# Generation of an iPSC line, INMi001-A, carrying the two most common USH2A mutations from a compound heterozygote with non-syndromic retinitis pigmentosa

Carla Sanjurjo-Soriano, Nejla Erkilic, Gael Manes, Grégor Dubois, Christian

P. Hamel, Isabelle Meunier, Vasiliki Kalatzis

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Carla Sanjurjo-Soriano, Nejla Erkilic, Gael Manes, Grégor Dubois, Christian P. Hamel, et al.. Generation of an iPSC line, INMi001-A, carrying the two most common USH2A mutations from a compound heterozygote with non-syndromic retinitis pigmentosa. Stem Cell Research, 2018, 33, pp.228-232. 10.1016/j.scr.2018.11.004. hal-02310858

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Lab Resource: Stem Cell Line

# Generation of an iPSC line, INMi001-A, carrying the two most common USH2A mutations from a compound heterozygote with non-syndromic retinitis pigmentosa

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Carla Sanjurjo-Soriano<sup>a,b</sup>, Nejla Erkilic<sup>a,b</sup>, Gaël Manes<sup>a,b</sup>, Gregor Dubois<sup>a,b</sup>, Christian P. Hamel<sup>a,b,c</sup>, Isabelle Meunier<sup>a,b,c</sup>, Vasiliki Kalatzis<sup>a,b,*</sup>

## ABSTRACT

We generated an induced pluripotent stem cell (iPSC) line from a patient with non-syndromic retinitis pigmentosa who is a compound heterozygote for the two most frequent USH2A variants, c.2276G > T and c.2299delG localized in exon 13. Patient fibroblasts were reprogrammed using the non-integrative Sendai virus re-programming method and the human OSKM transcription factor cocktail. The generated cells were pluripotent and genetically stable. This iPSC line will be an important tool for studying the pathogenesis of these USH2A mutations and for developing treatments that, due their high prevalence, will target a large patient population.

## Resource table

<table><tr><td>Unique stem cell line identifier</td><td>INMi001-A</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>USH2A-RP-iPSC</td></tr><tr><td>Institution</td><td>Institute for Neurosciences of Montpellier, Montpellier, France</td></tr><tr><td>Contact information of distributor</td><td>Vasiliki Kalatzis<br>vasiliki.kalatzis@inserm.fr</td></tr><tr><td>Type of cell line</td><td>iPSC</td></tr><tr><td>Origin</td><td>Human</td></tr><tr><td>Additional origin info</td><td>Age: 70 years old<br>Sex: Female<br>Ethnicity: Caucasian</td></tr><tr><td>Cell Source</td><td>Dermal fibroblasts</td></tr><tr><td>Clonality</td><td>Clonal</td></tr><tr><td>Method of reprogramming</td><td>Non-integrative Sendai virus vectors</td></tr><tr><td>Genetic Modification</td><td>Yes</td></tr><tr><td>Type of Modification</td><td>Congenital mutation</td></tr><tr><td>Associated disease</td><td>Non-syndromic retinitis pigmentosa</td></tr><tr><td>Gene/locus</td><td>USH2A, 1q41</td></tr><tr><td>Method of modification</td><td>N/A</td></tr><tr><td>Name of transgene or resistance</td><td>N/A</td></tr><tr><td>Inducible/constitutive system</td><td>N/A</td></tr><tr><td>Date archived/stock date</td><td>March 2018</td></tr><tr><td>Cell line repository/bank</td><td>https://hpsreg.eu/cell-line/INMi001-A</td></tr><tr><td>Ethical approval</td><td>Regional committee: CPP Southern<br>Mediterranean I (2014-A00549-38)<br>National committee: ANSM (140549B-62)</td></tr></table>

## Resource utility

This iPSC line carries two recurrent USH2A variants, the hypomorphic c.2276G > T and the deleterious c.2299delG, and is associated with autosomal recessive retinitis pigmentosa (RP). It will prove an invaluable tool for deciphering the pathophysiology of non-syndromic and syndromic RP, both caused by mutations in USH2A.

