del4 creates a new DraIII site, which was used to screen short, highly specific 811 bp FLG gene fragment, as described in more detail previously (Palmer et al., 2006).

Reverse transcription PCR analysis of SeV vectors
Total RNA was isolated from the cells 7, 45 and 60 days posttransduction (RNeasy mini Kit, Qiagen). SeV specific primers were used to assess the presence of remaining Sendai virus vectors (Table 2). RT-PCR for the detection of SeV transgenes was carried out using the Su-perScript IV First-strand cDNA synthesis reaction kit (Invitrogen).

Pluripotency markers
The pluripotency status of KCLi001-A line was evaluated by immunostaining for three germ layer markers in spontaneously differentiated cells (Table 1) as previously described (Petrova et al., 2014).

Alkaline phosphatase activity
Emerging iPSCs were analyzed for alkaline phosphatase activity by AP Live Stain (Thermo Fisher). After live staining, iPSCs were washed and fixed, and cytoskeletal actin filaments have been contrasted by labelling with rhodamine phalloidin (Molecular Probes).

Spontaneuous differentiation into three germ layers
To test the differentiation capacity of our iPSC line, in vitro embryonic body formation, as well as in vivo conventional teratoma assay were assessed (Table 1), as previously described (Petrova et al., 2014).

Molecular karyotyping
Array comparative genomic hybridization (aCGH) and short tandem repeat (STR) analysis of 17 STR loci were conducted at Viapath Genetics Centre.

Mycoplasma contamination detection
The absence of mycoplasma contamination was detected using LookOut® Mycoplasma PCR Detection Kit (Sigma-Aldrich).