Abstract
Purpose :
Peripherin-2 (PRPH2) is known to be expressed by photoreceptors. PRPH2 protein helps maintain photoreceptor disc outer segment curvature; consequently, disease mechanism studies have focused mainly on outer segment pathology. Our group of study have generated patient-derived induced pluripotent stem-cells (iPSCs), retinal pigment epithelial cells (RPE), and retinal organoids to better understand PRPH2-associated retinal disease. Therefore, the aim of this study was to evaluate if PRPH2 was expressed by these cells, which may help in understanding unexplained disease mechanisms and phenotypic variability, and in developing functional studies.
Methods :
We initially investigated mouse retinal cryosections for PRPH2 expression by immunostaining. Then, iPSCs were reprogrammed from peripheral blood mononuclear cells (PBMCs) from a healthy control and a patient with a PRPH2 pathogenic variant. iPSCs were differentiated to RPE and retinal organoids using our standardized protocols, and validated by immunofluorescence (IF) staining. iPSCs, iPSC-derived RPE and iPSC-derived organoids were initially assessed for PRPH2 mRNA expression by PCR and, subsequently, for PRPH2 protein expression by western blot and/or IF.
Results :
PRPH2 protein was strongly expressed in photoreceptor outer segments and focally in RPE of mouse retinal cryosections, suggesting that RPE may express PRPH2 (Fig. 1A). To determine if RPE expressed PRPH2independently of outer segment phagocytosis we first performed PCR, confirming PRPH2 expression in human iPSCs, iPSC-derived RPE and iPSC-derived retinal organoids. Western blot experiments detected protein expression in iPSCs, RPE and organoids (Fig. 2). IF staining localized PRPH2 to outer segments, as expected, in 9-month-old organoids (Fig. 1B). Interestingly, IF also detected PRPH2 staining in concentrated nuclear granules in RPE cells (Fig. 1C).
Conclusions :
Our findings suggest PRPH2 is expressed not only by photoreceptors, but also by iPSCs and RPE. The expression of PRPH2 in iPSCs reveals that this model could potentially be used to perform functional studies without the need to differentiate them to retinal organoids. While our protein studies suggest PRPH2 expression in RPE, photoreceptor-independently, further studies are needed to understand whether PRPH2 has a functional role in RPE and whether PRPH2 pathogenic variants in RPE contribute to disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.