## Resource details

Retinitis pigmentosa (RP) is an inherited retinal dystrophy (IRD) characterized by progressive retinal degeneration, which results in night blindness and subsequently peripheral vision loss. Mutations in over 80 genes have been described to cause RP (Verbakel et al., 2018). Interestingly, mutations in USH2A can either cause Usher syndrome type 2 (USH2), presenting with RP and hearing loss, or isolated RP, in which only the retina is affected (Kremer et al., 2006). The different phenotypes found in patients with mutations in USH2A, was suggested to be due to an allelic hierarchy of USH2A mutations. Retina-specific alleles in USH2A, are considered to segregate in patients presenting non-syndromic RP and normal hearing. The recurrent missense variant c.2276G > T; p.Cys759Phe is one of these retina-specific alleles (Lenassi et al., 2015). In the present study, we have generated an iPSC line from a patient presenting autosomal recessive RP carrying this

<footnote><sup>a</sup> Inserm U1051, Institute for Neurosciences of Montpellier, Montpellier, France</footnote>

<footnote><sup>b</sup> University of Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> Centre of Reference for Genetic Sensory Diseases, CHU, Montpellier, France</footnote>

<footnote>* Corresponding author at: Inserm U1051, INM, Hôpital St Eloi, BP 74103, 80 Avenue Augustin Fliche, 34091 Montpellier, France.
E-mail address: vasiliki.kalatzis@inserm.fr (V. Kalatzis).</footnote>
allele. Furthermore, this individual is compound heterozygous for an-
other recurrent **USH2A** variant, c.2299delG; p.Glu767Serfs*21, which,
in the homozygous state, is associated with USH2.

The human iPSC line, INMi001-A (USH2A-RP-iPSC), has been gen-
erated using a non-integrative method, the CytoTune™-iPS 2.0 Sendai
Reprogramming Kit. Human dermal fibroblasts were obtained from a
patient skin biopsy and reprogrammed using the Sendai virus vectors
containing the reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC,
which confer pluripotency to somatic cells (Takahashi et al., 2007).
After reprogramming, USH2A-RP-iPSC colonies displayed the typical
morphology of tightly packed cells surrounded by a distinct border
(Fig. 1A). We assessed the loss of the Sendai vectors over successive
passages (tested at P8 and P18) by reverse transcription (RT)-PCR using
specific primers for each transgene (Fig. 1B). By comparison, as a ne-
gative control, non-transduced patient fibroblasts (Fibro) did not carry
the Sendai vectors whereas, as a positive control, transduced fibroblasts
(Fibro + SeV) expressed all three vectors. The genetic integrity of the
USH2A-RP-iPSC line was determined by karyotype analysis performed
after 12 feeder-free passages, which showed a normal karyotype (46,
XX), ruling out major chromosome abnormalities as a result of the re-
programming process (Fig. 1C). The pluripotency of the generated iPSC
line was evaluated by the endogenous expression of the pluripotency-
associated transcription factors NANOG, OCT3/4 and LIN28a by
quantitative real time PCR (q-PCR). As shown in Fig. 1D, USH2A-RP-
iPSC expressed these genes in contrast to the fibroblast controls (Fibro;
Fibro + SeV), indicating the pluripotent state. This was further con-
firmed by immunofluorescence staining of antibodies specific to
NANOG, OCT3/4 and SOX2 (Fig. 1E). The ability of the USH2A-RP-iPSC
line to differentiate into the three germ layers was explored in vitro
using an embryoid body (EB) assay. The corresponding EBs expressed
markers of the three germ layers as assessed by immunofluorescence
staining of Glial Fibrillary Acidic Protein (GFAP) for ectoderm, Smooth
Muscle Actin (SMA) for mesoderm and α-Fetoprotein (AFP) for en-
doderm (Fig. 1E). We verified the presence of the two USH2A variants
c.2276G > T and c.2299delG, segregating with the retinal phenotype
in the patient, in the USH2A-RP-iPSC line by Sanger sequencing, in
comparison to a wild type control (Fig. 1F). Furthermore, 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
(Suppl. File 1). Lastly, the generated USH2A-RP-iPSC line was con-
firmed to be free of mycoplasma contamination (Suppl. File 2).

