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Lindsey Weed, Kamaljot Gill, Jean Bennett, Jason Mills; Modeling CRB1-associated LCA Using Patient-Specific Retinal Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):985.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the Crumbs homologue 1 (CRB1) gene account for 10-15% of all Leber's congenital amaurosis (LCA) cases. Understanding the disease pathology is pivotal for therapeutic development. However, in vivo functional studies of CRB1 are complicated by the differential expression pattern of CRB1 between human and murine retinas. Here we present our differentiation of CRB1-associated LCA patient-derived induced pluripotent stem cells (iPSCs) to retinal progenitor cells (RPCs) as a model to investigate the early stages of pathophysiology.
Two iPSC lines from CRB1-associated LCA patients and three healthy-sighted controls were derived from PBMCs. iPSC cultures were differentiated in retinal-specific conditions and samples were collected at various time points. Immunohistochemistry and qRT-PCR were used to assess expression of RPC markers and developmental signaling pathways, Wnt and Notch. Flow cytometry was used to quantify EdU labeling of proliferating RPCs.
RPCs were successfully generated from CRB1-/- and control iPSC lines. Early RPCs lost expression of pluripotency markers OCT4 and NANOG and began expressing eye field transcription factors RAX, PAX6, SIX3, and LHX2 by day 7 of the differentiations. Importantly, CRB1 expression was observed as early as day 7 and expression continued throughout retinal patterning. EdU staining revealed differences in the number of proliferating cells between CRB1-/- and control RPCs between day 14 and 21. We observed altered expression of several Wnt signaling pathway components including AXIN2, LEF1, TCF7, and WNT3a between CRB1-/- and control RPCs at the same time points. No significant differences were detected in expression of Notch signaling pathway components between CRB1-/- and control RPCs.
We have achieved highly enriched RPC differentiations from patient-derived iPSC lines characterized by the temporally appropriate expression of eye field transcription factors necessary for development of retinal cell lineages. Differences in the number of proliferating cells between CRB1-/- and control RPCs may partially be explained by alterations in the expression of Wnt signaling pathway components. The increased proliferative rates of CRB1-/- RPCs correlates with the murine models and human clinical phenotypes of hyperproliferation of the neural retina, establishing a model for studying the cellular and molecular phenotypes of CRB1-associated LCA.
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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