July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Phenotypic study of PAX6 haploinsufficient aniridic corneal stromal cells in a tissue equivalent
Author Affiliations & Notes
  • Carla Sanchez Martinez
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Victoria Tovell
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Stephen J. Tuft
    Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
  • Julie T Daniels
    Institute of Ophthalmology, UCL, London, United Kingdom
  • Footnotes
    Commercial Relationships   Carla Sanchez Martinez, None; Victoria Tovell, None; Stephen Tuft, None; Julie Daniels, None
  • Footnotes
    Support  Moorfields Eye Charity Studentship
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 523. doi:
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      Carla Sanchez Martinez, Victoria Tovell, Stephen J. Tuft, Julie T Daniels; Phenotypic study of PAX6 haploinsufficient aniridic corneal stromal cells in a tissue equivalent. Invest. Ophthalmol. Vis. Sci. 2018;59(9):523.

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

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Purpose : Aniridia related keratopathy (ARK), caused by PAX6 haploinsufficiency, leads to corneal opacification and subsequently, sight loss. The biological mechanisms of ARK progression are not yet fully understood. Most research studies focus on the corneal epithelium. We hypothesised that the aniridic corneal stromal cells are also affected and contribute to the development of ARK. For the first time, we cultured and studied the phenotype of human aniridic corneal stromal cells (ACSC) and control corneal stromal cells (CCSC) inside a tissue equivalent (TE) to identify any ARK specific changes.

Methods : ACSC were isolated from fresh corneas (N=3) and genotyped to identify their PAX6 variants. CCSC were isolated from organ cultured corneas (N=3) and both ACSC and CCSC were cultured inside a compressed collagen I gel for 21 days. Supernatant was collected at day 7,14 and 21 for assessment of MMP-1 and MMP-2 concentration by zymography and ELISA, as an indication of the cell capacity to remodel the extracellular matrix. At the end point, gels were stained and morphology was recorded. Circularity and alignment of the cells were calculated with Fiji ImageJ 1.49s software. Two-way repeated-measures ANOVA (ELISA) and two-tailed Student’s t-test (Image analysis) were used for the statistical analysis.

Results : ACSC and CCSC were successfully isolated, cultured and expanded. One run-on (NM_000280: p.*423Leuext*?) and two premature stops (NM_000280: p.(Gly194*), NM_000280: p.(Arg125Serfs*7)) variants were identified in the PAX6 gene of the three aniridic samples, respectively. When cultured inside the TE, ACSC secreted lower amounts of MMP-1 and MMP-2 than CCSC at day 14 (p<0.05) and 21 (p<0.001) (ELISA), in accordance with zymography results. Cell distribution and morphology also differed between ACSC and CCSC. While CCSC appeared to be thin and elongated (Circularity=0.135), ACSC had a wider range of morphologies with many cells presenting a rounded body with protrusions (Circularity= 0.321, p<0.001). CCSC presented a 75% of alignment with each other, whereas ACSC showed only 39%.

Conclusions : In the native cornea, stromal cells are quiescent and align inside the collagen matrix. Evident differences between the two cells phenotypes in culture indicate that the ability of ACSC to remodel the matrix might be impaired and this might contribute to the development of ARK.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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