Abstract
Purpose :
Retinitis pigmentosa (RP) leads to a progressive dysfunction and loss of rod and cone photoreceptors, resulting in defective dark adaptation, loss of peripheral vision and ultimately blindness. Mutations in rhodopsin, the light sensitive protein of rod photoreceptors, are the most common cause of autosomal dominant RP. Rhodopsin mutations can be classified on their biochemical and cellular properties; however, the mechanisms of rhodopsin-induced cell death in different classifications are still unclear. In this study, we aimed to produce induced pluripotent stem cells (iPSCs) from patients with M39R, R135W, and P347L mutations to understand of the mechanisms of rhodopsin mutation on retinal degeneration.
Methods :
Dermal fibroblasts were isolated from patient skin biopsies and reprogrammed into iPSCs with episomal Yamanaka factors after confirmation of mutations by Sanger sequencing. 12 colonies were picked and 2 were selected from each patient for detailed analysis. To characterize the selected clones, PCR and immunocytochemistry were performed to detect the expression of pluripotency markers, Oct4, Nanog, Tra1-60, and SSEA4.
Results :
Dermal fibroblast lines were established. The presence of mutations in fibroblasts isolated from patients were confirmed by Sanger sequencing. Dermal fibroblasts were successfully reprogrammed into iPSCs using episomal Yamanaka factors and 2 clones from each cell line studied in detail. This confirmed expression of pluripotency markers; Oct4, Nanog, Tra1-60, and SSEA4. PCR confirmed the loss of episomal vectors. In addition, iPSC will be differentiated to photoreceptors using methods to produce 3D retinal organoids and analysed with retinal markers and morphology.
Conclusions :
iPSC can be generated from patient fibroblasts with dominant rhodopsin mutations. 3D retinal organoids differentiated from patient iPSCs will be a good model to study RP caused by rhodopsin mutations in human photoreceptors with the mutations at the correct gene dosage and genomic context. They will provide a platform not only to have further understanding of disease mechanisms leading to blindness, but also to develop potential therapies for this incurable disease, especially any that are sequence or context dependent.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.