Abstract
Purpose :
Mutations in the MYO7A gene result in severe cases of Usher Syndrome type 1B (USH1B), which is characterized by profound deafness at birth, vestibular abnormalities, and early, progressive retinal degeneration. We sought to grow retinal organoids and retinal pigment epithelium (RPE) cells from healthy control and USH1B patient induced pluripotent stem cells (iPSCs) to better model USH1B retinal pathogenesis and to develop novel therapies.
Methods :
Peripheral blood mononuclear cells from three USH1B patients with a disease-causing mutation(s) in the MYO7A gene (MYO7AT165M/Y108I; MYO7AR1240Q/R1240Q; MYO7AR1240Q/R1861*) were reprogrammed to generate iPSCs. Pluripotency was confirmed in each iPSC line using immunocytochemistry and RT-PCR to detect the presence of OCT4, SOX2, and NANOG. In addition, Sanger sequencing was utilized to confirm the presence of the MYO7A mutation(s) for each line. Finally, karyotyping analysis was performed to ensure no chromosomal abnormalities were acquired during the reprogramming process. The characterized stem cells were differentiated to make retinal organoids and RPE cells, and immunofluorescence staining was used to identify key markers for RPE cells.
Results :
Each USH1B patient line has been successfully reprogrammed and expresses the desired markers indicating pluripotency. Healthy control RPE cells, grown for 109 days in suspension culture, express cell type-specific markers, including PAX6, OTX2, MITF, PMEL17, ZO-1, RPE65, and MYO7A, indicating RPE development and maturation. Direct iPSC-RPE differentiations have also revealed early RPE markers, including PAX6, MITF, and OTX2, and show pigmented foci at D25 of differentiation in a control line.
Conclusions :
This work will allow us to study the pathophysiology of USH1B, including the nature of the interactions between diseased and control photoreceptors and RPE cells, as well as the role that MYO7A plays in retinal disease features and progression. Importantly, this in vitro patient cell-derived model system will help us develop and test novel therapies to treat USH1B.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.