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

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

Hamel, Isabelle Meunier, Vasiliki Kalatzis

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## To cite this version:

Carla Sanjurjo-Soriano, Nejla Erkilic, Gael Manes, Grégor Dubois, Christian Hamel, et al. Generation of a human iPSC line, INMi002-A, carrying the most prevalent USH2A variant associated with Usher syndrome type 2. Stem Cell Research, 2018, 33, pp.247-250. 10.1016/j.scr.2018.11.007. hal-02307062

HAL Id: hal-02307062

https://hal.umontpellier.fr/hal-02307062

Submitted on 8 Oct 2019

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Lab resource: Stem Cell Line

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

<figure><img src="image_3.png" /></figure>

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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 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 table

Unique stem cell line identifier
Alternative name(s) of stem cell line
Institution

Contact information of distributor

Type of cell line

Origin

Additional origin info

Cell Source

Clonality

Method of reprogramming

Genetic modification

Type of modification

Associated disease

Gene/locus

Method of modification

Name of transgene or resistance

Inducible/constitutive system

Date archived/stock date

Cell line repository/bank

Ethical approval

INMi002-A
USH2A-USH-iPSC

Institute for Neurosciences of Montpellier,
Montpellier, France

Vasiliki Kalatzis vasiliki.kalatzis@inserm.fr

iPSC

Human

Age: 59 years old

Sex: Female

Ethnicity: Caucasian

Dermal fibroblasts

Clonal

Non-integrative Sendai virus vectors

Yes

Congenital mutation

Usher Syndrome type II

USH2A, 1q41

N/A

N/A

N/A

December 2017

https://hpscreg.eu/cell-line/INMi002-A

Regional committee: CPP Southern

Mediterranean I (2014-A00549-38)

National committee: ANSM (140549B-62)

## 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). The morphology of the iPSC line generated displayed a typical colony

<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>
<figure><img src="image_4.png" /><figcaption>Fig. 1. Characterization of the INMi002-A (USH2A-USH-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>Immunocytochemistry<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–1G<br>Fig. 1D</td></tr><tr><td>Genotype<br>Identity</td><td>Karyotype (G-banding) and resolution<br>Microsatellite PCR (mPCR) OR</td><td>46XX Resolution 400–500<br>DNA Profiling Performed<br>D3S306<br>D8S532<br>D11S4191<br>D17S787<br>D19S572</td><td>Fig. 1C<br>Available with authors</td></tr><tr><td>Mutation analysis</td><td>STR analysis<br>Sequencing<br>Southern Blot OR WGS</td><td>Not performed<br>Homozygous, USH2A c.2299delG<br>Not performed</td><td>N/A<br>Fig. 1K<br>N/A</td></tr><tr><td>Microbiology and virology<br>Differentiation potential</td><td>Mycoplasma<br>Embryoid body formation</td><td>Mycoplasma testing by luminescence. Negative<br>Expression of GFAP (ectoderm), SMA (mesoderm) and AFP<br>(endoderm) in iPSC-derived EBs</td><td>Supplementary File 1<br>Fig. 1H–1J</td></tr><tr><td>Donor screening (OPTIONAL)<br>Genotype additional info<br>(OPTIONAL)</td><td>Hepatitis A, Hepatitis B, Hepatitis C, HIV 1–2<br>Blood group genotyping<br>HLA tissue typing</td><td>Negative<br>N/A<br>N/A</td><td>Available with authors<br>N/A<br>N/A</td></tr></tbody></table>

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, non-transduced 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 (Fig. 1E), OCT3/4 (Fig. 1F) and SOX2 (Fig. 1G). The ability of the INMi002-A cell line to give rise to the three embryonic cell layers was determined by spontaneous differentiation of the iPSC into embryoid bodies (EBs). EBs were plated onto Matrigel-coated dishes and the expression of the three germ layer markers, Glial Fibrillary Acidic Protein (GFAP; ectoderm; Fig. 1H), Smooth Muscle Actin (SMA; mesoderm; Fig. 1I) and α-Fetoprotein (AFP; endoderm; Fig. 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).

## Materials and methods

### Human dermal fibroblast cell culture

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

### iPSC generation

Fibroblasts were seeded and reprogrammed using the integration-free CytoTune™-iPS 2.0 Sendai Reprogramming Kit as previously described (Torriano et al., 2017). iPSC colonies were cultured in Essential 8 (E8) medium (Gibco) under 5% CO₂ at 37 °C, and passaged (1:10 dilution; twice weekly) using Versene (Gibco).

### 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.

### Immunofluorescence staining

Cells were fixed using 4% PFA (20 min, RT), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich; 15 min, RT) and blocked (45 min, RT) in 1% BSA (Sigma-Aldrich)/10% donkey serum (Millipore). Primary antibodies were incubated overnight, 4 °C (no primary antibody for negative control), and fluorescence-conjugated secondary antibodies 1 h, 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
<table><caption>**Table 2**
Reagents details.</caption><thead><tr><th colspan="4">Antibodies used for immunocytochemistry/flow-cytometry</th></tr><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></tbody></table>

<table><thead><tr><th colspan="4">Primers</th></tr><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>SeV</td><td>GGATCACTAGGTGATATCGAGC<br>ACCAGACAAGAGTTTAAGAGATATGTATC</td><td>181</td></tr><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>CTGCTGGAGGCTGAGGTAT</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>GGGGAATCACCTACAAACCT<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>

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 (Gibco), 1% NEAA (Gibco) and 55 mM β-mercaptoethanol (Gibco). At day 7, embryoid bodies were seeded onto Matrigel-coated wells and cultured in the same medium for a further 10 days before 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.

### 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.2018.11.007.

## References

Kremer, H., van Wijk, E., Marker, T., Wolfrum, U., Roepman, R., 2006. Usher syndrome: molecular links of pathogenesis, proteins and pathways. Hum. Mol. Genet. 15 (2), 262–270. https://doi.org/10.1093/hmg/ddl205.

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.

Torriano, S., Erkilic, N., Faugère, V., Damodar, K., Hamel, C.P., Roux, A.-F., Kalatzis, V., 2017. Pathogenicity of a novel missense variant associated with choroideremia and its impact on gene replacement therapy. Hum. Mol. Genet. 26 (18), 3573–3584. https://doi.org/10.1093/hmg/ddx244.