Generation of human iPSC line (UCLi013-A) from a patient with microphthalmia and aniridia, carrying a heterozygous missense mutation c.372C>A p.(Asn124Lys) in PAX6

A human induced pluripotent stem cell (hiPSC) line (UCLi013-A) was generated from fibroblast cells of a 34-year-old donor with multiple ocular conditions including severe microphthalmia and aniridia. The patient had a heterozygous missense mutation in PAX6 c.372C>A, p.(Asn124Lys), validated in the fibroblasts through Sanger sequencing. Fibroblasts derived from a skin biopsy were reprogrammed using integration free episomal reprogramming. The established iPSC line was found to express pluripotency markers, exhibit differentiation potential in vitro and display a normal karyotype. This cell line will act as a tool for disease modelling of microphthalmia and aniridia, identification of therapeutic targets and drug screening.


Resource utility
The iPSC line UCLi013-A was established after reprogramming of fibroblasts isolated from a female individual with severe microphthalmia, aniridia and other ocular disorders caused by a heterozygous missense mutation in PAX6, c.373C>A p.(Asn124Lys). This line provides a valuable resource for in vitro eye development studies, disease modelling and drug screening.

Resource details
PAX6 (OMIM:607108) is a highly conserved transcriptional regulator of oculogenesis (Lima Cunha et al., 2019). Switched on early in eye development, PAX6 is expressed throughout the optic vesicle by 5 weeks development. Pathogenic heterozygous variants in PAX6 cause a variety of ocular disorders including microphthalmia (small eye), aniridia (absent iris), cataracts (clouded lens), nystagmus (uncontrolled eye movement) and coloboma (gap in eye structure).
An iPSC line was derived from fibroblasts of a 34-year-old female with severe microphthalmia, aniridia, cataracts, optic nerve coloboma and nystagmus, and genetically diagnosed with a heterozygous missense mutation in PAX6 c.372C>A p.(Asn124Lys) ( Table 1). Missense mutations in DNA binding domains of PAX6, including p.(Asn124Lys), can result in reduced DNA binding ability (Williamson et al., 2019). Patients carrying this mutation exhibit severe microphthalmia, alongside complex ocular features phenocopying SOX2-associated microphthalmia syndrome, including iris defects, coloboma, congenital corneal opacification and lens defects.
hiPSCs provide a resource to investigate congenital human diseases, such as microphthalmia and aniridia, which affect early eye development so are otherwise inaccessible to study. Generation of patientderived iPSCs with known PAX6 mutations may improve understanding of PAX6 function in eye development through in vitro human disease modelling. Consequently, researchers can clarify the molecular basis of aniridia (through modelling iris and optic nerve development), in addition to microphthalmia pathogenesis (by replicating early eye development). Additionally, these models may elucidate genotype/phenotype relationships observed in PAX6 patient cohorts, thereby improving diagnosis and management, and aiding development of novel treatments.
With ethical approval, a skin biopsy was taken and fibroblasts derived. DNA was extracted from fibroblasts, and the variant c.372C>A in PAX6 exon 7 was confirmed by Sanger sequencing (Fig. 1A). Fibroblasts were reprogrammed into iPSCs using non-integrating episomal plasmids encoding the reprogramming factors OCT4, KLF4, SOX2, L-MYC and LIN28 as well as transient transcription enhancer EBNA (Table S1) (Parfitt et al., 2016). Embryonic stem cell-like colonies were picked, and three iPSC clones were expanded and characterised for pluripotency. iPSC morphology was examined showing flat, compact colonies and cells with cobblestone appearance and large nuclei to cytoplasmic ratio (Fig. 1B). iPSCs were stained for alkaline phosphatase activity (Fig. 1C) and pluripotency markers OCT4 and SSEA3 (Fig. 1D). Gene expression of pluripotency markers OCT4, SOX2, L-MYC and LIN28 was validated using qRT-PCR analysis, which showed upregulation of these markers compared to fibroblast controls (Fig. 1E). In vitro differentiation ability after embryoid body formation showed positive staining for all three germ layers, using endoderm marker AFP, mesoderm marker Vimentin (VIM) and ectoderm marker PAX6 (Fig. 1F). Low-pass whole genome sequencing analysis of iPSCs revealed a normal female 46,XX karyotype (Fig. 1G). Genetic signature identity of fibroblasts and iPSCs was confirmed through STR analysis (submitted to journal). Absence of Mycoplasma was confirmed in iPSCs (Table S1).
In conclusion, iPSCs were generated from a patient with a heterozygous c.372C>A p.(Asn124Lys) missense mutation in PAX6, associated with severe microphthalmia, aniridia and other ocular disorders. This iPSC line could be used as a resource for disease modelling, therapeutic target identification and drug screening for various ocular conditions.

Alkaline phosphatase staining
Cells were stained using StemAb Alkaline Phosphatase Staining Kit II (Reprocell).

Immunocytochemistry
Cells were fixed using 4% PFA for 20 min at 4 • C, permeabilized and blocked for 1 h using 10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS at RT. Cells were incubated for 1 h at RT with primary antibodies diluted in 1% NGS (Table 2). Secondary antibodies and DAPI were added for 1 h at RT (Table 2). Cells were imaged using the EVOS M7000 Imaging System.

qRT-PCR
RNA was extracted from cell pellets using RNeasy Mini Kit (Qiagen) and 1 μg of cDNA synthesised using SuperScript III First-Strand Synthesis kit (Invitrogen). qRT-PCR was performed using SYBR green mastermix (Applied Biosystems), run on the StepOne Plus RealTime PCR System (Thermo Fisher) using standard cycle conditions ( Table 2). The relative expression of each target gene was normalised to housekeeper GAPDH and compared to fibroblast expression using the comparative CT method.

Embryoid body mediated spontaneous in vitro differentiation
Embryoid bodies were formed by cell dissociation with ReLeSR and culturing in Aggrewell media (Stemcell) supplemented with 10 µM Y27632 for 7-10 days. Embryoid bodies were plated in 0.1% gelatincoated plates for 11-15 days, where embryoid bodies attached and spontaneously differentiated. Cells were fixed and immunostained for AFP, Vimentin and PAX6 (Table 2).

Low-pass whole genome sequencing and STR analysis
DNA was extracted using QIAamp DNA Micro Kit (Qiagen). Low-pass WGS libraries were produced using the Illumina DNA Prep library prep kit and sequenced on the Illumina HiSeq 4000 with paired 100 bp reads. After alignment, copy number estimation was performed using the QDNASeq package (Scheinin et al., 2014). Short Tandem Repeat (STR) profiling of 16 sites was obtained for iPSC and fibroblast lines with the Promega PowerPlex16HS system and compared to any commercial cell banks (such as ATCC).

Mycoplasma testing
Absence of Mycoplasma contamination was confirmed using MycoAlert™ Mycoplasma Detection Kit (Lonza).

Funding
This research was funded by The Wellcome Trust, grant number 205174/Z/16/Z; and Moorfields Eye Charity.

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.