Generation of two human control iPS cell lines (UCLi016-A and UCLi017-A) from healthy donors with no known ocular conditions

Two human induced pluripotent stem cell (hiPSC) lines (UCLi016-A and UCLi017-A) were generated from fibroblast cells of 23- and 34-year-old healthy male donors with no known ocular conditions. Fibroblast cells were derived from skin biopsies and reprogrammed using integration free episomal reprogramming. The established iPSC lines were found to express pluripotency markers, exhibit differentiation potential in vitro and display a normal karyotype. These cell lines will act as a control lines for researchers studying ocular diseases.


Resource utility
Human induced pluripotent stem cell (hiPSCs) generated from two healthy male donors with no known ocular conditions using integrationfree episomal reprogramming from fibroblasts will act as control lines for studying cellular models of ocular disease.

Resource details
hiPSCs provide a resource to investigate human conditions which would otherwise be inaccessible to study. Patient-derived iPSCs with known pathogenic mutations may elucidate the molecular basis of genetic ocular disorders through in vitro human cellular disease modelling. This knowledge will aid in understanding the phenotypic variation observed in patient cohorts, improving diagnosis and management, in addition to allowing development of novel treatments. In order to study misregulated gene and protein function in disease lines, it is necessary to have stable control lines derived from healthy individuals.
In this study, two iPSC lines were derived from the fibroblasts of the healthy male donors with no known ocular conditions (Table 1). These iPSC lines can be used as control lines for research into cellular models of ocular disease.
With ethical approval, skin biopsies were taken and fibroblasts were derived. Fibroblasts were reprogrammed into iPSCs using nonintegrating 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 celllike colonies were picked, and three iPSC clones per line were expanded and characterised for pluripotency (Table 2). iPSC morphology was examined showing flat, compact colonies and cells with cobblestone appearance and large nuclei to cytoplasmic ratio (Fig. 1A). iPSCs were positively stained for alkaline phosphatase activity (Fig. 1B) and key pluripotency markers OCT4 and SSEA3 (Fig. 1C). 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. 1D). 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. 1E). Low-pass whole genome sequencing analysis of iPSCs revealed a normal male 46XY karyotypes (Fig. 1F). Genetic signature identity of fibroblasts and iPSCs was confirmed through STR analysis (submitted to journal). Absence of Mycoplasma was confirmed in iPSCs (Table S2).
In conclusion, two wild-type (WT) control hiPSCs were generated from fibroblasts of healthy male donors with no known ocular conditions. These iPSC lines will be used as control lines for disease modelling, to aid in understanding molecular pathology of ocular disease, identifying therapeutic targets and drug screening.

Fibroblast reprogramming and iPSC culture
1 × 10 6 fibroblast cells were electroporated (1700 V, 20 ms, 1 pulse) with 1 μg of each episomal plasmid (Table S1) using the Neon Transfection System (Parfitt et al., 2016). Transfected cells were plated in fibroblast media with 0.5 mM sodium butyrate in a 0.1% gelatin-coated 100 mm dish for 7 days. Cells were dissociated with TrypLE Express and 200,000 cells plated into each well of a Matrigel-coated (Corning) 6-well plate in mTeSR Plus (Stemcell Technologies). Colonies were picked manually for the first 4 passages, then passaged using ReLeSR (Stemcell Technologies) at 70% confluency.

Alkaline phosphatase staining
Cells were stained using the 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 3). Secondary antibodies and DAPI were added for 1 h at RT (Table 3). 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 with designed primers (Sigma Aldrich) (Table 3) (Ye et al., 2012). 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 Technologies) supplemented with 10 µM Y27632 (Abcam) for 7-10 days. Embryoid bodies were plated in 0.1% gelatin-coated plates for 11-15 days, where embryoid bodies attached and spontaneously differentiated. Cells were fixed and immunostained for AFP, Vimentin and PAX6 (Table 3).

Low-pass whole genome sequencing and STR analysis
DNA was extracted using QIAamp DNA Micro Kit (Qiagen). For lowpass 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 was compared back to any available on commercial cell banks (such as ATCC).

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

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.