Materials and methods

Human dermal fibroblast cell culture

Dermal fibroblast derived from a skin biopsy were cultured in AmnioMAX C100 basal media with GlutaMAX (Gibco) containing 10% decomplemented foetal calf serum (FCS; Lonza), 1% penicillin-streptomycin-amphotericin B (Lonza) and 2% AmnioMax-C100 supplement (Gibco).

Reprogramming of USH2A-RP-iPSC fibroblasts

Fibroblasts were reprogrammed using the integration-free
CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Fibroblasts were seeded
in high glucose DMEM containing GlutaMAX (Gibco) and supplemented
with 10% FCS (Lonza), 1% non-essential amino acids (NEAA) (Gibco)
and 55mM β-mercaptoethanol (Gibco). Cells were transduced ac-
cording to the manufacturer's instructions. The medium was refreshed
daily for 7 days before the cells were passaged onto plates coated with
Matrigel hESC-Qualified Matrix (Corning). On day 8, the medium was
changed to TeSR-E7 Basal Medium (Stemcell Technologies) and was

refreshed daily until iPSC colonies emerged. The colonies were me-
chanically passaged and cultured in Essential 8 (E8) medium (Gibco).
Passages were subsequently performed using Versene solution (Gibco).

Karyotype analysis

iPSC were grown to 50% confluence and incubated for 3 h in E8
containing 0.1 µg/ml Nocodazole (Sigma-Aldrich). Cells were dis-
sociated using TrypLE (Gibco), pelleted (200g, 10 min, RT), gently re-
suspended in buffered hypotonic solution (Genial Genetics) and in-
cubated 20 min at 37 °C. The reaction was stopped with ice-cold fixation
solution (3 vol. methanol: 1 vol. glacial acetic acid), prior to cen-
trifugation at 200g for 10 min at 4 °C. The pellet was washed, re-
suspended in ice-cold fixation solution and stored at -20 °C until
analysis. Twenty metaphase spreads were counted and analyses were
performed by the Chromostem facility (CHU Montpellier, France).

RT-PCR and qPCR analysis

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) and subjected to first strand cDNA synthesis 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 as follows: denaturation (95 °C, 10 min), amplification (95 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min for 35 cycles), and a final extension (72 °C, 10 min). For qPCR analysis, the cDNA was diluted to 2.5 ng/µl and 2 µl was used for each reaction with the FastStart SYBR Green I Master mix on a LightCycler 480 II thermal cycler (Roche). Gene expression was normalized to GAPDH expression. All primers used in this study are listed in Table 2.

Immunofluorescence staining

Cells were fixed using 4% PFA and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich). Non-specific binding was blocked with 1% BSA and 10% donkey serum (Millipore). Primary antibodies were incubated overnight at 4 °C (as a negative control, no primary antibody was used) and fluorescence-conjugated secondary antibodies 1 h at RT (Jackson ImmunoResearch). Nuclei were stained with 0.2 µg/ml bisBenzimide (Sigma-Aldrich). Cells were observed using a Zeiss ApoTome 2 Upright wide-field microscope.

In vitro differentiation assay

iPSC were dissociated with Accutase (Stemcell Technologies) and seeded on ultra-low attachment dishes for 2 days in E8 containing Y27632 StemMACS. At day 3, the medium was changed to DMEM/F12 (Gibco) supplemented with 20% Knockout serum replacement (Gibco), 1% penicillin-streptomycin (Gibco), 1% GlutaMax, 55 mM β-mercaptoethanol and 1% NEAA. At day 7, the embryoid bodies were seeded onto Matrigel-coated wells and culture for a further 10 days before immunofluorescence staining.

