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Jessica Eason, Gary Smith, Brenda L Bohnsack; Differentiation and characterization of PAX6 mutant human embryonic stem cells derived from a family affected with aniridia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6056.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in PAX6 are primarily associated with aniridia, a congenital disease that affects almost all structures of the eye. Animal studies have demonstrated that PAX6 is expressed in the optic cup and surface ectoderm in the developing eye. Studies in humans have been limited due to access to tissue. In these studies, we used human embryonic stem cells (ESCs) to develop a model to further understand the role of PAX6 in eye development.
Human ESCs were derived from a family affected with aniridia that had a known 250kB deletion in one copy of the PAX6 gene. Both wildtype and PAX6 mutant hESCs were directed towards retinal cells through a stepwise protocol using defined mediums (neural induction medium and retinal differentiation medium). Cells were analyzed for defined gene markers at each time point using cell morphology, end point RT-PCR, western blot and immunocytochemistry.
Human ESCs from both wildtype and PAX6 mutant lines differentiated into primitive neuroepithelial cells in less than 14 days. Analysis by RT-PCR demonstrated equal expression of neural markers PAX6 and SOX1 as well as OTX2, RAX, SIX3 and LHX2 in both lines. The primitive neuroepithelial cells from both lines also expressed PAX6 as shown by immunocytochemistry. Retinal pigment epithelial cells formed in approximately 110 days and were easily identified by their hexagonal morphology and pigmentation in both wildtype and PAX6 mutant lines. RPE65, OTX2, PAX6, and CHX10 were expressed in the wildtype and the PAX6 mutant cells as demonstrated by RT-PCR. Immunocytochemistry showed expression of ZO-1 and OTX2 and Western blot demonstrated expression of PAX6 in both lines.
Human ESCs derived from blastocysts containing gene mutations associated with congenital eye anomalies can be used to understand human eye development and disease pathogenesis. We have taken the first steps in characterizing PAX6 mutant human ESCs. Further research is ongoing to analyze interactions between PAX6 mutant cells and their ability to form 3-dimensional optic-cup structures. The lack of difference in gene and protein expression between wildtype and PAX6 mutant human ESC lines suggests that additional post-transcriptional and translational regulatory mechanisms are important in eye development.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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