Abstract
Purpose :
Albinism is a genetic disorder characterized by absent/reduced melanin pigment in eyes, skin and hair. In addition, the retina exhibits foveal hypoplasia and misrouting of the axonal tracts to the brain, resulting in blurred vision. In (ocular) albinism, the RPE has a melanosomal pigment deficiency. The molecular mechanisms that link pigmentation to neural retinal development and disease are yet unclear. To model the disease in vitro, we compared stem cell derived iRPE of OCA1 patients and controls.
Methods :
Human oculocutaneous albinism 1 (OCA1) patient derived iPSCs and controls were embedded in Matrigel for 5 days and cultured in N2B27 medium. The formed embryoid bodies were subsequently plated as single cells on growth factor reduced Matrigel coated trans-well inserts and medium changed to 20% KOSR medium. Produced iRPE was analyzed using RT-PCR and immunohistochemistry (IHC).
Results :
As expected, OCA1 patient stem cell-derived RPE showed no pigmentation at all, while control WT iRPE cell lines started to pigment gradually from day 24 onward. To confirm the RPE identity of the differentiated cells, two general mature RPE markers were analyzed: BEST1 and RPE65. BEST1 is a regulator of fluid flux across the RPE and peaks earlier in patient derived iRPE but is lower than controls in later stages. RPE65 is an enzyme involved in the visual cycle and its expression is lower in later stage patient-derived iRPE. At day 60, expression of a number key disease genes involved in human albinism was different between the OCA1 iRPE control derived iRPE. These results were confirmed using IHC.
Conclusions :
We created and characterized a new stem cell-derived in vitro model for OCA1. This model has been validated and can be used to study molecular mechanisms and signaling pathways of human albinism in vitro.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.