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.
A

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B

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C

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D

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<figure><img src="image_7.png" /></figure>

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E

DAPI/NANOG

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DAPI/OCT3/4

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DAPI/SOX2

<figure><img src="image_11.png" /></figure>

F

DAPI/GFAP

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DAPI/SMA

<figure><img src="image_13.png" /></figure>

DAPI/AFP

<figure><img src="image_14.png" /></figure>

G

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<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>Fig. 1. Characterization of the INMi001-A (USH2A-RP-iPSC) line</figcaption></figure>
<table><caption>**Table 1**

Characterization and validation.</caption><thead><tr><th>Classification</th><th>Test</th><th>Result</th><th>Data</th></tr></thead><tbody><tr><td>Morphology</td><td>Photography<br>Microscopy</td><td>Normal morphology</td><td>Fig. 1A</td></tr><tr><td>Phenotype</td><td>Qualitative analysis<br>Immunofluorescence staining<br>Quantitative analysis<br>qPCR</td><td>Positive for pluripotency markers: NANOG, OCT3/4 and SOX2<br>Expression of pluripotency markers: NANOG, OCT3/4, LIN28a</td><td>Fig. 1E<br>Fig. 1D</td></tr><tr><td>Genotype</td><td>Karyotype (G-banding) and resolution</td><td>46XX, Resolution 400-500</td><td>Fig. 1C</td></tr><tr><td>Identity</td><td>Microsatellite PCR (mPCR) OR</td><td>DNA Profiling Performed<br>D3S306<br>D8S532<br>D11S4191<br>D17S787<br>D19S572</td><td>Suppl. file 1</td></tr><tr><td>Mutation analysis</td><td>STR analysis<br>Sequencing</td><td>Not performed<br>Compound heterozygous, USH2A c.2276G > T and c.2299delG</td><td>N/A<br>Fig. 1G</td></tr><tr><td>Microbiology and virology</td><td>Southern Blot OR WGS</td><td>Not performed</td><td>N/A</td></tr><tr><td>Differentiation potential</td><td>Mycoplasma<br>Embryoid body formation</td><td>Mycoplasma testing luminescence. Negative<br>Expression of GFAP (ectoderm), SMA (mesoderm) and AFP (endoderm) in iPSC-derived EBs</td><td>Suppl. file 2<br>Fig. 1F</td></tr><tr><td>Donor screening (OPTIONAL)</td><td>Hepatitis A, Hepatitis B, Hepatitis C, HIV 1-2</td><td>Negative</td><td>Not shown but available with authors</td></tr><tr><td>Genotype additional info (OPTIONAL)</td><td>Blood group genotyping<br>HLA tissue typing</td><td>N/A<br>N/A</td><td>N/A<br>N/A</td></tr></tbody></table>

<table><caption>**Table 2**

Reagents details.</caption><caption>Antibodies used for immunocytochemistry/flow-cytometry</caption><thead><tr><th></th><th>Antibody</th><th>Dilution</th><th>Company cat # and RRID</th></tr></thead><tbody><tr><td>Pluripotency Markers</td><td>Rabbit anti-SOX2</td><td>1/200</td><td>Thermo Fisher Scientific Cat# 48-1400, RRID:AB_2533841</td></tr><tr><td>Pluripotency Markers</td><td>Rabbit anti-NANOG</td><td>1/200</td><td>Abcam Cat# ab21624, RRID:AB_446437</td></tr><tr><td>Pluripotency Markers</td><td>Mouse anti-OCT3/4</td><td>1/200</td><td>Santa Cruz Biotechnology Cat# sc-5279, RRID:AB_628051</td></tr><tr><td>Differentiation Markers</td><td>Rabbit anti-GFAP</td><td>1/200</td><td>Dako Cat# Z0334, RRID:AB_10013382</td></tr><tr><td>Differentiation Markers</td><td>Mouse anti-SMA</td><td>1/200</td><td>Dako Cat# M0851, RRID:AB_2223500</td></tr><tr><td>Differentiation Markers</td><td>Mouse anti-AFP</td><td>1/200</td><td>Sigma Aldrich Cat# WH0000174M1, RRID:AB_1839587</td></tr><tr><td>Secondary antibodies</td><td>Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG (H + L)</td><td>1/500</td><td>Jackson ImmunoResearch, Cat# 711-545-152, RRID: AB_2313584</td></tr><tr><td>Secondary antibodies</td><td>Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H + L)</td><td>1/500</td><td>Jackson ImmunoResearch, Cat# 715-545-150, RRID: AB_2340846</td></tr><tr><td>Secondary antibodies</td><td>Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H + L)</td><td>1/500</td><td>Jackson ImmunoResearch, Cat# 711-606-152, RRID: AB_2340625</td></tr></tbody></table>

