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Yue Li, Ricardo F Frausto, Doug D Chung, E. Maryam Maryam Hanser, Austin Kassels, Marina Zakharevich, Anthony J Aldave; The functional impact of CRISPR-Cas9-mediated ZEB1 deficiency in human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5652. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the impact of ZEB1 deficiency on corneal endothelial cell (CEnC) proliferation, migration and barrier function in a CEnC culture model of posterior polymorphous corneal dystrophy 3 (PPCD3).
ZEB1+/+, ZEB1+/- and ZEB1-/- HCEnC-21T cell lines, generated using CRISPR-Cas9, were used to test the functional impact of ZEB1 deficiency on various cellular processes (cell proliferation, cell migration and cell barrier). To measure cell proliferation, each HCEnC-21T line was seeded at 10% confluency. The number of cells (N) was counted at 3 hours (defined as N0) and compared to the number of cells at various time points (Nt: 24, 48, 72 and 96 hours) post-seeding and graphed as a ratio (Nt/N0). A non-wounding (electric fence) protocol using a single electrode array was used to evaluate cell migration by electric cell-substrate impedance sensing (ECIS). Cell barrier function was measured using a 40-electrode array designed, in part, for measuring the resistance to an applied electrical field through intercellular junctions.
ZEB1+/- and ZEB1-/- HCEnC-21T cells demonstrated significantly impaired cell proliferation at 72 and 96 hours post-seeding compared with ZEB1+/+ HCEnC-21T cells (p<0.0001). No statistically significant difference in cell proliferation was observed between ZEB1+/- and ZEB1-/- at any of the time points tested. Cell migration assays demonstrated significantly slower migration for ZEB1+/- and ZEB1-/- compared with ZEB1+/+ (p<0.05). The ZEB1-/- cells demonstrated significantly greater cell barrier function compared with both ZEB1+/- and ZEB1+/+ beginning at approximately 60 hours post-seeding. By 120 hours, ZEB1+/- established a significantly increased cell barrier function compared with ZEB1+/+, but significantly reduced compared with ZEB1-/-.
We have established a cell-based model of PPCD3 using CRISPR-Cas9. Herein, we provide the first experimental evidence that human corneal endothelial cells deficient in ZEB1 manifest altered cellular proliferation, migration and barrier function. However, additional investigation is necessary to clarify the relevance of these alterations to the development of PPCD3.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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