June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Transcriptomic analysis of aniridia iPSC-derived limbal epithelial stem cells reveals early changes linked to the limbal niche
Author Affiliations & Notes
  • Dulce Lima Cunha
    Radboud Universiteit, Nijmegen, Gelderland, Netherlands
    University College London, London, London, United Kingdom
  • Jos Smits
    Radboud Universiteit, Nijmegen, Gelderland, Netherlands
  • Huiqing Zhou
    Radboud Universiteit, Nijmegen, Gelderland, Netherlands
    Radboudumc, Nijmegen, Gelderland, Netherlands
  • Mariya Moosajee
    University College London, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Dulce Lima Cunha None; Jos Smits None; Huiqing Zhou None; Mariya Moosajee None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 2821. doi:
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      Dulce Lima Cunha, Jos Smits, Huiqing Zhou, Mariya Moosajee; Transcriptomic analysis of aniridia iPSC-derived limbal epithelial stem cells reveals early changes linked to the limbal niche. Invest. Ophthalmol. Vis. Sci. 2023;64(8):2821.

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

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Purpose : Aniridia is a pan-ocular disease caused by mutations affecting the transcription factor PAX6. Patients suffer from congenital and progressive ocular problems, the most vision-threatening one being aniridia-related keratopathy (ARK), where deficient function of limbal epithelial stem cells (LESC) in the periphery of the cornea lead to central cornea opacification and vascularisation. However, the exact molecular and cellular mechanisms behind ARK are not yet known. Our purpose is to use induced pluripotent stem cells (iPSC) technology to investigate how limbal and corneal development might be affected by PAX6 defects in aniridia patients.

Methods : We have generated iPSCs from 2 independent aniridia patients carrying PAX6 nonsense mutations and 2 unaffected controls and differentiated these lines into LESCs (iLESCs). Bulk RNAsequencing was performed in triplicate for each line at different stages of differentiation: day 0, day 15 and day 25. Data pre-processing was performed using seq2science, and differentially expressed genes were called using DESEQ2. Gene Ontology (GO)-term and pathway enrichment were run using Clusterprofiler and Progeny.

Results : Analysis of differentiation day 15 show that both control and aniridia iLESCs express early limbal epithelial markers, like TP63 or ABCG2, and downregulation of pluripotency genes compared to day 0 (iPSC). However, compared to control iLESCs, aniridia iLESCs show enrichment of neuronal-related GO-terms and marked downregulation of genes linked to processes involved in the limbal niche, like TGFβ pathway, extracellular matrix or neural crest markers. By differentiation day 25, control cells show a clear epithelial signature but aniridia iLESCs seem to arrest within the differentiation process and present overall downregulation of epithelial genes.

Conclusions : Our results suggest that PAX6 defects may disrupt limbal epithelial stem cell development by first affecting the limbal niche, which in turn may impair LESC identity. These results can have an important impact in therapy development for ARK so further validation studies are ongoing.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.


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