July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Development of patient-derived iPSC disease models of TGFBI corneal dystrophies
Author Affiliations & Notes
  • Beatriz Sanchez
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Alice E Davidson
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Kirithika Muthusamy
    Institute of Ophthalmology, University College London, London, United Kingdom
    Moorfields Eye Hospital, London, United Kingdom
  • Stephen J Tuft
    Moorfields Eye Hospital, London, United Kingdom
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Alison J Hardcastle
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Beatriz Sanchez, None; Alice Davidson, None; Kirithika Muthusamy, None; Stephen Tuft, None; Alison Hardcastle, None
  • Footnotes
    Support  Fight for sight
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4151. doi:
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    • Get Citation

      Beatriz Sanchez, Alice E Davidson, Kirithika Muthusamy, Stephen J Tuft, Alison J Hardcastle; Development of patient-derived iPSC disease models of TGFBI corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4151.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : New models and therapies are needed for inherited corneal dystrophies. Mutations in TGFBI are of special interest since they are the most common cause of dominant lattice, Reis-Bücklers, granular and Thiel-Behnke corneal dystrophies. We aim to use patient-derived induced pluripotent stem cells (iPSC) as an in vitro model to replicate the disease in its cellular and genomic context, to test therapeutic approaches.

Methods : Skin biopsies were obtained from patients with heterozygous c.1664G>A, p.Arg555Gln and c.1663C>T, p.Arg555Trp mutations to generate fibroblast lines. The genotypes were confirmed by gDNA extraction and Sanger Sequencing. Patient-derived iPSC lines were generated by nucleofection of fibroblasts with the Yamanaka factors. In addition, heterozygous and homozygous TGFBI knockout lines were generated by simultaneously using CRISPR-Cas9 technology and reprogramming with Yamanaka factors. Corneal epithelial-like cell lines were generated from each iPSC line, with an initial induction with small molecules followed by culture in a commercial medium to favour epithelial cell growth and differentiation in feeder-free conditions. Immunocytochemistry (ICC) and RT-PCR were used to characterise iPSC and epithelial-like cells.

Results : Endogenous expression of the transcription factors Oct3/4, Sox2, L-myc and Lin28 was detected by RT-PCR in the stem cell lines prior to differentiation. We used immunofluorescence to confirm the expression of the stem cell markers Oct4, Nanog, SSEA4, Tra-1-61 and Tra-1-81 in all iPSC lines. Differentiated corneal epithelial-like cells showed mRNA expression of ΔNp63, KRT14, KRT15 from differentiation day (D) 14; KRT3 at D 30, and no expression of KRT12. Expression of the proteins E-Cadherin, KRT14 and ABCG2 were also confirmed at different points during differentiation.

Conclusions : We have developed disease-associated (p.Arg555Gln and p.Arg555Trp) and knock-out TGFBI cell models to investigate the molecular mechanisms of TGFBI-associated dystrophies in corneal epithelial-like cells and test therapeutic approaches. Our next step is to use gene editing tools to design drugs that can molecularly target the specific genetic mutations, which in conjunction with genotyping, could be used for personalised treatment of these corneal dystrophies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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