Generation of two human iPSC lines from patients with autosomal dominant retinitis pigmentosa (UCLi014-A) and autosomal recessive Leber congenital amaurosis (UCLi015-A), associated with RDH12 variants

Induced pluripotent stem cell (iPSC) lines were generated from two patients with RDH12 variants. UCLi014-A is from a patient with heterozygous frameshift mutation c.759del p.(Phe254Leufs*24), associated with autosomal dominant retinitis pigmentosa. UCLi015-A is from a patient with homozygous missense mutation c.619A > G p.(Asn207Asp), associated with Leber congenital amaurosis. Fibroblasts were derived from skin biopsies and reprogrammed using integration free episomal reprogramming plasmids. The iPSC lines expressed pluripotency markers, exhibited differentiation potential in vitro and displayed normal karyotypes. These cell lines will act as a tool for disease modelling, enabling comparison of disease mechanisms, identification of therapeutic targets and drug screening.

Induced pluripotent stem cell (iPSC) lines were generated from two patients with RDH12 variants. UCLi014-A is from a patient with heterozygous frameshift mutation c.759del p.(Phe254Leufs*24), associated with autosomal dominant retinitis pigmentosa. UCLi015-A is from a patient with homozygous missense mutation c.619A > G p.(Asn207Asp), associated with Leber congenital amaurosis. Fibroblasts were derived from skin biopsies and reprogrammed using integration free episomal reprogramming plasmids. The iPSC lines expressed pluripotency markers, exhibited differentiation potential in vitro and displayed normal karyotypes. These cell lines will act as a tool for disease modelling, enabling comparison of disease mechanisms, identification of therapeutic targets and drug screening.

Resource utility
Autosomal dominant variants in RDH12 are associated with mild retinitis pigmentosa, and autosomal recessive variants are associated with Leber congenital amaurosis. The iPSC lines generated can be used to create disease models, enabling comparison of disease mechanisms between the two conditions and identification of therapeutic targets.

Resource details
Variants in the retinol dehydrogenase 12 (RDH12) gene are commonly associated with Leber congenital amaurosis (LCA), a severe retinal dystrophy characterised by night blindness, nystagmus and central loss of vision in early childhood, eventually leading to complete blindness in adulthood (Fahim et al., 2019). However, in rare cases, heterozygous variants in RDH12 have been associated with an autosomal dominant late onset mild retinitis pigmentosa phenotype, characterised by nyctalopia and visual field loss, but relatively preserved central vision (Fingert et al., 2008, Sarkar et al., 2020. RDH12 is an NADPH-dependent retinal reductase, expressed in the inner segments of photoreceptors. Loss of functional RDH12 is thought to result in build-up of toxic retinoids, although the exact disease mechanisms are not yet fully understood (Sarkar and Moosajee, 2019). Induced pluripotent stem cells (iPSCs) provide a useful resource to investigate inherited retinal dystrophies in cell types that would otherwise be inaccessible for study. iPSCs derived from patients with RDH12 variants can be used to create retinal organoids to study the differences in disease mechanisms between autosomal dominant and autosomal recessive mutations. Understanding the molecular pathogenesis of RDH12-related retinopathies will enable the identification of therapeutic targets and development of novel therapies.
Two iPSC lines were generated from patients with mutations in RDH12. The first (UCLi014-A) is from a 32-year old male with autosomal dominant retinitis pigmentosa, carrying a heterozygous frameshift mutation c.759del p.(Phe254Leufs*24). This variant is predicted to result in premature termination and expression of a truncated protein. The second (UCLi015-A) is from a 40 year old female with Leber congenital amaurosis, carrying a homozygous missense mutation c.619A > G p. (Asn207Asp). Fibroblasts were reprogrammed into iPSCs using nonintegrating episomal plasmids encoding the reprogramming factors OCT4, KLF4, SOX2, L-MYC and LIN28. Stem cell-like colonies were picked, and three iPSC clones were expanded and characterised for pluripotency. Mutations were confirmed in iPSCs by Sanger sequencing (Fig. 1D). The morphology of colonies were examined for characteristics of iPSCs, including flat, compact colonies with a cobblestone appearance and large nuclei to cytoplasmic ratio (Fig. 1A). Colonies stained red for alkaline phosphatase, indicating cells are undifferentiated (Fig. 1B). Colonies stained positive for pluripotency markers, OCT4 and SSEA3 (Fig. 1C). Expression of pluripotency markers OCT4, SOX2, L-MYC and LIN28 were validated using qRT-PCR analysis, which showed upregulation of these markers compared to fibroblast controls (Fig. 1E). Gbanding karyotyping revealed a normal male 46,XY karyotype for UCLi014-A and low-pass whole genome sequencing analysis revealed normal female 46,XX karyotype for UCLi015-A (Fig. 1G). Random differentiation of embryoid bodies stained positive for markers of endoderm (AFP), mesoderm (Vimentin) and ectoderm (PAX6), confirming differentiation potential to the three germ layers (Fig. 1F). iPSC identity was confirmed by STR analysis (Table S2). Absence of mycoplasma was confirmed in iPSCs (Table S3).

Fig. 1.
In conclusion, two human iPSCs lines were generated from patients with RDH12-related retinopathies. These iPSC lines provide a valuable resource for disease modelling, comparison of disease mechanisms, therapeutic target identification and drug screening.

Validation of mutation
DNA was extracted using QIAamp DNA Micro Kit (Qiagen). RDH12 was amplified using MyTaq PCR (Bioline) ( Table 3). Mutations were confirmed by Sanger sequencing.

Fibroblast reprogramming and iPSC culture
1 × 10 6 fibroblast cells were electroporated with 1 μg of each episomal plasmid (Table S1) using Neon Transfection System (1700 V, 20 ms, 1 pulse). Cells were plated into 1 well of a Matrigel-coated (Corning) 6-well plate in fibroblast media. On day 5, medium was changed to 3:1 fibroblast medium:mTeSR Plus (Stemcell). On day 7, medium was changed to 1:1 fibroblast medium:mTeSR Plus, from day 9 medium was changed daily with mTESR Plus. Colonies were expanded manually up to passage 4, then passaged using ReLeSR (Stemcell) at a 1:10 split ratio. iPSCs under passage 15 were used for all further characterisations (See Table 2).

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, permeabilised and blocked for 1 h at room temperature (RT) in 10% normal goat serum (NGS), 0.1% X-100, PBS. Cells were incubated for 1 h with primary antibodies diluted in 1% NGS at RT (Table 3). Secondary antibodies and DAPI were added for 1 h at RT. Cells were washed and imaged using the EVOS M7000 Imaging System.

qRT-PCR
RNA was extracted using RNeasy Mini Kit (Qiagen). cDNA was synthesised from 1 μg of RNA using Superscript II First Strand cDNA synthesis kit (Invitrogen). Transcript levels were analysed using SYBR Green MasterMix on StepOne Plus RealTime PCR System (Table 3). Relative expression of each target gene was normalised to GAPDH and compared to fibroblast expression.

Karyotyping
iPSCs were sent to Cell Guidance Systems for karyotyping and 20 metaphases were counted.

Low-pass whole genome sequencing and STR analysis
DNA was extracted using QIAamp DNA Micro Kit (Qiagen). For lowpass WGS, libraries were produced using Illumina DNA Prep library prep kit and sequenced on 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 was obtained for each cell line with Promega PowerPlex16HS system and was compared back to any available on commercial cell banks.

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.