Abstract
Purpose :
Retinitis Punctata Albescens (RPA) is caused by mutations in the RLBP1 gene, which codes for the visual cycle protein CRALBP. A CRALBP-deficiency results in a dysfunction of the retinal pigment epithelium (RPE) that leads to RPE and eventually photoreceptor death. An Rlbp1-deficient mouse model has been previously generated but it does not fully reproduce the disease course. As a complement to this in vivo model, we generated a human retinal model for RPA via the intermediate of patient induced pluripotent stem cells (iPSc).
Methods :
We performed punch skin biopsies of three RPA patients carrying different mutations in the RLBP1 gene. The CRALBP-deficient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSc) using initially, retroviral and, subsequently, sendai viral vectors, carrying the Yamanaka transcription factor cocktail. The RPA iPSc were then differentiated into RPE using a spontaneous differentiation protocol.
Results :
We obtained iPSc from the 3 RPA patients. We determined their pluripotency by qPCR analysis for the expression of Nanog, Sox-2, Lin 28 and Oct3-4 and by their ability to produce teratomas. We determined their genetic stability by karyotype analysis and confirmed the presence of the original RLBP1 mutations by sequencing. The iPSc-derived RPE from RPA patients expresses characteristic RPE markers but lacks CRALBP. We mimicked the visual cycle by incubating the cells with vitamin A and showed that the RPA iPSc-derived RPE contains an accumulation of all-transretinyl esters within the cells and a decrease in the production of 11 cis-retinal in the supernatant, as compared to control cells. In addition, transmission electron microscopy studies detected morphological differences between patient and control RPE.
Conclusions :
This is the first iPSc-derived RPE model described for RPA. This model is a unique tool, as it will allow i) the direct study of disease pathophysiology and ii) the testing of novel therapies on the tissue actually affected in patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.