Generation of a human iPSC line, INMi002-A, carrying the most prevalent USH2A variant associated with Usher syndrome type 2

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a patient with Usher syndrome type 2 (USH2). This individual was homozygous for the most prevalent variant reported in the USH2A gene, c.2299delG localized in exon 13. Reprogramming was performed using the non-integrative Sendai virus reprogramming method and the human OSKM transcription factor cocktail under feeder-free culture conditions. This iPSC line will be an invaluable tool for studying the pathophysiology of USH2 and for testing the efficacy of novel treatments.


Resource utility
We established an iPSC line from a patient with Usher syndrome type 2 (USH2), characterized by retinitis pigmentosa and hearing loss, homozygous for the recurrent USH2A variant, c.2299delG. This line will allow modelling of the USH2 retinal and inner ear defects, and the development of novel gene and cell therapies.

Resource details
Mutations in USH2A cause a syndromic inherited retinal dystrophy (IRD) known as Usher syndrome type 2 (USH2), which is characterized by progressive hearing and vision loss. In addition, mutations in USH2A also cause a wide majority of autosomal recessive retinitis pigmentosa (RP) cases (Kremer et al., 2006). In the present study, we have generated an iPSC line from a patient with USH2 carrying a homozygous variant in exon 13 of the USH2A gene, c.2299delG; p.Glu767Serfs*21. This is a recurrent mutation originating from a common ancestor. Therefore, c.2299delG is observed more frequently in the population compared to the > 600 USH2A mutations identified (Lenassi et al., 2015), conferring a great deal of interest from a clinical perspective.
Human dermal fibroblasts were isolated and cultured from a patient skin biopsy sample and reprogrammed using an integration-free method, the CytoTune™-iPS 2.0 Sendai Reprogramming Kit. This method is based on transient overexpression of the four Yamanaka factors: OCT3/4, SOX2, KLF4 and c-MYC (Takahashi et al., 2007). C. Sanjurjo-Soriano et al. Stem Cell Research 33 (2018) 247-250 appearance comprised of tightly packed cells (Fig. 1A). The absence of the Sendai reprogramming vectors in the INMi002-A clones was confirmed using reverse transcription (RT)-PCR. As a negative control, nontransduced patient fibroblasts (Fibro) did not carry the Sendai vectors. By contrast, the transduced fibroblasts (Fibro + SeV) expressed all three vectors that were used for reprogramming. Due to their non-integrative nature, the INMi002-A cell line had lost these vectors by passage 12 (P12) (Fig. 1B). The iPSC line generated showed a normal 46,XX karyotype at P12, which excluded major chromosomal abnormalities as a result of the reprogramming process ( Fig. 1C).
Using real time polymerase chain reaction (qPCR), we showed expression of the endogenous pluripotency markers, NANOG, OCT3/4 and LIN28a in the INMi002-A cell line when compared to non-transduced (Fibro) or transduced (Fibro + SeV; Fig. 1D) fibroblasts. The pluripotency state was further confirmed by immunofluorescence staining of NANOG ( 1J) was verified by immunofluorescence staining. We verified the presence of the homozygous causative mutation in exon 13 of USH2A (c.2299delG) in the INMi002-A iPSC, as compared to wild type iPSC, by Sanger sequencing (Fig. 1K). The identity of the patient iPSC line was confirmed by microsatellite PCR analysis in comparison to fibroblasts of the same individual and wild type iPSC (available with the authors). Lastly, the generated INMi002-A cell line was confirmed to be free of mycoplasma contamination (Supplementary File 1).

Karyotype analysis
Preparation of the iPSC for karyotype analysis was performed as previously described without modification (Torriano et al., 2017). Twenty metaphase spreads were counted and analyses were performed using standard G-banding procedures by the Chromostem facility (CHU Montpellier, France).

RT-PCR and qPCR analysis
RNA was extracted using the RNeasy Mini Kit (QIAGEN) and cDNA synthesis performed using the SuperScript® III First-Strand Synthesis System (Life Technologies) and random hexamers (Life Technologies), according to the manufacturer's protocols. RT-PCR was performed using a standard protocol on an Applied Biosystems Veriti 96-well thermal cycler. qPCR analysis was performed using the FastStart SYBR Green I Master mix on a LightCycler 480 II thermal cycler (Roche). Gene expression was normalized to GAPDH; see Table 2 for all primers.

Mutation analysis
Genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) and PCR-amplified using USH2A-specific primers ( Table 2). The dNTPs were removed using the ExoSAP-IT PCR Clean-up kit (GE Healthcare) and the amplicon sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit V3.1 on an Applied Biosystems 3130xL Genetic Analyzer.

Microsatellite PCR analysis
Genomic DNA was amplified using primers for informative markers ( Table 1). The PCR products were mixed with Genescan 400HD ROX size standard and subsequently analyzed on an Applied Biosystems 3130xL genetic analyzer.

Mycoplasma analysis
Mycoplasma detection was performed on cell culture supernatant using the MycoAlert Mycoplasma Detection Kit (Lonza), according to the manufacturer's instructions, and a CLARIOstar microplate reader (BMG Labtech).  Sanjurjo-Soriano et al. Stem Cell Research 33 (2018) 247-250