<table><caption>Primers</caption><thead><tr><th></th><th>Target</th><th>Forward/Reverse primer (5'-3')</th><th>Product size (bp)</th></tr></thead><tbody><tr><td>Exogenous reprogramming transgene (RT-PCR)</td><td>KOS</td><td>ATGCACCGCTACGACGTGAGCGC<br>ACCTTGACAATCCTGATGTGG</td><td>528</td></tr><tr><td>Exogenous reprogramming transgene (RT-PCR)</td><td>KLF4</td><td>TTCCTGCATGCCAGAGGAGCCC<br>AATGTATCGAAGGTGCTCAA</td><td>410</td></tr><tr><td>Exogenous reprogramming transgene (RT-PCR)</td><td>c-MYC</td><td>TAACTGACTAGCAGGCTTGTCG<br>TCCACATACAGTCCTGGATGATGATG</td><td>532</td></tr><tr><td>Pluripotency marker (RT-qPCR)</td><td>NANOG</td><td>CAAAGGCAAACAACCCACTT<br>TCTGCTGGAGGCTGAGGTAT</td><td>158</td></tr><tr><td>Pluripotency marker (RT-qPCR)</td><td>OCT3/4</td><td>GTACTCCTCGGTCCCTTTCC<br>CAAAAACCCTGGCACAAACT</td><td>168</td></tr><tr><td>Pluripotency marker (RT-qPCR)</td><td>LIN28a</td><td>GGGGAATCACCTACAACCT<br>CTTGGCTCCATGAATCTGGT</td><td>166</td></tr><tr><td>GAPDH (RT-qPCR and RT-PCR)</td><td>GAPDH</td><td>AACCATGAGAAGTATGACAAC<br>CTTCCACGATACCAAAGTT</td><td>112</td></tr><tr><td>USH2A exon 13</td><td>USH2A</td><td>GAAGTTCATCGCAAACAGTTG<br>CACTGATTACAGCGAAGACCTG</td><td>686</td></tr></tbody></table>

### 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).
## Acknowledgements

The authors are grateful to the patient who contributed to this study. We thank Audrey Senechal (Neurogenetic facility, INM, Montpellier) for help with sequencing analysis, Pauline Bouret and Franck Pellestor (Chromostem facility, CHU Montpellier) for karyotype analyses, and Daria Mamaeva and Stephanie Venteo (INM) for helpful discussions. This work was supported by Aviesan-Unadev, Vaincre Usher 2 and SOS retinite.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scr.2018.11.004.

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Lenassi, E., Vincent, A., Li, Z., Saihan, Z., Coffey, A.J., Steele-Stallard, H.B., ... Webster, A.R., 2015. A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants. Eur. J. Hum. Genet. 23 (10), 1318–1327. https://doi.org/10.1038/ejhg.2014.283.

Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S., 2007. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131 (5), 861–872. https://doi.org/10.1016/j.cell.2007.11.019.

Verbakel, S.K., van Huet, R.A.C., Boon, C.J.F., den Hollander, A.I., Collin, R.W.J., Klaver, C.C.W., ... Klevering, B.J., 2018. Non-syndromic retinitis pigmentosa. Prog. Retin. Eye Res. 17, S1350–S9462. https://doi.org/10.1016/j.preteyeres.2018.03.005.