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An in vitro investigation of the impact of PAX6 haploinsufficiency on human corneal stromal cell phenotype

Sanchez Martinez, Carla; (2020) An in vitro investigation of the impact of PAX6 haploinsufficiency on human corneal stromal cell phenotype. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Aniridia related keratopathy (ARK), caused by PAX6 haploinsufficiency, leads to corneal opacification and sight loss. ARK is still an unmet clinical need, as treatments are not always available or successful. Therefore, it is essential to clarify the biological mechanisms behind ARK to develop new efficient clinical treatments. It was hypothesized that aniridic corneal stromal cells are affected and contribute to the development of ARK. To test this hypothesis, stromal cells were isolated from central human aniridic corneas and compared to normal corneal stromal cells isolated from healthy donors. Cells were cultured in 2D and in 3D tissue equivalent culture models to identify ARK-associated features. For the first time, aniridic and normal corneal stromal cells were successfully cultured and expanded in vitro. Unexpectedly, aniridic and normal corneal stromal cells showed similar phenotype in 2D. They presented similar stem cell-like characteristics when cultured in 2% FBS containing media and showed the potential to differentiate into keratocyte-like cells when differentiated in serum-free medium. Aniridic corneal stromal cells showed reduced proliferation capacity when cultured both in 2D and in 3D. Moreover, when cultured inside the 3D tissue equivalent, aniridic corneal stromal cells showed different morphology and distinct spatial distribution when compared to normal cells. RNA sequencing data showed major differences between aniridic and normal corneal stromal cells transcriptomes (with 153 differentially expressed genes being identified), with significant differences in expression levels of genes involved in several biological processes, including matrix remodelling. Real-Time qPCR and ELISA confirmed lower expression and secretion levels of MMP1 and MMP2 (proteins involved on matrix remodelling) by aniridic corneal stromal cells when compared to their normal counterparts. In conclusion, it was demonstrated for the first time that aniridic corneal stromal cells can be successfully cultured in 2D and 3D environments. The differences observed between aniridic and normal corneal stromal cell phenotypes in vitro, suggest that aniridic corneal stromal cells have impaired functionality in vivo and therefore might contribute to the development of ARK.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: An in vitro investigation of the impact of PAX6 haploinsufficiency on human corneal stromal cell phenotype
Event: UCL (University College London)
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10091456
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